POLYNUCLEOTIDE AGENTS TARGETING COMPLEMENT COMPONENT C5 AND METHODS OF USE THEREOF

Abstract

The invention relates to polynucleotide agents targeting the complement component C5 gene, and methods of using such polynucleotide agents to inhibit expression of C5 and to treat subjects having a complement component C5-associated disease, e.g., paroxysmal nocturnal hemoglobinuria.

Description

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 15/450,326, filed on Mar. 6, 2017, which is a 35 .sctn. U.S.C. 111(a) continuation application which claims the benefit of priority to PCT/US2015/049368, filed on Sep. 10, 2015, which, in turn, claims the benefit of priority to U.S. Provisional Patent Application No. 62/049,775, filed on Sep. 12, 2014. The entire contents of each of the aforementioned applications are incorporated herein by reference.

SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 28, 2019, is named 121301_02203_SL.txt and is 240,658 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Complement was first discovered in the 1890s when it was found to aid or "complement" the killing of bacteria by heat-stable antibodies present in normal serum (Walport, M. J. (2001) N Engl J Med. 344:1058). The complement system consists of more than 30 proteins that are either present as soluble proteins in the blood or are present as membrane-associated proteins. Activation of complement leads to a sequential cascade of enzymatic reactions, known as complement activation pathways, resulting in the formation of the potent anaphylatoxins C3a and C5a that elicit a plethora of physiological responses that range from chemoattraction to apoptosis. Initially, complement was thought to play a major role in innate immunity where a robust and rapid response is mounted against invading pathogens. However, recently it is becoming increasingly evident that complement also plays an important role in adaptive immunity involving T and B cells that help in elimination of pathogens (Dunkelberger J R and Song W C. (2010) Cell Res. 20:34; Molina H, et al. (1996) Proc Natl Acad Sci USA. 93:3357), in maintaining immunologic memory preventing pathogenic re-invasion, and is involved in numerous human pathological states (Qu, H, et al. (2009) Mol Immunol. 47:185; Wagner, E. and Frank M M. (2010) Nat Rev Drug Discov. 9:43).

[0004] Complement activation is known to occur through three different pathways: alternate, classical, and lectin (FIG. 1), involving proteins that mostly exist as inactive zymogens that are then sequentially cleaved and activated. All pathways of complement activation lead to cleavage of the C5 molecule generating the anaphylatoxin C5a and, C5b that subsequently forms the terminal complement complex (C5b-9). C5a exerts a predominant pro-inflammatory activity through interactions with the classical G-protein coupled receptor C5aR (CD88) as well as with the non-G protein coupled receptor C5L2 (GPR77), expressed on various immune and non-immune cells. C5b-9 causes cytolysis through the formation of the membrane attack complex (MAC), and sub-lytic MAC and soluble C5b-9 also possess a multitude of non-cytolytic immune functions. These two complement effectors, C5a and C5b-9, generated from C5 cleavage, are key components of the complement system responsible for propagating and/or initiating pathology in different diseases, including paroxysmal nocturnal hemoglobinuria, rheumatoid arthritis, ischemia-reperfusion injuries and neurodegenerative diseases.

[0005] To date, only one therapeutic that targets the C5-C5a axis is available for the treatment of complement component C5-associated diseases, the anti-C5 antibody, eculizumab (Soliris.RTM.). Although eculizumab has been shown to be effective for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) and is currently being evaluated in clinical trials for additional complement component C5-associated diseases, eculizumab therapy requires weekly high dose infusions followed by biweekly maintenance infusions at a yearly cost of about $400,000. Accordingly, there is a need in the art for alternative therapies for subjects having a complement component C5-associated disease.

SUMMARY OF THE INVENTION

[0006] The present invention provides antisense polynucleotide agents and compositions comprising such agents which target nucleic acids encoding complement component C5 and interfere with the normal function of the targeted nucleic acid. The C5 nucleic acid may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods and combination therapies for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a C5 mRNA, e.g., a complement component C5-associated disease, such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) using the antisense polynucleotide agents and compositions of the invention.

[0007] Accordingly, in one aspect, the present invention provides antisense polynucleotide agents for inhibiting expression of complement component C5. The agents comprise about 4 to about 50 contiguous nucleotides, wherein at least one of the contiguous nucleotides is a modified nucleotide, and wherein the nucleotide sequence of the agent is about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs:1-4.

[0008] In one embodiment, the equivalent region is one of the target regions of SEQ ID NO:1 provided in Table 3, e.g., residues 1-20, 12-31, 23-42, 34-53, 188-207, 287-306, 430-449, 716-735, 969-988, 1244-1263, 1695-1714, 2025-2044, 2289-2308, 2531-2550, 2817-2836, 3092-3111, or 3884-3903 of SEQ ID NO:1.

[0009] In another aspect, the present invention provides antisense polynucleotide agents for inhibiting expression of complement component C5, wherein the agent comprises at least 8 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences listed in Table 3.

[0010] In some embodiments, substantially all of the nucleotides of the antisense polynucleotide agentas of the invention are modified nucleotides. In other embodiment, all of the nucleotides of the antisense polynucleotide agent are modified nucleotides.

[0011] The antisense polynucleotide agent may be 10 to 40 nucleotides in length; 10 to 30 nucleotides in length; 18 to 30 nucleotides in length; 10 to 24 nucleotides in length; 18 to 24 nucleotides in length; or 20 nucleotides in length.

[0012] In one embodiment, the modified nucleotide comprises a modified sugar moiety selected from the group consisting of: a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, and a bicyclic sugar moiety.

[0013] In one embodiment, the bicyclic sugar moiety has a (--CH2-)n group forming a bridge between the 2' oxygen and the 4' carbon atoms of the sugar ring, wherein n is 1 or 2.

[0014] In another embodiment, the modified nucleotide is a 5-methylcytosine.

[0015] In one embodiment, the modified nucleotide comprises a modified internucleoside linkage, such as a phosphorothioate internucleoside linkage.

[0016] In one embodiment, an agent of the invention comprises a plurality of 2'-deoxynucleotides flanked on each side by at least one nucleotide having a modified sugar moiety.

[0017] In one embodiment, the agent is a gapmer comprising a gap segment comprised of linked 2'-deoxynucleotides positioned between a 5' and a 3' wing segment.

[0018] In one embodiment, the modified sugar moiety is selected from the group consisting of a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, and a bicyclic sugar moiety.

[0019] In one embodiment, the 5'-wing segment is 1 to 6 nucleotides in length, e.g., 2, 3, 4, or 5 nucleotides in length.

[0020] In one embodiment, the 3'-wing segment is 1 to 6 nucleotides in length, e.g., 2, 3, 4, or 5 nucleotides in length.

[0021] In one embodiment, the gap segment is 5 to 14 nucleotides in length, e.g., 10 nucleotides in length.

[0022] In one aspect, the present invention provides antisense polynucleotide agents for inhibiting complement component C5 expression, comprising a gap segment consisting of linked deoxynucleotides; a 5'-wing segment consisting of linked nucleotides; a 3'-wing segment consisting of linked nucleotides; wherein the gap segment is positioned between the 5'-wing segment and the 3'-wing segment and wherein each nucleotide of each wing segment comprises a modified sugar.

[0023] In one embodiment, the gap segment is ten 2'-deoxynucleotides in length and each of the wing segments is five nucleotides in length.

[0024] In another embodiment, the gap segment is ten 2'-deoxynucleotides in length and each of the wing segments is four nucleotides in length.

[0025] In yet another embodiment, the gap segment is ten 2'-deoxynucleotides in length and each of the wing segments is three nucleotides in length.

[0026] In another embodiment, the gap segment is ten 2'-deoxynucleotides in length and each of the wing segments is two nucleotides in length.

[0027] In one embodiment, the modified sugar moiety is selected from the group consisting of a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, and a bicyclic sugar moiety.

[0028] In some embodiments, the agents of the invention further comprise a ligand.

[0029] In one embodiment, the agent is conjugated to the ligand at the 3'-terminus.

[0030] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0031] In one embodiment, the ligand is

##STR00001##

[0032] In one aspect, the present invention provides pharmaceutical compositions for inhibiting expression of a complement component C5 gene comprising the agents of the invention.

[0033] In one embodiment, the agent is present in an unbuffered solution, such as saline or water.

[0034] In another embodiment, the agent is present in a buffer solution, such as a buffer comprising acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.

[0035] In one embodiment, the buffer solution is phosphate buffered saline (PBS).

[0036] In another aspect, the present invention provides pharmaceutical composition comprising an agent of the invention and a lipid formulation, such as a lipid formulation comprising an LNP or a MC3.

[0037] In one aspect, the present invention provides methods of inhibiting complement component C5 expression in a cell. The methods include contacting the cell with the agent of the invention or a pharmaceutical composition of the invention; and maintaining the cell for a time sufficient to obtain antisense inhibition of a complement component C5 gene, thereby inhibiting expression of the complement component C5 gene in the cell.

[0038] In one embodiment, the cell is within a subject.

[0039] In one embodiment, the subject is a human.

[0040] In one embodiment, the complement component C5 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

[0041] In another aspect, the present invention provides methods of treating a subject having a disease or disorder that would benefit from reduction in complement component C5 expression. The methods include administering to the subject a therapeutically effective amount of an agent of the invention or a pharmaceutical composition of the invention, thereby treating the subject.

[0042] In yet another aspect, the present invention provides methods of preventing at least one symptom in a subject having a disease or disorder that would benefit from reduction in complement component C5 expression. The methods include administering to the subject a prophylactically effective amount of the agent of the invention or a pharmaceutical composition of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in C5 expression.

[0043] In one embodiment, the administration of the antisense polynucleotide agent to the subject causes a decrease in intravascular hemolysis, a stabilization of hemoglobin levels and/or a decrease in C5 protein levels.

[0044] In one embodiment, the disorder is a complement component C5-associated disease.

[0045] In another embodiment, the complement component C5-associated disease is selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemia-reperfusion injury; typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TITP); spontaneous fetal loss; Pauci-immune vasculitis; epidermolysis bullosa; recurrent fetal loss; pre-eclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis bullous pemphigoid, Shiga toxin E. coli-related hemolytic uremic syndrome, C3 nephropathy, anti-neutrophil cytoplasmic antibody-associated vasculitis, humoral and vascular transplant rejection, graft dysfunction, myocardial infarction, an allogenic transplant, sepsis, Coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture syndrome, Degos disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), a cardiovascular disorder, myocarditis, a cerebrovascular disorder, a peripheral vascular disorder, a renovascular disorder, a mesenteric/enteric vascular disorder, vasculitis, Henoch-Schinlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki's disease (arteritis), venous gas embolus (VGE), and restenosis following stent placement, rotational atherectomy, membraneous nephropathy, Guillain-Barre syndrome, and percutaneous transluminal coronary angioplasty (PTCA).

[0046] In one embodiment, the complement component C5-associated disease is paroxysmal nocturnal hemoglobinuria (PNH).

[0047] In another embodiment, the complement component C5-associated disease is atypical hemolytic uremic syndrome (aHUS).

[0048] In one embodiment the subject is human.

[0049] In one embodiment, the methods of the invention further include administering an anti-complement component C5 antibody, or antigen-binding fragment thereof, to the subject.

[0050] In one embodiment, the agent is administered at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.

[0051] In one embodiment, the agent is administered at a dose of about 10 mg/kg to about 30 mg/kg.

[0052] In one embodiment, the agent is administered to the subject once a week.

[0053] In another embodiment, the agent is administered to the subject twice a week.

[0054] In yet another embodiment, the agent is administered to the subject twice a month.

[0055] In one embodiment, the agent is administered to the subject subcutaneously.

[0056] In one embodiment, the methods of the invention further include measuring hemoglobin and/or LDH levels in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1 is a schematic of the three complement pathways: alternative, classical and lectin.

DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention provides antisense polynucleotide agents and compositions comprising such agents which target nucleic acids encoding complement component C5 (e.g., mRNA encoding C5 as provided in, for example, any one of SEQ ID NOs:1-4). The antisense polynucleotide agents bind to nucleic acids encoding C5 via, e.g., Watson-Crick base pairing, and interfere with the normal function of the targeted nucleic acid.

[0059] The antisense polynucleotide agents of the invention include a nucleotide sequence which is about 4 to about 50 nucleotides or less in length and which is about 80% complementary to at least part of an mRNA transcript of a C5 gene. The use of these antisense polynucleotide agents enables the targeted inhibition of RNA expression and/or activity of a C5 gene in mammals.

[0060] The present inventors have demonstrated that antisense polynucleotide agents targeting C5 can mediate antisense inhibition in vitro resulting in significant inhibition of expression of a C5 gene. Thus, methods and compositions including these antisense polynucleotide agents are useful for treating a subject who would benefit by a reduction in the levels and/or activity of a C5 protein, such as a subject having a complement component C5-associated disease, such as paroxysmal nocturnal hemoglobinuria (PNH).

[0061] The present invention also provides methods and combination therapies for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a C5 gene, e.g., a complement component C5-associated disease, such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) using the antisense polynucleotide agents and compositions of the invention.

[0062] The present invention also provides methods for preventing at least one symptom, e.g., hemolysis, in a subject having a disorder that would benefit from inhibiting or reducing the expression of a C5 gene, e.g., a complement component C5-associated disease, such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The present invention further provides compositions comprising antisense polynucleotide agents which effect antisense inhibition of a complement component C5 gene. The C5 gene may be within a cell, e.g., a cell within a subject, such as a human.

[0063] The combination therapies of the present invention include administering to a subject having a complement component C5-associated disease, an antisense polynucleotide agent of the invention and an additional therapeutic, such as anti-complement component C5 antibody, or antigen-binding fragment thereof, e.g., eculizumab. The combination therapies of the invention reduce C5 levels in the subject (e.g., by about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 99%) by targeting C5 mRNA with an antisense polynucleotide agent of the invention and, accordingly, allow the therapeutically (or prophylactically) effective amount of eculizumab required to treat the subject to be reduced, thereby decreasing the costs of treatment and permitting easier and more convenient ways of administering eculizumab, such as subcutaneous administration.

[0064] The following detailed description discloses how to make and use antisense polynucleotide agents to inhibit the mRNA and/or protein expression of a C5 gene, as well as compositions, uses, and methods for treating subjects having diseases and disorders that would benefit from inhibition and/or reduction of the expression of this gene.

I. Definitions

[0065] In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

[0066] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0067] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

[0068] The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.

[0069] As used herein, "complement component C5," used interchangeably with the term "C5" refers to the well-known gene and polypeptide, also known in the art as CPAMD4, C3 and PZP-like alpha-2-macroglobulin domain-containing protein, anaphtlatoxin C5a analog, hemolytic complement (Hc), and complement C5. The sequence of a human C5 mRNA transcript can be found at, for example, GenBank Accession No. GI:38016946 (NM_001735.2; SEQ ID NO:1). The sequence of rhesus C5 mRNA can be found at, for example, GenBank Accession No. GI:297270262 (XM_001095750.2; SEQ ID NO:2). The sequence of mouse C5 mRNA can be found at, for example, GenBank Accession No. GI:291575171 (NM_010406.2; SEQ ID NO:3). The sequence of rat C5 mRNA can be found at, for example, GenBank Accession No. GI:392346248 (XM_345342.4; SEQ ID NO:4). Additional examples of C5 mRNA sequences are readily available using publicly available databases, e.g., GenBank.

[0070] The term "C5," as used herein, also refers to naturally occurring DNA sequence variations of the C5 gene, such as a single nucleotide polymorphism in the C5 gene. Numerous SNPs within the C5 gene have been identified and may be found at, for example, NCBI dbSNP (see, e.g., ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within the C5 gene may be found at, NCBI dbSNP Accession Nos. rs121909588 and rs121909587.

[0071] The terms "antisense polynucleotide agent" "antisense compound", and "agent" as used interchangeably herein, refer to an agent comprising a single-stranded oligonucleotide that contains RNA as that term is defined herein, and which targets nucleic acid molecules encoding complement component C5 (e.g., mRNA encoding C5 as provided in, for example, any one of SEQ ID NOs: 1-4). The antisense polynucleotide agents specifically bind to the target nucleic acid molecules via hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) and interfere with the normal function of the targeted nucleic acid (e.g., by an antisense mechanism of action). This interference with or modulation of the function of a target nucleic acid by the polynucleotide agents of the present invention is referred to as "antisense inhibition."

[0072] The functions of the target nucleic acid molecule to be interfered with may include functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA.

[0073] In some embodiments, antisense inhibition refers to "inhibiting the expression" of target nucleic acid levels and/or target protein levels in a cell, e.g., a cell within a subject, such as a mammalian subject, in the presence of the antisense polynucleotide agent complementary to a target nucleic acid as compared to target nucleic acid levels and/or target protein levels in the absence of the antisense polynucleotide agent. For example, the antisense polynucleotide agents of the invention can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355.

[0074] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a C5 gene, including mRNA that is a product of RNA processing of a primary transcription product.

[0075] As used herein, "target nucleic acid" refers to a nucleic acid molecule to which an antisense polynucleotide agent specifically hybridizes.

[0076] As used herein, the term "specifically hybridizes" refers to an antisense polynucleotide agent having a sufficient degree of complementarity between the antisense polynucleotide agent and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays and therapeutic treatments.

[0077] A target sequence may be from about 4-50 nucleotides in length, e.g., 8-45, 10-45, 10-40, 10-35, 10-30, 10-20, 11-45, 11-40, 11-35, 11-30, 11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35, 13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 16-25, 16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35, 18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19-30, 19-25, 19-20, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous nucleotides of the nucleotide sequence of an mRNA molecule formed during the transcription of a C5 gene. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

[0078] The terms "complementary," "fully complementary" and "substantially complementary" are used herein with respect to the base matching between an antisense polynucleotide agent and a target sequence. The term"complementarity" refers to the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

[0079] As used herein, an antisense polynucleotide agent that is "substantially complementary to at least part of" a messenger RNA (mRNA) refers to an antisense polynucleotide agent that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding C5). For example, a polynucleotide is complementary to at least a part of a C5 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding C5.

[0080] As used herein, the term "region of complementarity" refers to the region of the antisense polynucleotide agent that is substantially complementary to a sequence, for example a target sequence, e.g., a C5 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5'- and/or 3'-terminus of the antisense polynucleotide.

[0081] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of a polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. for 12-16 hours followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the nucleotides.

[0082] Complementary sequences include those nucleotide sequences of an antisense polynucleotide agent of the invention that base-pair to a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., antisense inhibition of target gene expression.

[0083] "Complementary" sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

[0084] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

[0085] "G," "C," "A," `T` and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the terms "deoxyribonucleotide", "ribonucleotide" and "nucleotide" can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 2). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of the agents featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

[0086] A "nucleoside" is a base-sugar combination. The "nucleobase" (also known as "base") portion of the nucleoside is normally a heterocyclic base moiety. "Nucleotides" are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. "Polynucleotides," also referred to as "oligonucleotides," are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the polynucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the polynucleotide.

[0087] In general, the majority of nucleotides of the antisense polynucleotide agents are ribonucleotides, but as described in detail herein, the agents may also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide. In addition, as used in this specification, an "antisense polynucleotide agent" may include nucleotides (e.g., ribonucleotides or deoxyribonucleotides) with chemical modifications; an antisense polynucleotide agent may include substantial modifications at multiple nucleotides.

[0088] As used herein, the term "modified nucleotide" refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, and/or modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the antisense polynucleotide agents of the invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in nucleotides, are encompassed by "antisense polynucleotide agent" for the purposes of this specification and claims.

[0089] As used herein, a "subject" is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in C5 expression; a human at risk for a disease, disorder or condition that would benefit from reduction in C5 expression; a human having a disease, disorder or condition that would benefit from reduction in C5 expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in C5 expression as described herein.

[0090] As used herein, the terms "treating" or "treatment" refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms associated with unwanted complement pathway activation (e.g., hemolysis and/or chronic inflammation); diminishing the extent of unwanted complement pathway activation; stabilization (i.e., not worsening) of the state of chronic inflammation and/or hemolysis; amelioration or palliation of unwanted complement pathway activation (e.g., chronic inflammation and/or hemolysis) whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0091] The term "lower" in the context of the level of a complement component C5 in a subject or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more and is preferably down to a level accepted as within the range of normal for an individual without such disorder.

[0092] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of a C5 gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of unwanted complement activation, such as a chronic inflammation, hemolysis and/or thrombosis. The likelihood of developing a thrombosis is reduced, for example, when an individual having one or more risk factors for a thrombosis either fails to develop a thrombosis or develops a thrombosis with less severity relative to a population having the same risk factors and not receiving treatment as described herein. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.

[0093] As used herein, the term "complement component C5-associated disease" is a disease or disorder that is caused by, or associated with complement activation. Such diseases are typically associated with inflammation and/or immune system activation, e.g., membrane attack complex-mediated lysis, anaphylaxis, and/or hemolysis. Non-limiting examples of complement component C5-associated diseases include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemia-reperfusion injury; typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; Pauci-immune vasculitis; epidermolysis bullosa; recurrent fetal loss; pre-eclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis bullous pemphigoid, Shiga toxin E. coli-related hemolytic uremic syndrome, C3 nephropathy, anti-neutrophil cytoplasmic antibody-associated vasculitis (e.g., granulomatosis with polyangiitis (previously known as Wegener granulomatosis), Churg-Strauss syndrome, and microscopic polyangiitis), humoral and vascular transplant rejection, graft dysfunction, myocardial infarction (e.g., tissue damage and ischemia in myocardial infarction), an allogenic transplant, sepsis (e.g., poor outcome in sepsis), Coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture syndrome, Degos disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), a cardiovascular disorder, myocarditis, a cerebrovascular disorder, a peripheral (e.g., musculoskeletal) vascular disorder, a renovascular disorder, a mesenteric/enteric vascular disorder, vasculitis, Henoch-Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki's disease (arteritis), venous gas embolus (VGE), and restenosis following stent placement, rotational atherectomy, membraneous nephropathy, Guillain-Barre syndrome, and percutaneous transluminal coronary angioplasty (PTCA) (see, e.g., Holers (2008) Immunological Reviews 223:300-316; Holers and Thurman (2004) Molecular Immunology 41:147-152; U.S. Patent Publication No. 20070172483).

[0094] In one embodiment, a complement component C5-associated disease is paroxysmal nocturnal hemoglobinuria (PNH). The PNH may be classical PNH or PNH in the setting of another bone marrow failure syndrome and/or myelodysplastic syndromes (MDS), e.g., cytopenias. In another embodiment, a complement component C5-associated disease is atypical hemolytic uremic syndrome (aHUS).

II. Antisense Polynucleotide Agents of the Invention

[0095] The present invention provides antisense polynucleotide agents, and compositions comprising such agents, which target a complement component C5 gene and inhibit the expression of the C5 gene. In one embodiment, the antisense polynucleotide agents inhibit the expression of a C5 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having a complement component C5-associated disease, e.g., PNH.

[0096] The antisense polynucleotide agents of the invention include a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a C5 gene. The region of complementarity may be about 50 nucleotides or less in length (e.g., about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides or less in length). Upon contact with a cell expressing the C5 gene, the antisense polynucleotide agent inhibits the expression of the C5 gene (e.g., a human, a primate, a non-primate, or a bird C5 gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western Blotting or flow cytometric techniques.

[0097] The region of complementarity between an antisense polynucleotide agent and a target sequence may be substantially complementary (e.g., there is a sufficient degree of complementarity between the antisense polynucleotide agent and a target nucleic acid to so that they specifically hybridize and induce a desired effect), but is generally fully complementary to the target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of a C5 gene.

[0098] Accordingly, in one aspect, an antisense polynucleotide agent of the invention specifically hybridizes to a target nucleic acid molecule, such as the mRNA encoding complement component C5, and comprises a contiguous nucleotide sequence which corresponds to the reverse complement of a nucleotide sequence of any one of SEQ ID NOs:1-4, or a fragment of any one of SEQ ID NOs:1-4.

[0099] In some embodiments, the antisense polynucleotide agents of the invention may be substantially complementary to the target sequence. For example, an antisense polynucleotide agent that is substantially complementary to the target sequence may include a contiguous nucleotide sequence comprising no more than 5 mismatches (e.g., no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches) when hybridizing to a target sequence, such as to the corresponding region of a nucleic acid which encodes a mammalian C5 mRNA. In some embodiments, the contiguous nucleotide sequence comprises no more than a single mismatch when hybridizing to the target sequence, such as the corresponding region of a nucleic acid which encodes a mammalian C5 mRNA.

[0100] In some embodiments, the antisense polynucleotide agents of the invention that are substantially complementary to the target sequence comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1-4, or a fragment of any one of SEQ ID NOs:1-4, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0101] In some embodiments, an antisense polynucleotide agent comprises a contiguous nucleotide sequence which is fully complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1-4 (or a fragment of any one of SEQ ID NOs:1-4). For example, the nucleotide sequence of Sequence ID A-128563.1 is fully complementary over its entire length to the equivalent region of nucleotides 1-20 of NM_001735.2 (SEQ ID NO:1) (see, e.g., Table 3). Similarly, the nucleotide sequence of Sequence ID A-128637.1 is fully complementary over its entire length to the equivalent region of nucleotides 815-834 of NM_001735.2 (SEQ ID NO:1) (see, e.g., Table 3) and the nucleotide sequence of Sequence ID A-128915.1 is fully complementary over its entire length to the equivalent region of nucleotides 3873-3892 of NM_001735.2 (SEQ ID NO:1) (see, e.g., Table 3).

[0102] An antisense polynucleotide agent may comprise a contiguous nucleotide sequence of about 4 to about 50 nucleotides in length, e.g., 8-49, 8-48, 8-47, 8-46, 8-45, 8-44, 8-43, 8-42, 8-41, 8-40, 8-39, 8-38, 8-37, 8-36, 8-35, 8-34, 8-33, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 10-49, 10-48, 10-47, 10-46, 10-45, 10-44, 10-43, 10-42, 10-41, 10-40, 10-39, 10-38, 10-37, 10-36, 10-35, 10-34, 10-33, 10-32, 10-31, 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-49, 11-48, 11-47, 11-46, 11-45, 11-44, 11-43, 11-42, 11-41, 11-40, 11-39, 11-38, 11-37, 11-36, 11-35, 11-34, 11-33, 11-32, 11-31, 11-30, 11-29, 11-28, 11-27, 11-26, 11-25, 11-24, 11-23, 11-22, 11-21, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-49, 12-48, 12-47, 12-46, 12-45, 12-44, 12-43, 12-42, 12-41, 12-40, 12-39, 12-38, 12-37, 12-36, 12-35, 12-34, 12-33, 12-32, 12-31, 12-30, 12-29, 12-28, 12-27, 12-26, 12-25, 12-24, 12-23, 12-22, 12-21, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-49, 13-48, 13-47, 13-46, 13-45, 13-44, 13-43, 13-42, 13-41, 13-40, 13-39, 13-38, 13-37, 13-36, 13-35, 13-34, 13-33, 13-32, 13-31, 13-30, 13-29, 13-28, 13-27, 13-26, 13-25, 13-24, 13-23, 13-22, 13-21, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-49, 14-48, 14-47, 14-46, 14-45, 14-44, 14-43, 14-42, 14-41, 14-40, 14-39, 14-38, 14-37, 14-36, 14-35, 14-34, 14-33, 14-32, 14-31, 14-30, 14-29, 14-28, 14-27, 14-26, 14-25, 14-24, 14-23, 14-22, 14-21, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-49, 15-48, 15-47, 15-46, 15-45, 15-44, 15-43, 15-42, 15-41, 15-40, 15-39, 15-38, 15-37, 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 15-16, 16-49, 16-48, 16-47, 16-46, 16-45, 16-44, 16-43, 16-42, 16-41, 16-40, 16-39, 16-38, 16-37, 16-36, 16-35, 16-34, 16-33, 16-32, 16-31, 16-30, 16-29, 16-28, 16-27, 16-26, 16-25, 16-24, 16-23, 16-22, 16-21, 16-20, 16-19, 16-18, 16-17, 17-49, 17-48, 17-47, 17-46, 17-45, 17-44, 17-43, 17-42, 17-41, 17-40, 17-39, 17-38, 17-37, 17-36, 17-35, 17-34, 17-33, 17-32, 17-31, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24, 17-23, 17-22, 17-21, 17-20, 17-19, 17-18, 18-49, 18-48, 18-47, 18-46, 18-45, 18-44, 18-43, 18-42, 18-41, 18-40, 18-39, 18-38, 18-37, 18-36, 18-35, 18-34, 18-33, 18-32, 18-31, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-49, 19-48, 19-47, 19-46, 19-45, 19-44, 19-43, 19-42, 19-41, 19-40, 19-39, 19-38, 19-37, 19-36, 19-35, 19-34, 19-33, 19-32, 19-31, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-49, 20-48, 20-47, 20-46, 20-45, 20-44, 20-43, 20-42, 20-41, 20-40, 20-39, 20-38, 20-37, 20-36, 20-35, 20-34, 20-33, 20-32, 20-31, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-49, 21-48, 21-47, 21-46, 21-45, 21-44, 21-43, 21-42, 21-41, 21-40, 21-39, 21-38, 21-37, 21-36, 21-35, 21-34, 21-33, 21-32, 21-31, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 22-49, 22-48, 22-47, 22-46, 22-45, 22-44, 22-43, 22-42, 22-41, 22-40, 22-39, 22-38, 22-37, 22-36, 22-35, 22-34, 22-33, 22-32, 22-31, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 22-23, 23-49, 23-48, 23-47, 23-46, 23-45, 23-44, 23-43, 23-42, 23-41, 23-40, 23-39, 23-38, 23-37, 23-36, 23-35, 23-34, 23-33, 23-32, 23-31, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 23-24, 24-49, 24-48, 24-47, 24-46, 24-45, 24-44, 24-43, 24-42, 24-41, 24-40, 24-39, 24-38, 24-37, 24-36, 24-35, 24-34, 24-33, 24-32, 24-31, 24-30, 24-29, 24-28, 24-27, 24-26, 24-25, 25-49, 25-48, 25-47, 25-46, 25-45, 25-44, 25-43, 25-42, 25-41, 25-40, 25-39, 25-38, 25-37, 25-36, 25-35, 25-34, 25-33, 25-32, 25-31, 25-30, 25-29, 25-28, 25-27, 25-26, 26-49, 26-48, 26-47, 26-46, 26-45, 26-44, 26-43, 26-42, 26-41, 26-40, 26-39, 26-38, 26-37, 26-36, 26-35, 26-34, 26-33, 26-32, 26-31, 26-30, 26-29, 26-28, 26-27, 27-49, 27-48, 27-47, 27-46, 27-45, 27-44, 27-43, 27-42, 27-41, 27-40, 27-39, 27-38, 27-37, 27-36, 27-35, 27-34, 27-33, 27-32, 27-31, 27-30, 27-29, 27-28, 28-49, 28-48, 28-47, 28-46, 28-45, 28-44, 28-43, 28-42, 28-41, 28-40, 28-39, 28-38, 28-37, 28-36, 28-35, 28-34, 28-33, 28-32, 28-31, 28-30, 28-29, 29-49, 29-48, 29-47, 29-46, 29-45, 29-44, 29-43, 29-42, 29-41, 29-40, 29-39, 29-38, 29-37, 29-36, 29-35, 29-34, 29-33, 29-32, 29-31, 29-30, 30-49, 30-48, 30-47, 30-46, 30-45, 30-44, 30-43, 30-42, 30-41, 30-40, 30-39, 30-38, 30-37, 30-36, 30-35, 30-34, 30-33, 30-32, or 30-31 nucleotides in length, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0103] In some embodiments, an antisense polynucleotide agent may comprise a contiguous nucleotide sequence of no more than 22 nucleotides, such as no more than 21 nucleotides, 20 nucleotides, 19 nucleotides, or no more than 18 nucleotides. In some embodiments the antisense polynucleotide agents of the invention comprises less than 20 nucleotides. In other embodiments, the antisense polynucleotide agents of the invention comprise 20 nucleotides.

[0104] In one aspect, an antisense polynucleotide agent of the invention includes a sequence selected from the group of sequences provided in Table 3. It will be understood that, although some of the sequences in Table 3 are described as modified and/or conjugated sequences, an antisense polynucleotide agent of the invention, may also comprise any one of the sequences set forth in Table 3 that is un-modified, un-conjugated, and/or modified and/or conjugated differently than described therein.

[0105] By virtue of the nature of the nucleotide sequences provided in Table 3, antisense polynucleotide agents of the invention may include one of the sequences of Table 3 minus only a few nucleotides on one or both ends and yet remain similarly effective as compared to the antisense polynucleotide agents described above. Hence, antisense polynucleotide agents having a sequence of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of Table 3 and differing in their ability to inhibit the expression of a C5 gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from an antisense polynucleotide agent comprising the full sequence, are contemplated to be within the scope of the present invention.

[0106] In addition, the antisense polynucleotide agents provided in Table 3 identify a region(s) in a C5 transcript that is susceptible to antisense inhibition (e.g., the regions encompassed by the start and end positions relative to NM_001735.2 in Table 3). As such, the present invention further features antisense polynucleotide agents that target within one of these sites. As used herein, an antisense polynucleotide agent is said to target within a particular site of an RNA transcript if the antisense polynucleotide agent promotes antisense inhibition of the target at that site. Such an antisense polynucleotide agent will generally include at least about 15 contiguous nucleotides from one of the sequences provided in Table 3 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a C5 gene.

[0107] While a target sequence is generally about 4-50 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing antisense inhibition of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a "window" or "mask" of a given size (as a non-limiting example, 20 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence "window" progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an antisense polynucleotide agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Table 3 represent effective target sequences, it is contemplated that further optimization of antisense inhibition efficiency can be achieved by progressively "walking the window" one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

[0108] Further, it is contemplated that for any sequence identified, e.g., in Table 3, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of antisense polynucleotide agents based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in length, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

III. Modified Antisense Polynucleotide Agents of the Invention

[0109] In one embodiment, the nucleotides of an antisense polynucleotide agent of the invention are un-modified, and do not comprise, e.g., chemical modifications and/or conjugations known in the art and described herein. In another embodiment, at least one of the nucleotides of an antisense polynucleotide agent of the invention is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an antisense polynucleotide agent of the invention are modified. In other embodiments of the invention, all of the nucleotides of an antisense polynucleotide agent of the invention are modified. Antisense polynucleotide agents of the invention in which "substantially all of the nucleotides are modified" are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0110] The nucleic acids featured in the invention can be synthesized and/or modified by standard methods known in the art as further discussed below, e.g., solution-phase or solid-phase organic synthesis or both, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. Well-established methods for the synthesis and/or modification of the nucleic acids featured in the invention are described in, for example, "Current protocols in nucleic acid chemistry," Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5'-end modifications (phosphorylation, conjugation, inverted linkages) or 3'-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2'-position or 4'-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages.

[0111] Specific examples of modified nucleotides useful in the embodiments described herein include, but are not limited to nucleotides containing modified backbones or no natural internucleoside linkages. Nucleotides having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified nucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified antisense polynucleotide agent will have a phosphorus atom in its internucleoside backbone.

[0112] Modified nucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5'-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

[0113] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

[0114] Modified nucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts.

[0115] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

[0116] In other embodiments, suitable nucleotide mimetics are contemplated for use in antisense polynucleotide agents, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the antisense polynucleotide agents of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0117] Some embodiments featured in the invention include polynucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH.sub.2--NH--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene (methylimino) or MMI backbone], --CH.sub.2--O--N(CH.sub.3)--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and --N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the antisense polynucleotide agents featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0118] Modified nucleotides can also contain one or more modified or substituted sugar moieties. The antisense polynucleotide agents featured herein can include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2)..sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m are from 1 to about 10.

[0119] In other embodiments, antisense polynucleotide agents include one of the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an antisense polynucleotide, or a group for improving the pharmacodynamic properties of an antisense polynucleotide agent, and other substituents having similar properties. In some embodiments, the modification includes a 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2.

[0120] Other modifications include 2'-methoxy (2'-OCH.sub.3), 2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on a nucleotide of an antisense polynucleotide agent, particularly the 3' position of the sugar on the 3' terminal nucleotide. Antisense polynucleotide agents can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0121] Additional nucleotides having modified or substituted sugar moieties for use in the polynucleotide agents of the invention include nucleotides comprising a bicyclic sugar. A "bicyclic sugar" is a furanosyl ring modified by the bridging of two atoms. A"bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring. Thus, in some embodiments an antisense polynucleotide agent may include one or more locked nucleic acids. A "locked nucleic acid" ("LNA") is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4'-CH.sub.2--O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to santisense polynucleotide agents has been shown to increase santisense polynucleotide agent stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0122] Examples of bicyclic nucleosides for use in the polynucleotides of the invention include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides, include but are not limited to 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4'-CH2-O--N(CH3)-2' (see, e.g., U.S. Patent Publication No. 2004/0171570); 4'-CH2-N(R)--O-2', wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(.dbd.CH2)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0123] Additional representative U.S. Patents and US Patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

[0124] Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example .alpha.-L-ribofuranose and .beta.-D-ribofuranose (see WO 99/14226).

[0125] In one particular embodiment of the invention, an antisense polynucleotide agent can include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4'-CH(CH.sub.3)--O-2' bridge. In one embodiment, a constrained ethyl nucleotide is in an S conformation and is referred to as an "S-constrained ethyl nucleotide" or "S-cEt."

[0126] Modified nucleotides included in the antisense polynucleotide agents of the invention can also contain one or more sugar mimetics. For example, the antisense polynucleotide agent may include a "modified tetrahydropyran nucleotide" or "modified THP nucleotide." A "modified tetrahydropyran nucleotide" has a six-membered tetrahydropyran "sugar" substituted in for the pentofuranosyl residue in normal nucleotides (a sugar surrogate). Modified THP nucleotides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see, e.g., Leumann, Bioorg. Med. Chem., 2002, 10, 841-854), or fluoro HNA (F-HNA).

[0127] In some embodiments of the invention, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleotides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). Morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholinos."

[0128] Combinations of modifications are also provided without limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008/101157 published on Aug. 21, 2008 for other disclosed 5', 2'-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4'-CH2-O-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

[0129] In certain embodiments, antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety).

[0130] An antisense polynucleotide agent can also include nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in "Modified Nucleosides in Biochemistry," Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, antisense polynucleotide agent Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the agents featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., antisense polynucleotide agent Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0131] Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

[0132] One or more of the nucleotides of an iRNA of the invention may also include a hydroxymethyl substituted nucleotide. A "hydroxymethyl substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also referred to as an "unlocked nucleic acid" ("UNA") modification. Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

[0133] Additional modification which may potentially stabilize the ends of antisense polynucleotide agents can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3''-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in US Patent Publication No. 2012/0142101.

[0134] Any of the antisense polynucleotide agents of the invention may be optionally conjugated with a GalNAc derivative ligand, as described in Section IV, below.

[0135] As described in more detail below, an agent that contains conjugations of one or more carbohydrate moieties to an antisense polynucleotide agent can optimize one or more properties of the agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the antisense polynucleotide agent. For example, the ribose sugar of one or more ribonucleotide subunits of an agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

[0136] The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one "backbone attachment point," preferably two "backbone attachment points" and (ii) at least one "tethering attachment point." A "backbone attachment point" as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A "tethering attachment point" (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

[0137] The antisense polynucleotide agents may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

[0138] In certain specific embodiments, the antisense polynucleotide agent for use in the methods of the invention is an agent selected from the group of agents listed in Table 3. These agents may further comprise a ligand, as described in Section IV, below.

[0139] A. Antisense Polynucleotide Agents Comprising Motifs

[0140] In certain embodiments of the invention, at least one of the contiguous nucleotides of the antisense polynucleotide agents of the invention may be a modified nucleotide. In one embodiment, the modified nucleotide comprises one or more modified sugars. In other embodiments, the modified nucleotide comprises one or more modified nucleobases. In yet other embodiments, the modified nucleotide comprises one or more modified internucleoside linkages. In some embodiments, the modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif. In one embodiment, the patterns of modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another.

[0141] Antisense polynucleotide agents having modified oligonucleotides arranged in patterns, or motifs may, for example, confer to the agents properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases. For example, such agents may contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of such agents may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

[0142] An exemplary antisense polynucleotide agent having modified oligonucleotides arranged in patterns, or motifs is a gapmer. In a "gapmer", an internal region or "gap" having a plurality of linked nucleotides that supports RNaseH cleavage is positioned between two external flanking regions or "wings" having a plurality of linked nucleotides that are chemically distinct from the linked nucleotides of the internal region. The gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleotides.

[0143] The three regions of a gapmer motif (the 5'-wing, the gap, and the 3'-wing) form a contiguous sequence of nucleotides and may be described as "X--Y-Z", wherein "X" represents the length of the 5-wing, "Y" represents the length of the gap, and "Z" represents the length of the 3'-wing. In one embodiment, a gapmer described as "X--Y-Z" has a configuration such that the gap segment is positioned immediately adjacent to each of the 5' wing segment and the 3' wing segment. Thus, no intervening nucleotides exist between the 5' wing segment and gap segment, or the gap segment and the 3' wing segment. Any of the antisense compounds described herein can have a gapmer motif. In some embodiments, X and Z are the same, in other embodiments they are different.

[0144] In certain embodiments, the regions of a gapmer are differentiated by the types of modified nucleotides in the region. The types of modified nucleotides that may be used to differentiate the regions of a gapmer, in some embodiments, include .beta.-D-ribonucleotides, .beta.-D-deoxyribonucleotides, 2'-modified nucleotides, e.g., 2'-modified nucleotides (e.g., 2'-MOE, and 2'-O--CH3), and bicyclic sugar modified nucleotides (e.g., those having a 4'-(CH2)n-O-2' bridge, where n=1 or n=2).

[0145] In one embodiment, at least some of the modified nucleotides of each of the wings may differ from at least some of the modified nucleotides of the gap. For example, at least some of the modified nucleotides of each wing that are closest to the gap (the 3'-most nucleotide of the 5'-wing and the 5'-most nucleotide of the 3-wing) differ from the modified nucleotides of the neighboring gap nucleotides, thus defining the boundary between the wings and the gap. In certain embodiments, the modified nucleotides within the gap are the same as one another. In certain embodiments, the gap includes one or more modified nucleotides that differ from the modified nucleotides of one or more other nucleotides of the gap.

[0146] The length of the 5'-wing (X) of a gapmer may be 1 to 6 nucleotides in length, e.g., 2 to 6, 2 to 5, 3 to 6, 3 to 5, 1 to 5, 1 to 4, 1 to 3, 2 to 4 nucleotides in length, e.g., 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0147] The length of the 3'-wing (Z) of a gapmer may be 1 to 6 nucleotides in length, e.g., 2 to 6, 2-5, 3 to 6, 3 to 5, 1 to 5, 1 to 4, 1 to 3, 2 to 4 nucleotides in length, e.g., 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0148] The length of the gap (Y) of a gapmer may be 5 to 14 nucleotides in length, e.g., 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 7 to 8, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 9 to 10, 10 to 14, 10 to 13, 10 to 12, 10 to 1, 11 to 14, 11 to 13, 11 to 12, 12 to 14, 12 to 13, or 13 to 14 nucleotides in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length.

[0149] In some embodiments of the invention X consists of 2, 3, 4, 5 or 6 nucleotides, Y consists of 7, 8, 9, 10, 11, or 12 nucleotides, and Z consists of 2, 3, 4, 5 or 6 nucleotides. Such gapmers include (X--Y--Z) 2-7-2, 2-7-3, 2-7-4, 2-7-5, 2-7-6, 3-7-2, 3-7-3, 3-7-4, 3-7-5, 3-7-6, 4-7-3, 4-7-4, 4-7-5, 4-7-6, 5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4, 6-7-5, 6-7-6, 3-7-3, 3-7-4, 3-7-5, 3-7-6, 4-7-3, 4-7-4, 4-7-5, 4-7-6, 5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4, 6-7-5, 6-7-6, 2-8-2, 2-8-3, 2-8-4, 2-8-5, 2-8-6, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 3-8-6, 4-8-3, 4-8-4, 4-8-5, 4-8-6, 5-8-3, 5-8-4, 5-8-5, 5-8-6, 6-8-3, 6-8-4, 6-8-5, 6-8-6, 2-9-2, 2-9-3, 2-9-4, 2-9-5, 2-9-6, 3-9-2, 3-9-3, 3-9-4, 3-9-5, 3-9-6, 4-9-3, 4-9-4, 4-9-5, 4-9-6, 5-9-3, 5-9-4, 5-9-5, 5-9-6, 6-9-3, 6-9-4, 6-9-5, 6-9-6, 2-10-2, 2-10-3, 2-10-4, 2-10-5, 2-10-6, 3-10-2, 3-10-3, 3-10-4, 3-10-5, 3-10-6, 4-10-3, 4-10-4, 4-10-5, 4-10-6, 5-10-3, 5-10-4, 5-10-5, 5-10-6, 6-10-3, 6-10-4, 6-10-5, 6-10-6, 2-11-2, 2-11-3, 2-11-4, 2-11-5, 2-11-6, 3-11-2, 3-11-3, 3-11-4, 3-11-5, 3-11-6, 4-11-3, 4-11-4, 4-11-5, 4-11-6, 5-11-3, 5-11-4, 5-11-5, 5-11-6, 6-11-3, 6-11-4, 6-11-5, 6-11-6, 2- 12-2, 2-12-3, 2-12-4, 2-12-5, 2-12-6, 3-12-2, 3-12-3, 3-12-4, 3-12-5, 3-12-6, 4-12-3, 4-12-4, 4-12-5, 4-12-6, 5-12-3, 5-12-4, 5-12-5, 5-12-6, 6-12-3, 6-12-4, 6-12-5, or 6-12-6.

[0150] In some embodiments of the invention, antisense polynucleotide agents targeting C5 include a 5-10-5 gapmer motif. In other embodiments of the invention, antisense polynucleotide agents targeting C5 include a 4-10-4 gapmer motif. In another embodiment of the invention, antisense polynucleotide agents targeting C5 include a 3-10-3 gapmer motif. In yet other embodiments of the invention, antisense polynucleotide agents targeting C5 include a 2-10-2 gapmer motif.

[0151] The 5'-wing and/or 3'-wing of a gapmer may independently include 1-6 modified nucleotides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleotides.

[0152] In some embodiment, the 5'-wing of a gapmer includes at least one modified nucleotide. In one embodiment, the 5'-wing of a gapmer comprises at least two modified nucleotides. In another embodiment, the 5'-wing of a gapmer comprises at least three modified nucleotides. In yet another embodiment, the 5'-wing of a gapmer comprises at least four modified nucleotides. In another embodiment, the 5'-wing of a gapmer comprises at least five modified nucleotides. In certain embodiments, each nucleotide of the 5'-wing of a gapmer is a modified nucleotide.

[0153] In some embodiments, the 3'-wing of a gapmer includes at least one modified nucleotide. In one embodiment, the 3'-wing of a gapmer comprises at least two modified nucleotides. In another embodiment, the 3'-wing of a gapmer comprises at least three modified nucleotides. In yet another embodiment, the 3'-wing of a gapmer comprises at least four modified nucleotides. In another embodiment, the 3'-wing of a gapmer comprises at least five modified nucleotides. In certain embodiments, each nucleotide of the 3'-wing of a gapmer is a modified nucleotide.

[0154] In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties of the nucleotides. In one embodiment, the nucleotides of each distinct region comprise uniform sugar moieties. In other embodiments, the nucleotides of each distinct region comprise different sugar moieties. In certain embodiments, the sugar nucleotide modification motifs of the two wings are the same as one another. In certain embodiments, the sugar nucleotide modification motifs of the 5'-wing differs from the sugar nucleotide modification motif of the 3'-wing.

[0155] The 5'-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleotides.

[0156] In one embodiment, at least one modified nucleotide of the 5'-wing of a gapmer is a bicyclic nucleotide, such as a constrained ethyl nucleotide, or an LNA. In another embodiment, the 5'-wing of a gapmer includes 2, 3, 4, or 5 bicyclic nucleotides. In some embodiments, each nucleotide of the 5'-wing of a gapmer is a bicyclic nucleotide.

[0157] In one embodiment, the 5'-wing of a gapmer includes at least 1, 2, 3, 4, or 5 constrained ethyl nucleotides. In some embodiments, each nucleotide of the 5'-wing of a gapmer is a constrained ethyl nucleotide.

[0158] In one embodiment, the 5'-wing of a gapmer comprises at least one LNA nucleotide. In another embodiment, the 5'-wing of a gapmer includes 2, 3, 4, or 5 LNA nucleotides. In other embodiments, each nucleotide of the 5'-wing of a gapmer is an LNA nucleotide.

[0159] In certain embodiments, at least one modified nucleotide of the 5'-wing of a gapmer is a non-bicyclic modified nucleotide, e.g., a 2'-substituted nucleotide. A "2'-substituted nucleotide" is a nucleotide comprising a modification at the 2'-position which is other than H or OH, such as a 2'-OMe nucleotide, or a 2'-MOE nucleotide. In one embodiment, the 5'-wing of a gapmer comprises 2, 3, 4, or 5 2'-substituted nucleotides. In one embodiment, each nucleotide of the 5'-wing of a gapmer is a 2'-substituted nucleotide.

[0160] In one embodiment, the 5'-wing of a gapmer comprises at least one 2'-OMe nucleotide. In one embodiment, the 5'-wing of a gapmer comprises at least 2, 3, 4, or 5 2'-OMe nucleotides. In one embodiment, each of the nucleotides of the 5'-wing of a gapmer comprises a 2'-OMe nucleotide.

[0161] In one embodiment, the 5'-wing of a gapmer comprises at least one 2'-MOE nucleotide. In one embodiment, the 5'-wing of a gapmer comprises at least 2, 3, 4, or 5 2'-MOE nucleotides. In one embodiment, each of the nucleotides of the 5'-wing of a gapmer comprises a 2'-MOE nucleotide.

[0162] In certain embodiments, the 5'-wing of a gapmer comprises at least one 2'-deoxynucleotide. In certain embodiments, each nucleotide of the 5'-wing of a gapmer is a 2'-deoxynucleotide. In a certain embodiments, the 5'-wing of a gapmer comprises at least one ribonucleotide. In certain embodiments, each nucleotide of the 5'-wing of a gapmer is a ribonucleotide.

[0163] The 3'-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleotides.

[0164] In one embodiment, at least one modified nucleotide of the 3'-wing of a gapmer is a bicyclic nucleotide, such as a constrained ethyl nucleotide, or an LNA. In another embodiment, the 3'-wing of a gapmer includes 2, 3, 4, or 5 bicyclic nucleotides. In some embodiments, each nucleotide of the 3'-wing of a gapmer is a bicyclic nucleotide.

[0165] In one embodiment, the 3'-wing of a gapmer includes at least one constrained ethyl nucleotide. In another embodiment, the 3'-wing of a gapmer includes 2, 3, 4, or 5 constrained ethyl nucleotides. In some embodiments, each nucleotide of the 3'-wing of a gapmer is a constrained ethyl nucleotide.

[0166] In one embodiment, the 3'-wing of a gapmer comprises at least one LNA nucleotide. In another embodiment, the 3'-wing of a gapmer includes 2, 3, 4, or 5 LNA nucleotides. In other embodiments, each nucleotide of the 3'-wing of a gapmer is an LNA nucleotide.

[0167] In certain embodiments, at least one modified nucleotide of the 3'-wing of a gapmer is a non-bicyclic modified nucleotide, e.g., a 2'-substituted nucleotide. In one embodiment, the 3'-wing of a gapmer comprises 2, 3, 4, or 5 2'-substituted nucleotides. In one embodiment, each nucleotide of the 3'-wing of a gapmer is a 2'-substituted nucleotide.

[0168] In one embodiment, the 3'-wing of a gapmer comprises at least one 2'-OMe nucleotide. In one embodiment, the 3'-wing of a gapmer comprises at least 2, 3, 4, or 5 2'-OMe nucleotides. In one embodiment, each of the nucleotides of the 3'-wing of a gapmer comprises a 2'-OMe nucleotide.

[0169] In one embodiment, the 3'-wing of a gapmer comprises at least one 2'-MOE nucleotide. In one embodiment, the 3'-wing of a gapmer comprises at least 2, 3, 4, or 5 2'-MOE nucleotides. In one embodiment, each of the nucleotides of the 3'-wing of a gapmer comprises a 2'-MOE nucleotide.

[0170] In certain embodiments, the 3'-wing of a gapmer comprises at least one 2'-deoxynucleotide. In certain embodiments, each nucleotide of the 3'-wing of a gapmer is a 2'-deoxynucleotide. In a certain embodiments, the 3'-wing of a gapmer comprises at least one ribonucleotide. In certain embodiments, each nucleotide of the 3'-wing of a gapmer is a ribonucleotide.

[0171] The gap of a gapmer may include 5-14 modified nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 modified nucleotides.

[0172] In one embodiment, the gap of a gapmer comprises at least one 5-methylcytosine. In one embodiment, the gap of a gapmer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 5-methylcytosines. In one embodiment, all of the nucleotides of the gap of a gapmer are 5-methylcytosines.

[0173] In one embodiment, the gap of a gapmer comprises at least one 2'-deoxynucleotide. In one embodiment, the gap of a gapmer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 2'-deoxynucleotides. In one embodiment, all of the nucleotides of the gap of a gapmer are 2'-deoxynucleotides.

[0174] A gapmer may include one or more modified internucleotide linkages. In some embodiments, a gapmer includes one or more phosphodiester internucleotide linkages. In other embodiments, a gapmer includes one or more phosphorothioate internucleotide linkages.

[0175] In one embodiment, each nucleotide of a 5'-wing of a gapmer are linked via a phosphorothioate internucleotide linkage. In another embodiment, each nucleotide of a 3'-wing of a gapmer are linked via a phosphorothioate internucleotide linkage. In yet another embodiment, each nucleotide of a gap segment of a gapmer is linked via a phosphorothioate internucleotide linkage. In one embodiment, all of the nucleotides in a gapmer are linked via phosphorothioate internucleotide linkages.

[0176] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising five nucleotides and a 3'-wing segment comprising 5 nucleotides.

[0177] In another embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising four nucleotides and a 3'-wing segment comprising four nucleotides.

[0178] In another embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising three nucleotides and a 3'-wing segment comprising three nucleotides.

[0179] In another embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising two nucleotides and a 3'-wing segment comprising two nucleotides.

[0180] In one embodiment, each nucleotide of a 5-wing flanking a gap segment of 10 2'-deoxyribonucleotides comprises a modified nucleotide. In another embodiment, each nucleotide of a 3-wing flanking a gap segment of 10 2'-deoxyribonucleotides comprises a modified nucleotide. In one embodiment, each of the modified 5'-wing nucleotides and each of the modified 3'-wing nucleotides comprise a 2'-sugar modification. In one embodiment, the 2'-sugar modification is a 2'-OMe modification. In another embodiment, the 2'-sugar modification is a 2'-MOE modification. In one embodiment, each of the modified 5'-wing nucleotides and each of the modified 3'-wing nucleotides comprise a bicyclic nucleotide. In one embodiment, the bicyclic nucleotide is a constrained ethyl nucleotide. In another embodiment, the bicyclic nucleotide is an LNA nucleotide. In one embodiment, each cytosine in an antisense polynucleotide agent targeting a C5 gene is a 5-methylcytosine.

[0181] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising five nucleotides comprising a 2'OMe modification and a 3'-wing segment comprising five nucleotides comprising a 2'OMe modification, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine. In one embodiment, the agent further comprises a ligand.

[0182] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising five nucleotides comprising a 2'MOE modification and a 3'-wing segment comprising five nucleotides comprising a 2'MOE modification, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine. In one embodiment, the agent further comprises a ligand.

[0183] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising five constrained ethyl nucleotides and a 3'-wing segment comprising five constrained ethyl nucleotides, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0184] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising five LNA nucleotides and a 3'-wing segment comprising five LNA nucleotides, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0185] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising four nucleotides comprising a 2'OMe modification and a 3'-wing segment comprising four nucleotides comprising a 2'OMe modification, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0186] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising four nucleotides comprising a 2'MOE modification and a 3'-wing segment comprising four nucleotides comprising a 2'MOE modification, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0187] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising four constrained ethyl nucleotides and a 3'-wing segment comprising four constrained ethyl nucleotides, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0188] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising four LNA nucleotides and a 3'-wing segment comprising four LNA nucleotides, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0189] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising three nucleotides comprising a 2'OMe modification and a 3'-wing segment comprising three nucleotides comprising a 2'OMe modification, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0190] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising three nucleotides comprising a 2'MOE modification and a 3'-wing segment comprising three nucleotides comprising a 2'MOE modification, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0191] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising three constrained ethyl nucleotides and a 3'-wing segment comprising three constrained ethyl nucleotides, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0192] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising three LNA nucleotides and a 3'-wing segment comprising three LNA nucleotides, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0193] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising two nucleotides comprising a 2'OMe modification and a 3'-wing segment comprising two nucleotides comprising a 2'OMe modification, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0194] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising two nucleotides comprising a 2'MOE modification and a 3'-wing segment comprising two nucleotides comprising a 2'MOE modification, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0195] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising two constrained ethyl nucleotides and a 3'-wing segment comprising two constrained ethyl nucleotides, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0196] In one embodiment, an antisense polynucleotide agent targeting a C5 gene comprises a gap segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising two LNA nucleotides and a 3'-wing segment comprising two LNA nucleotides, wherein each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine.

[0197] Further gapmer designs suitable for use in the agents, compositions, and methods of the invention are disclosed in, for example, U.S. Pat. Nos. 7,687,617 and 8,580,756; U.S. Patent Publication Nos. 20060128646, 20090209748, 20140128586, 20140128591, 20100210712, and 20080015162A1; and International Publication No. WO 2013/159108, the entire content of each of which are incorporated herein by reference.

IV. Antisense Polynucleotide Agents Conjugated to Ligands

[0198] Another modification of the polynucleotide agents of the invention involves chemically linking to the agent one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the antisense polynucleotide agent. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0199] In one embodiment, a ligand alters the distribution, targeting or lifetime of an antisense polynucleotide agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in hybridization of an antisense polynucleotide agent to the targeted mRNA.

[0200] Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[0201] Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucoseamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0202] Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG].sub.2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0203] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-.kappa.B.

[0204] The ligand can be a substance, e.g., a drug, which can increase the uptake of the antisense polynucleotide agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0205] In some embodiments, a ligand attached to an antisense polynucleotide agent as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

[0206] Ligand-conjugated polynucleotides of the invention may be synthesized by the use of a polynucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive polynucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

[0207] The polynucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other polynucleotides, such as the phosphorothioates and alkylated derivatives.

[0208] In the ligand-conjugated polynucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the polynucleotides and polynucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0209] When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the polynucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0210] A. Lipid Conjugates

[0211] In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

[0212] A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

[0213] In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

[0214] In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

[0215] In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

[0216] B. Cell Permeation Agents

[0217] In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

[0218] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to antisense polynucleotide agents can affect pharmacokinetic distribution of the agent, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0219] A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to an antisense polynucleotide agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0220] An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0221] A "cell permeation peptide" is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an .alpha.-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., .alpha.-defensin, .beta.-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0222] C. Carbohydrate Conjugates

[0223] In some embodiments of the compositions and methods of the invention, an antisense polynucleotide agent further comprises a carbohydrate. The carbohydrate conjugated agents are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein (see, e.g., Prakash, et al. (2014) Nuc Acid Res doi 10.1093/nar/gku531). As used herein, "carbohydrate" refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0224] In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as

##STR00002##

[0225] In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

##STR00003## ##STR00004## ##STR00005## ##STR00006## ##STR00007##

[0226] Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to

##STR00008##

when one of X or Y is an oligonucleotide, the other is a hydrogen.

[0227] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

[0228] D. Linkers

[0229] In some embodiments, the conjugate or ligand described herein can be attached to an antisense polynucleotide agent with various linkers that can be cleavable or non-cleavable.

[0230] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO.sub.2, SO.sub.2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO.sub.2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

[0231] A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

[0232] Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

[0233] A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

[0234] A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

[0235] Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

[0236] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0237] i. Redox Cleavable Linking Groups

[0238] In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (--S--S--). To determine if a candidate cleavable linking group is a suitable "reductively cleavable linking group," or for example is suitable for use with a particular antisense polynucleotide agent moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

[0239] ii. Phosphate-Based Cleavable Linking Groups

[0240] In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are --O--P(O)(ORk)-O--, --O--P(S)(ORk)-O--, --O--P(S)(SRk)-O--, --S--P(O)(ORk)-O--, --O--P(O)(ORk)-S--, --S--P(O)(ORk)-S--, --O--P(S)(ORk)-S--, --S--P(S)(ORk)-O--, --O--P(O)(Rk)-O--, --O--P(S)(Rk)-O--, --S--P(O)(Rk)-O--, --S--P(S)(Rk)-O--, --S--P(O)(Rk)-S--, --O--P(S)(Rk)-S--. Preferred embodiments are --O--P(O)(OH)--O--, --O--P(S)(OH)--O--, --O--P(S)(SH)--O--, --S--P(O)(OH)--O--, --O--P(O)(OH)--S--, --S--P(O)(OH)--S--, --O--P(S)(OH)--S--, --S--P(S)(OH)--O--, --O--P(O)(H)--O--, --O--P(S)(H)--O--, --S--P(O)(H)--O, --S--P(S)(H)--O--, --S--P(O)(H)--S--, --O--P(S)(H)--S--. A preferred embodiment is --O--P(O)(OH)--O--. These candidates can be evaluated using methods analogous to those described above.

[0241] iii. Acid Cleavable Linking Groups

[0242] In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula --C.dbd.NN--, C(O)O, or --OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

[0243] iv. Ester-Based Linking Groups

[0244] In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula --C(O)O--, or --OC(O)--. These candidates can be evaluated using methods analogous to those described above.

[0245] v. Peptide-Based Cleaving Groups

[0246] In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (--C(O)NH--). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula --NHCHRAC(O)NHCHRBC(O)--, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

[0247] In one embodiment, an antisense polynucleotide agent of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of antisense polynucleotide agent carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

##STR00009## ##STR00010## ##STR00011##

[0248] when one of X or Y is an oligonucleotide, the other is a hydrogen.

[0249] In certain embodiments of the compositions and methods of the invention, a ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

[0250] In one embodiment, a antisense polynucleotide agent of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXII)-(XXXV):

##STR00012##

wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P.sup.2A, P.sup.2B, P.sup.3A, P.sup.3B, P.sup.4A, P.sup.4B, P.sup.5A, P.sup.5B, P.sup.5C, T.sup.2A, T.sup.2B, T.sup.3A, T.sup.3B, T.sup.4A, T.sup.4B, T.sup.4A, T.sup.5B, T.sup.5B, are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH.sub.2, CH.sub.2NH or CH.sub.2O; Q.sup.2A, Q.sup.2B, Q.sup.3A, Q.sup.3B, Q.sup.4A, Q.sup.4B, Q.sup.5A, Q.sup.5B, Q.sup.5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO.sub.2, N(R.sup.N), C(R').dbd.C(R''), C.ident.C or C(O); R.sup.2A, R.sup.2B, R.sup.3A, R.sup.3B, R.sup.4A, R.sup.4B, R.sup.5A, R.sup.5B, R.sup.5C are each independently for each occurrence absent, NH, O, S, CH.sub.2, C(O)O, C(O)NH, NHCH(R.sup.a)C(O), --C(O)--CH(R.sup.a)--NH--, CO, CH.dbd.N--

##STR00013##

[0251] L.sup.2A, L.sup.2B, L.sup.3A, L.sup.3B, L.sup.4A, L.sup.4B, L.sup.5A, L.sup.5B and L.sup.5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R.sup.a is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with antisense polynucleotide agents for inhibiting the expression of a target gene, such as those of formula (XXXVI):

[0252] Formula XXXVI

[0252] ##STR00014##

[0253] wherein L.sup.5A, L.sup.5B and L.sup.5C represent a monosaccharide, such as GalNAc derivative.

[0254] Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

[0255] Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

[0256] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an antisense polynucleotide agent. The present invention also includes antisense polynucleotide agents that are chimeric compounds.

[0257] "Chimeric" antisense polynucleotide agents or "chimeras," in the context of this invention, are antisense polynucleotide agent compounds, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an antisense polynucleotide agent. These antisense polynucleotide agents typically contain at least one region wherein the RNA is modified so as to confer upon the antisense polynucleotide agent increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the antisense polynucleotide agent can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense polynucleotide agent inhibition of gene expression. Consequently, comparable results can often be obtained with shorter antisense polynucleotide agents when chimeric antisense polynucleotide agents are used, compared to phosphorothioate deoxy antisense polynucleotide agents hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0258] In certain instances, the nucleotide of an antisense polynucleotide agent can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to antisense polynucleotide agents in order to enhance the activity, cellular distribution or cellular uptake of the antisense polynucleotide agent, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

V. Delivery of Antisense Polynucleotide Agents of the Invention

[0259] The delivery of an antisense polynucleotide agent of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a complement component C5-associated disease) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an antisense polynucleotide agent of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an antisense polynucleotide agent to a subject.

[0260] In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an antisense polynucleotide agent of the invention (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an antisense polynucleotide agent include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an antisense polynucleotide agent can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the antisense polynucleotide agent to be administered. Several studies have shown successful knockdown of gene products when an antisense polynucleotide agent is administered locally. For example, intraocular delivery of a VEGF antisense polynucleotide agent by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a antisense polynucleotide agent in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an antisense polynucleotide agent systemically for the treatment of a disease, the agent can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the antisense polynucleotide agent by endo- and exo-nucleases in vivo. Modification of the agent or the pharmaceutical carrier can also permit targeting of the antisense polynucleotide agent composition to the target tissue and avoid undesirable off-target effects. Antisense polynucleotide agent can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. In an alternative embodiment, the antisense polynucleotide agent can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an antisense polynucleotide agent molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an antisense polynucleotide agent by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an antisense polynucleotide agent, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an antisense polynucleotide agent. The formation of vesicles or micelles further prevents degradation of the antisense polynucleotide agent when administered systemically. Methods for making and administering cationic-antisense polynucleotide agent complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N, et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of antisense polynucleotide agents include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an antisense polynucleotide agent forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of antisense polynucleotide agents and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

VI. Pharmaceutical Compositions of the Invention

[0261] The present invention also includes pharmaceutical compositions and formulations which include the antisense polynucleotide agents of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an antisense polynucleotide agent, as described herein, and a pharmaceutically acceptable carrier.

[0262] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0263] The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum components, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0264] The pharmaceutical compositions containing the antisense polynucleotide agents are useful for treating a disease or disorder associated with the expression or activity of a C5 gene, e.g. a complement component C5-associated disease. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by subcutaneous (SC) or intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a C5 gene. In general, a suitable dose of an antisense polynucleotide agent of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. For example, the antisense polynucleotide agent can be administered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per single dose.

[0265] For example, the antisense polynucleotide agent may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0266] In another embodiment, the antisense polynucleotide agent is administered at a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0267] For example, the antisense polynucleotide agent may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0268] In another embodiment, the antisense polynucleotide agent is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kgb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment, the antisense polynucleotide agent is administered at a dose of about 10 mg/kg to about 30 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0269] For example, subjects can be administered, e.g., subcutaneously or intravenously, a single therapeutic amount of antisense polynucleotide agent, such as about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0270] In some embodiments, subjects are administered, e.g., subcutaneously or intravenously, multiple doses of a therapeutic amount of antisense polynucleotide agent, such as a dose about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. A multi-dose regimen may include administration of a therapeutic amount of antisense polynucleotide agent daily, such as for two days, three days, four days, five days, six days, seven days, or longer.

[0271] In other embodiments, subjects are administered, e.g., subcutaneously or intravenously, a repeat dose of a therapeutic amount of antisense polynucleotide agent, such as a dose about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. A repeat-dose regimen may include administration of a therapeutic amount of antisense polynucleotide agent on a regular basis, such as every other day, every third day, every fourth day, twice a week, once a week, every other week, or once a month.

[0272] The pharmaceutical composition can be administered by intravenous infusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25 minute period. The administration may be repeated, for example, on a regular basis, such as weekly, biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months or a year or longer.

[0273] The pharmaceutical composition can be administered once daily, or the antisense polynucleotide agent can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the antisense polynucleotide agent contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the antisense polynucleotide agent over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0274] In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals. In some embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered once per week. In other embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered bi-monthly.

[0275] The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual antisense polynucleotide agents encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

[0276] Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as a disorder that would benefit from reduction in the expression of C5. Such models can be used for in vivo testing of an antisense polynucleotide agent, as well as for determining a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, collagen-induced arthritis mouse model (Courtenay, J. S., et al. (1980) Nature 283, 666-668), myocardial ischemia (Homeister J W and Lucchesi B R (1994) Annu Rev Pharmacol Toxicol 34:17-40), ovalbumin induced asthma mouse models (e.g., Tomkinson A., et al. (2001). J. Immunol. 166, 5792-5800), (NZB.times.NZW)F1, MRL/Fas.sup.lpr (MRL/lpr) and BXSB mouse models (Theofilopoulos, A. N. and Kono, D. H. 1999. Murine lupus models: gene-specific and genome-wide studies. In Lahita R. G., ed., Systemic Lupus Erythematosus, 3rd edn, p. 145. Academic Press, San Diego, Calif.), mouse aHUS model (Goicoechea de Jorge et al. (2011) The development of atypical hemolytic uremic syndrome depends on complement C5, J Am Soc Nephrol 22:137-145.

[0277] The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

[0278] The antisense polynucleotide agent can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).

[0279] Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the antisense polynucleotide agents featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Antisense polynucleotide agents featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, antisense polynucleotide agents can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C.sub.1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof). Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

[0280] A. Antisense Polynucleotide Agent Formulations Comprising Membranous Molecular Assemblies

[0281] An antisense polynucleotide agent for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the antisense polynucleotide agent composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the antisense polynucleotide agent composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the antisense polynucleotide agent are delivered into the cell where the antisense polynucleotide agent can specifically bind to a target RNA and can mediate antisense inhibition. In some cases the liposomes are also specifically targeted, e.g., to direct the antisense polynucleotide agent to particular cell types.

[0282] A liposome containing an antisense polynucleotide agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The antisense polynucleotide agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the antisense polynucleotide agent and condense around the antisense polynucleotide agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of antisense polynucleotide agent.

[0283] If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to favor condensation.

[0284] Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging antisense polynucleotide agent preparations into liposomes.

[0285] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0286] Liposomes which are pH-sensitive or negatively-charged, entrap nucleic acids rather than complex with it. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0287] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0288] Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0289] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome.TM. I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome.TM. II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

[0290] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G.sub.M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0291] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

[0292] In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver antisense polynucleotide agents to macrophages.

[0293] Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated antisense polynucleotide agents in their internal compartments from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0294] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of Antisense polynucleotide agent (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

[0295] A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin.TM. Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

[0296] Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam.TM., Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0297] Another cationic lipid conjugate includes derivatization of the lipid with cholesterol ("DC-Chol") which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

[0298] Liposomal formulations are particularly suited for topical administration; liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer an antisense polynucleotide agent into the skin. In some implementations, liposomes are used for delivering antisense polynucleotide agents to epidermal cells and also to enhance the penetration of antisense polynucleotide agents into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0299] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with antisense polynucleotide agents are useful for treating a dermatological disorder.

[0300] Liposomes that include antisense polynucleotide agent can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include antisense polynucleotide agents can be delivered, for example, subcutaneously by infection in order to deliver antisense polynucleotide agents to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

[0301] Other formulations amenable to the present invention are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations that are amenable to the present invention.

[0302] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0303] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0304] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0305] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0306] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0307] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0308] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0309] The antisense polynucleotide agent for use in the compositions and methods of the invention can also be provided as micellar formulations. "Micelles" are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

[0310] A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the antisense polynucleotide agent composition, an alkali metal C.sub.8 to C.sub.22 alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

[0311] In one method a first micellar composition is prepared which contains the antisense polynucleotide agent composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the antisense polynucleotide agent composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

[0312] Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

[0313] For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

[0314] Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

[0315] The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

[0316] B. Lipid Particles

[0317] Antisense polynucleotide agents of in the invention may be fully encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

[0318] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle comprising a lipid layer encapsulating a pharmaceutically active molecule. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include "pSPLP," which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; 6,858,225; 8,158,601; and 8,058,069; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

[0319] In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to antisense polynucleotide agent ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

[0320] The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N--(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N--(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)--N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydr- o-3aH-cyclopenta[d][l1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)ami- no)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech GI), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

[0321] In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-santisense polynucleotide agent nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

[0322] In one embodiment, the lipid-antisense polynucleotide agent particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0.+-.20 nm and a 0.027 antisense polynucleotide agent/Lipid Ratio.

[0323] The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

[0324] The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci.sub.2), a PEG-dimyristyloxypropyl (Ci.sub.4), a PEG-dipalmityloxypropyl (Ci.sub.6), or a PEG-distearyloxypropyl (C].sub.8). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

[0325] In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0326] In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-antisense polynucleotide agent nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous antisense polynucleotide agent (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-antisense polynucleotide agent nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

##STR00015##

[0327] LNP01 formulations are described, e.g., in International Application Publication No. WO 2008A042973, which is hereby incorporated by reference.

[0328] Additional exemplary lipid-antisense polynucleotide agent formulations are described in Table 1.

TABLE-US-00001 TABLE 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Ionizable/Cationic Lipid Lipid:santisense polynucleotide agent ratio SNALP-1 1,2-Dilinolenyloxy-N,N-dimethylaminopropane DLinDMA/DPPC/Cholesterol/PEG-cDMA (DLinDMA) (57.1/7.1/34.4/1.4) lipid:santisense polynucleotide agent ~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:santisense polynucleotide agent ~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:santisense polynucleotide agent ~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:santisense polynucleotide agent ~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:santisense polynucleotide agent ~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:santisense polynucleotide agent ~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:santisense polynucleotide agent 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH- 50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100) Lipid:santisense polynucleotide agent 10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl 4-(dimethylamino)butanoate 50/10/38.5/1.5 (MC3) Lipid:santisense polynucleotide agent 10:1 LNP12 1,1'-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:santisense polynucleotide agent 10:1 yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:santisense polynucleotide agent: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:santisense polynucleotide agent: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:santisense polynucleotide agent: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:santisense polynucleotide agent: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:santisense polynucleotide agent: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:santisense polynucleotide agent: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:santisense polynucleotide agent: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:santisense polynucleotide agent: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:santisense polynucleotide agent: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:santisense polynucleotide agent: 10:1

DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000) SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.

[0329] XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.

[0330] MC3 comprising formulations are described, e.g., in U.S. Publication No. 2010/0324120, filed Jun. 10, 2010, the entire contents of which are hereby incorporated by reference.

[0331] ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.

[0332] C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Synthesis of Ionizable/Cationic Lipids

[0333] Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention can be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.

[0334] "Alkyl" means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

[0335] "Alkenyl" means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

[0336] "Alkynyl" means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

[0337] "Acyl" means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, --C(.dbd.O)alkyl, --C(.dbd.O)alkenyl, and --C(.dbd.O)alkynyl are acyl groups.

[0338] "Heterocycle" means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle can be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0339] The terms "optionally substituted alkyl", "optionally substituted alkenyl", "optionally substituted alkynyl", "optionally substituted acyl", and "optionally substituted heterocycle" means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (.dbd.O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, --CN, --ORx, --NRxRy, --NRxC(.dbd.O)Ry, --NRxSO2Ry, --C(.dbd.O)Rx, --C(.dbd.O)ORx, --C(.dbd.O)NRxRy, --SOnRx and --SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents can be further substituted with one or more of oxo, halogen, --OH, --CN, alkyl, --ORx, heterocycle, --NRxRy, --NRxC(.dbd.O)Ry, --NRxSO2Ry, --C(.dbd.O)Rx, --C(.dbd.O)ORx, --C(.dbd.O)NRxRy, --SOnRx and --SOnNRxRy.

[0340] "Halogen" means fluoro, chloro, bromo and iodo.

[0341] In some embodiments, protecting groups can be used. Protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an "alcohol protecting group" is used. An "alcohol protecting group" is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

Synthesis of Formula A

[0342] In some embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:

##STR00016##

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above can be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.

##STR00017##

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.

##STR00018##

[0343] Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

[0344] Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g). Synthesis of ALNY-100

[0345] Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:

##STR00019##

Synthesis of 515

[0346] To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0.degree. C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0.degree. C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): .delta.=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516

[0347] To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0.degree. C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1.times.100 mL) and saturated NaHCO3 solution (1.times.50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): .delta.=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H] -232.3 (96.94%).

Synthesis of 517A and 517B

[0348] The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (.about.3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2.times.100 mL) followed by saturated NaHCO3(1.times.50 mL) solution, water (1.times.30 mL) and finally with brine (lx 50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: --6 g crude

[0349] 517A--Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): .delta.=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS--[M+H]-266.3, [M+NH4+]-283.5 present, HPLC--97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

[0350] Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): .delta.=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC--98.65%.

General Procedure for the Synthesis of Compound 519

[0351] A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40.degree. C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR .delta.=130.2, 130.1 (.times.2), 127.9 (.times.3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (.times.2), 29.7, 29.6 (.times.2), 29.5 (.times.3), 29.3 (.times.2), 27.2 (.times.3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.

[0352] Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total antisense polynucleotide agent concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated antisense polynucleotide agent can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total antisense polynucleotide agent in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the "free" antisense polynucleotide agent content (as measured by the signal in the absence of surfactant) from the total antisense polynucleotide agent content. Percent entrapped antisense polynucleotide agent is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

[0353] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which the antisense polynucleotide agents featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Antisense polynucleotide agents featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Antisense polynucleotide agent complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for antisense polynucleotide agents and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0354] Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0355] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver, e.g., when treating hepatic disorders, e.g., hepatic carcinoma.

[0356] The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0357] The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

[0358] C. Additional Formulations

[0359] i. Emulsions

[0360] The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 .mu.m in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

[0361] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0362] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0363] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0364] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0365] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0366] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0367] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0368] ii. Microemulsions

[0369] In one embodiment of the present invention, the compositions of antisense polynucleotide agents are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0370] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0371] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[0372] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or antisense polynucleotide agents. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of antisense polynucleotide agents from the gastrointestinal tract, as well as improve the local cellular uptake of antisense polynucleotide agents and nucleic acids.

[0373] Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the antisense polynucleotide agents of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories--surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0374] iii. Microparticles

[0375] An antisense polynucleotide agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

[0376] iv. Penetration Enhancers

[0377] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly antisense polynucleotide agents, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0378] Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[0379] Surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of antisense polynucleotide agents through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0380] Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C.sub.1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0381] The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0382] Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of antisense polynucleotide agents through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0383] As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of antisense polynucleotide agents through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0384] Agents that enhance uptake of antisense polynucleotide agents at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of antisense polynucleotide agents. Examples of commercially available transfection reagents include, for example Lipofectamine.TM. (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000.TM. (Invitrogen; Carlsbad, Calif.), 293fectin.TM. (Invitrogen; Carlsbad, Calif.), Cellfectin.TM. (Invitrogen; Carlsbad, Calif.), DMRIE-C.TM. (Invitrogen; Carlsbad, Calif.), FreeStyle.TM. (Invitrogen; Carlsbad, Calif.), Lipofectamine.TM. 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine.TM. (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine.TM. (Invitrogen; Carlsbad, Calif.), Optifect.TM. (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam.RTM. Reagent (Promega; Madison, Wis.), TransFast.TM. Transfection Reagent (Promega; Madison, Wis.), Tfx.TM.-20 Reagent (Promega; Madison, Wis.), Tfx.TM.-50 Reagent (Promega; Madison, Wis.), DreamFect.TM. (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass.sup.a DI Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec.TM./LipoGen.TM. (Invitrogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER.TM. transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect.TM. (B-Bridge International, Mountain View, Calif., USA), among others.

[0385] Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

[0386] v. Carriers

[0387] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioated antisense polynucleotide agent in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., Antisense polynucleotide agent Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense polynucleotide agent & Nucl. Acid Drug Dev., 1996, 6, 177-183.

[0388] vi. Excipients

[0389] In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

[0390] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0391] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0392] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0393] vii. Other Components

[0394] The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0395] Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

[0396] In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more antisense polynucleotide agents and (b) one or more agents which function by a non-antisense inhibition mechanism and which are useful in treating a hemolytic disorder. Examples of such agents include, but are not limited to an anti-inflammatory agent, anti-steatosis agent, anti-viral, and/or anti-fibrosis agent. In addition, other substances commonly used to protect the liver, such as silymarin, can also be used in conjunction with the antisense polynucleotide agents described herein. Other agents useful for treating liver diseases include telbivudine, entecavir, and protease inhibitors such as telaprevir and other disclosed, for example, in Tung et al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and in Hale et al., U.S. Application Publication No. 2004/0127488.

[0397] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds that exhibit high therapeutic indices are preferred.

[0398] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC.sub.50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0399] In addition to their administration, as discussed above, the antisense polynucleotide agents featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by C5 expression. In any event, the administering physician can adjust the amount and timing of antisense polynucleotide agent administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VII. Methods for Inhibiting C5 Expression

[0400] The present invention provides methods of inhibiting expression of C5 in a cell. The methods include contacting a cell with an antisense polynucleotide agent of the invention in an amount effective to inhibit expression of the C5 in the cell, thereby inhibiting expression of the C5 in the cell.

[0401] Contacting of a cell with an antisense polynucleotide agent may be done in vitro or in vivo. Contacting a cell in vivo with the antisense polynucleotide agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the antisense polynucleotide agent. Combinations of in vitro and in vivo methods of contacting are also possible. Contacting may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc.sub.3 ligand, or any other ligand that directs the antisense polynucleotide agent to a site of interest, e.g., the liver of a subject.

[0402] The term "inhibiting," as used herein, is used interchangeably with "reducing," "silencing," "downregulating" and other similar terms, and includes any level of inhibition.

[0403] The phrase "inhibiting expression of a C5" is intended to refer to inhibition of expression of any C5 gene (such as, e.g., a mouse C5 gene, a rat C5 gene, a monkey C5 gene, or a human C5 gene) as well as variants or mutants of a C5 gene. Thus, the C5 gene may be a wild-type C5 gene, a mutant C5 gene, or a transgenic C5 gene in the context of a genetically manipulated cell, group of cells, or organism.

[0404] "Inhibiting expression of a C5 gene" includes any level of inhibition of a C5 gene, e.g., at least partial suppression of the expression of a C5 gene. The expression of the C5 gene may be assessed based on the level, or the change in the level, of any variable associated with C5 gene expression, e.g., C5 mRNA level, C5 protein level, or for example, CH.sub.50 activity as a measure of total hemolytic complement, AH.sub.50 to measure the hemolytic activity of the alternate pathway of complement, and/or lactate dehydrogenase (LDH) levels as a measure of intravascular hemolysis, and/or hemoglobin levels. Levels of C5a, C5b, and soluble C5b-9 complex may also be measured to assess C5 expression. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.

[0405] Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with C5 expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

[0406] In some embodiments of the methods of the invention, expression of a C5 gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0407] Inhibition of the expression of a C5 gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a C5 gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an antisense polynucleotide agent of the invention, or by administering an antisense polynucleotide agent of the invention to a subject in which the cells are or were present) such that the expression of a C5 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s)). In preferred embodiments, the inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells, using the following formula:

( mRNA in control cells ) - ( mRNA in treated cells ) ( mRNA in control cells ) 100 % ##EQU00001##

[0408] Alternatively, inhibition of the expression of a C5 gene may be assessed in terms of a reduction of a parameter that is functionally linked to C5 gene expression, e.g., C5 protein expression, CH.sub.50 activity, AH.sub.50, lactate dehydrogenase (LDH) levels, hemoglobin levels, mRNA or protein levels of C5a, C5b, and soluble C5b-9 complex in tissues or serum. C5 gene silencing may be determined in any cell expressing C5, either constitutively or by genomic engineering, and by any assay known in the art. The liver is the major site of C5 expression. Other significant sites of expression include the kidneys and the uterus.

[0409] Inhibition of the expression of a C5 protein may be manifested by a reduction in the level of the C5 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above for the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

[0410] A control cell or group of cells that may be used to assess the inhibition of the expression of a C5 gene includes a cell or group of cells that has not yet been contacted with an antisense polynucleotide agent of the invention. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an antisense polynucleotide agent.

[0411] The level of C5 mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of C5 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the C5 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis.

[0412] In one embodiment, the level of expression of C5 is determined using a nucleic acid probe. The term "probe", as used herein, refers to any molecule that is capable of selectively binding to a specific C5. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0413] Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to C5 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of C5 mRNA.

[0414] An alternative method for determining the level of expression of C5 in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of C5 is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan.TM. System).

[0415] The expression levels of C5 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of C5 expression level may also comprise using nucleic acid probes in solution.

[0416] In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein.

[0417] The level of C5 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.

[0418] The term "sample" as used herein refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, lymph, urine, cerebrospinal fluid, saliva, ocular fluids, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In preferred embodiments, a "sample derived from a subject" refers to blood or plasma drawn from the subject. In further embodiments, a "sample derived from a subject" refers to liver tissue derived from the subject.

[0419] In some embodiments of the methods of the invention, the antisense polynucleotide agent is administered to a subject such that the antisense polynucleotide agent is delivered to a specific site within the subject. The inhibition of expression of C5 may be assessed using measurements of the level or change in the level of C5 mRNA or C5 protein in a sample derived from fluid or tissue from the specific site within the subject. In preferred embodiments, the site is the liver. The site may also be a subsection or subgroup of cells from any one of the aforementioned sites. The site may also include cells that express a particular type of receptor.

[0420] The phrase "contacting a cell with an antisense polynucleotide agent," as used herein, includes contacting a cell by any possible means. Contacting a cell with an antisense polynucleotide agent includes contacting a cell in vitro with the antisense polynucleotide agent or contacting a cell in vivo with the antisense polynucleotide agent. The contacting may be done directly or indirectly. Thus, for example, the antisense polynucleotide agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the antisense polynucleotide agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

[0421] Contacting a cell in vitro may be done, for example, by incubating the cell with the antisense polynucleotide agent. Contacting a cell in vivo may be done, for example, by injecting the antisense polynucleotide agent into or near the tissue where the cell is located, or by injecting the antisense polynucleotide agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the antisense polynucleotide agent may contain and/or be coupled to a ligand, e.g., GalNAc3, that directs the antisense polynucleotide agent to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an antisense polynucleotide agent and subsequently transplanted into a subject.

[0422] In one embodiment, contacting a cell with an antisense polynucleotide agent includes "introducing" or "delivering the antisense polynucleotide agent into the cell" by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an antisense polynucleotide agent can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an antisense polynucleotide agent into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, antisense polynucleotide agent can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

VIII. Methods for Treating or Preventing a Complement Component C5-Associated Disorder

[0423] The present invention also provides therapeutic and prophylactic methods which include administering to a subject having a complement component C5-associated disease, e.g., PNH or aHUS, an antisense polynucleotide agent or pharmaceutical compositions comprising an antisense polynucleotide agent of the invention. In some aspects of the invention, the methods further include administering to the subject an additional therapeutic agent, such as an anti-complement component C5 antibody, or antigen-binding fragment thereof (e.g., eculizumab).

[0424] In one aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in C5 expression, e.g., a complement component C5-associated disease, e.g., PNH or aHUS. The treatment methods (and uses) of the invention include administering to the subject, e.g., a human, a therapeutically effective amount of an antisense polynucleotide agent targeting a C5 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a C5 gene, thereby treating the subject having a disorder that would benefit from reduction in C5 expression.

[0425] In another aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in C5 expression, e.g., a complement component C5-associated disease, e.g., PNH or aHUS, which include administering to the subject, e.g., a human, a therapeutically effective amount of an antisense polynucleotide agent targeting a C5 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a C5 gene, and an additional therapeutic agent, such as an anti-complement component C5 antibody, or antigen-binding fragment thereof (e.g., eculizumab), thereby treating the subject having a disorder that would benefit from reduction in C5 expression.

[0426] In one aspect, the invention provides methods of preventing at least one symptom in a subject having a disorder that would benefit from reduction in C5 expression, e.g., a complement component C5-associated disease, e.g., PNH or aHUS. The methods include administering to the subject a prohpylactically effective amount of an antisense polynucleotide agent targeting a C5 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a C5 gene, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in C5 expression. For example, the invention provides methods for preventing hemolysis in a subject suffering from a disorder that would benefit from reduction in C5 expression, e.g., a complement component C5-associated disease, e.g., PNH or aHUS.

[0427] In another aspect, the invention provides methods of preventing at least one symptom in a subject having a disorder that would benefit from reduction in C5 expression, e.g., a complement component C5-associated disease, e.g., PNH or aHUS. The methods include administering to the subject a prohpylactically effective amount of an antisense polynucleotide agent targeting a C5 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a C5 gene, and an additional therapeutic agent, such as an anti-complement component C5 antibody, or antigen-binding fragment thereof (e.g., eculizumab), thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in C5 expression.

[0428] "Therapeutically effective amount," as used herein, is intended to include the amount of an antisense polynucleotide agent or anti-complement component C5 antibody, or antigen-binding fragment thereof (e.g., eculizumab), that, when administered to a subject having a complement component C5-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective amount" may vary depending on the antisense polynucleotide agent or antibody, or antigen-binding fragment thereof, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

[0429] "Prophylactically effective amount," as used herein, is intended to include the amount of an antisense polynucleotide agent or anti-complement component C5 antibody, or antigen-binding fragment thereof (e.g., eculizumab), that, when administered to a subject having a complement component C5-associate disease but not yet (or currently) experiencing or displaying symptoms of the disease, and/or a subject at risk of developing a complement component C5-associated disease, e.g., a subject having a graft and/or transplant, e.g., a sensitized or allogenic recipient, a subject having sepsis, and/or a subject having a myocardial infarction, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The "prophylactically effective amount" may vary depending on the antisense polynucleotide agent or anti-complement component C5 antibody, or antigen-binding fragment thereof, how the agent or anti-complement component C5 antibody, or antigen-binding fragment thereof, is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

[0430] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an antisense polynucleotide agent or anti-complement component C5 antibody, or antigen-binding fragment thereof (e.g., eculizumab), that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. antisense polynucleotide agents employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

[0431] In another aspect, the present invention provides uses of a therapeutically effective amount of an antisense polynucleotide agent of the invention for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of C5 expression.

[0432] In another aspect, the present invention provides uses of a therapeutically effective amount of an antisense polynucleotide agent of the invention and an additional therapeutic agent, such as an anti-complement component C5 antibody, or antigen-binding fragment thereof (e.g., eculizumab), for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of C5 expression.

[0433] In yet another aspect, the present invention provides use of an antisense polynucleotide agent of the invention targeting a C5 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a C5 gene in the manufacture of a medicament for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of C5 expression, such as a subject having a disorder that would benefit from reduction in C5 expression, e.g., a complement component C5-associated disease, e.g., PNH or aHUS.

[0434] In another aspect, the present invention provides uses of an antisense polynucleotide agent of the invention targeting a C5 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a C5 gene in the manufacture of a medicament for use in combination with an additional therapeutic agent, such as an anti-complement component C5 antibody, or antigen-binding fragment thereof (e.g., eculizumab), for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of C5 expression, e.g., a complement component C5-associated disease, e.g., PNH or aHUS.

[0435] In another aspect, the invention provides uses of an antisense polynucleotide agent of the invention for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of C5 expression, such as a complement component C5-associated disease, e.g., PNH or aHUS.

[0436] In yet another aspect, the invention provides uses of an antisense polynucleotide agent of the invention, and an additional therapeutic agent, such as an anti-complement component C5 antibody, or antigen-binding fragment thereof (e.g., eculizumab), for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of C5 expression, such as a complement component C5-associated disease, e.g., PNH or aHUS.

[0437] In a further aspect, the present invention provides uses of an antisense polynucleotide agent of the invention in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of C5 expression, such as a complement component C5-associated disease, e.g., PNH or aHUS.

[0438] In a further aspect, the present invention provides uses of an antisense polynucleotide agent of the invention in the manufacture of a medicament for use in combination with an additional therapeutic agent, such as an anti-complement component C5 antibody, or antigen-binding fragment thereof (e.g., eculizumab), for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of C5 expression, such as a complement component C5-associated disease, e.g., PNH or aHUS.

[0439] In one embodiment, an antisense polynucleotide agent targeting C5 is administered to a subject having a complement component C5-associated disease such that C5 levels, e.g., in a cell, tissue, blood, urine or other tissue or fluid of the subject are reduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more and, subsequently, an additional therapeutic (as described below) is administered to the subject.

[0440] The additional therapeutic may be an anti-complement component C5 antibody, or antigen-binding fragment or derivative thereof. In one embodiment, the anti-complement component C5 antibody is eculizumab (SOLIRIS.RTM.), or antigen-binding fragment or derivative thereof. Eculizumab is a humanized monoclonal IgG2/4, kappa light chain antibody that specifically binds complement component C5 with high affinity and inhibits cleavage of C5 to C5a and C5b, thereby inhibiting the generation of the terminal complement complex C5b-9. Eculizumab is described in U.S. Pat. No. 6,355,245, the entire contents of which are incorporated herein by reference.

[0441] The methods of the invention comprising administration of an antisense polynucleotide agent of the invention and eculizumab to a subject may further comprise administration of a meningococcal vaccine to the subject.

[0442] The additional therapeutic, e.g., eculizumab and/or a meningococcal vaccine, may be administered to the subject at the same time as the antisense polynucleotide agent targeting C5 or at a different time.

[0443] Moreover, the additional therapeutic, e.g., eculizumab, may be administered to the subject in the same formulation as the antisense polynucleotide agent targeting C5 or in a different formulation as the antisense polynucleotide agent targeting C5.

[0444] Eculizumab dosage regimens are described in, for example, the product insert for eculizumab (SOLIRIS.RTM.) and in U.S. Patent Application No. 2012/0225056, the entire contents of each of which are incorporated herein by reference. In exemplary methods of the invention for treating a complement component C5-associated disease, e.g., PNH or aHUS, an antisense polynucleotide agent targeting C5 is administered (e.g., subcutaneously) to the subject first, such that the C5 levels in the subject are reduced (e.g., by at least about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more) and subsequently eculizumab is administered at doses lower than the ones described in the product insert for SOLIRIS.RTM.. For example, eculizumab may be administered to the subject weekly at a dose less than about 600 mg for 4 weeks followed by a fifth dose at about one week later of less than about 900 mg, followed by a dose less than about 900 mg about every two weeks thereafter. Eculizumab may also be administered to the subject weekly at a dose less than about 900 mg for 4 weeks followed by a fifth dose at about one week later of less than about 1200 mg, followed by a dose less than about 1200 mg about every two weeks thereafter. If the subject is less than 18 years of age, eculizumab may be administered to the subject weekly at a dose less than about 900 mg for 4 weeks followed by a fifth dose at about one week later of less than about 1200 mg, followed by a dose less than about 1200 mg about every two weeks thereafter; or if the subject is less than 18 years of age, eculizumab may be administered to the subject weekly at a dose less than about 600 mg for 2 weeks followed by a third dose at about one week later of less than about 900 mg, followed by a dose less than about 900 mg about every two weeks thereafter; or if the subject is less than 18 years of age, eculizumab may be administered to the subject weekly at a dose less than about 600 mg for 2 weeks followed by a third dose at about one week later of less than about 600 mg, followed by a dose less than about 600 mg about every two weeks thereafter; or if the subject is less than 18 years of age, eculizumab may be administered to the subject weekly at a dose less than about 600 mg for 1 week followed by a second dose at about one week later of less than about 300 mg, followed by a dose less than about 300 mg about every two weeks thereafter; or if the subject is less than 18 years of age, eculizumab may be administered to the subject weekly at a dose less than about 300 mg for 1 week followed by a second dose at about one week later of less than about 300 mg, followed by a dose less than about 300 mg about every two weeks thereafter. If the subject is receiving plamapheresis or plasma exchange, eculizumab may be administered to the subject at a dose less than about 300 mg (e.g., if the most recent does of eculizumab was about 300 mg) or less than about 600 mg (e.g., if the most recent does of eculizumab was about 600 mg or more). If the subject is receiving plasma infusion, eculizumab may be administered to the subject at a dose less than about 300 mg (e.g., if the most recent does of eculizumab was about 300 mg or more). The lower doses of eculizumab allow for either subcutaneous or intravenous administration of eculizumab.

[0445] In the combination therapies of the present invention comprising eculizumab, eculizumab may be administered to the subject, e.g., subcutaneously, at a dose of about 0.01 mg/kg to about 10 mg/kg, or about 5 mg/kg to about 10 mg/kg, or about 0.5 mg/kg to about 15 mg/kg. For example, eculizumab may be administered to the subject, e.g., subcutaneously, at a dose of 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 10.5 mg/kg, 11 mg/kg, 11.5 mg/kg, 12 mg/kg, 12.5 mg/kg, 13 mg/kg, 13.5 mg/kg, 14 mg/kg, 14.5 mg/kg, or 15 mg/kg.

[0446] The methods and uses of the invention include administering a composition described herein such that expression of the target C5 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, expression of the target C5 gene is decreased for an extended duration, e.g., at least about two, three, four, five, six, seven days or more, e.g., about one week, two weeks, three weeks, or about four weeks or longer.

[0447] Administration of the antisense polynucleotide agent according to the methods and uses of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a complement component C5-associated disease. By "reduction" in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

[0448] Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a hemolytic disorder may be assessed, for example, by periodic monitoring of LDH and CH.sub.50 levels. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an antisense polynucleotide agent targeting C5 or pharmaceutical composition thereof, "effective against" a complement component C5-associated disease indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating a complement component C5-associated disease and the related causes.

[0449] A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given antisense polynucleotide agent drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

[0450] Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale, as but one example the Rheumatoid Arthritis Severity Scale (RASS). Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an antisense polynucleotide agent or antisense polynucleotide agent formulation as described herein.

[0451] Subjects can be administered a therapeutic amount of antisense polynucleotide agent, such as about 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg, 3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0 mg/kg, 4.1 mg/kg, 4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg, 4.7 mg/kg, 4.8 mg/kg, 4.9 mg/kg, 5.0 mg/kg, 5.1 mg/kg, 5.2 mg/kg, 5.3 mg/kg, 5.4 mg/kg, 5.5 mg/kg, 5.6 mg/kg, 5.7 mg/kg, 5.8 mg/kg, 5.9 mg/kg, 6.0 mg/kg, 6.1 mg/kg, 6.2 mg/kg, 6.3 mg/kg, 6.4 mg/kg, 6.5 mg/kg, 6.6 mg/kg, 6.7 mg/kg, 6.8 mg/kg, 6.9 mg/kg, 7.0 mg/kg, 7.1 mg/kg, 7.2 mg/kg, 7.3 mg/kg, 7.4 mg/kg, 7.5 mg/kg, 7.6 mg/kg, 7.7 mg/kg, 7.8 mg/kg, 7.9 mg/kg, 8.0 mg/kg, 8.1 mg/kg, 8.2 mg/kg, 8.3 mg/kg, 8.4 mg/kg, 8.5 mg/kg, 8.6 mg/kg, 8.7 mg/kg, 8.8 mg/kg, 8.9 mg/kg, 9.0 mg/kg, 9.1 mg/kg, 9.2 mg/kg, 9.3 mg/kg, 9.4 mg/kg, 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg, 9.9 mg/kg, 9.0 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0452] In certain embodiments, for example, when a composition of the invention comprises a antisense polynucleotide agent as described herein and a lipid, subjects can be administered a therapeutic amount of antisense polynucleotide agent, such as about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0453] For example, the antisense polynucleotide agent may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0454] In other embodiments, for example, when a composition of the invention comprises a antisense polynucleotide agent as described herein and an N-acetylgalactosamine, subjects can be administered a therapeutic amount of antisense polynucleotide agent, such as a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment, when a composition of the invention comprises a antisense polynucleotide agent as described herein and an N-acetylgalactosamine, subjects can be administered a therapeutic amount of about 10 to about 30 mg/kg of antisense polynucleotide agent. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0455] For example, subjects can be administered a therapeutic amount of antisense polynucleotide agent, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0456] The antisense polynucleotide agent can be administered by intravenous infusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about a 25 minute period. The administration may be repeated, for example, on a regular basis, such as weekly, biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months or a year or longer.

[0457] Administration of the antisense polynucleotide agent can reduce C5 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

[0458] Before administration of a full dose of the antisense polynucleotide agent, patients can be administered a smaller dose, such as a 5% infusion, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

[0459] Owing to the inhibitory effects on C5 expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.

[0460] An antisense polynucleotide agent of the invention may be administered in "naked" form, or as a "free antisense polynucleotide agent." A naked antisense polynucleotide agent is administered in the absence of a pharmaceutical composition. The naked antisense polynucleotide agent may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the antisense polynucleotide agent can be adjusted such that it is suitable for administering to a subject.

[0461] Alternatively, an antisense polynucleotide agent of the invention may be administered as a pharmaceutical composition, such as an antisense polynucleotide agent liposomal formulation.

[0462] Subjects that would benefit from a reduction and/or inhibition of C5 gene expression are those having a complement component C5-associated disease or disorder as described herein. In one embodiment, a subject having a complement component C5-associated disease has paroxysmal nocturnal hemoglobinuria (PNH). In another embodiment, a subject having a complement component C5-associated disease has asthma. In another embodiment, a subject having a complement component C5-associated disease has rheumatoid arthritis. In yet another embodiment, a subject having a complement component C5-associated disease has systemic lupus erythmatosis. In one embodiment, a subject having a complement component C5-associated disease has glomerulonephritis. In another embodiment, a subject having a complement component C5-associated disease has psoriasis. In yet another embodiment, a subject having a complement component C5-associated disease has dermatomyositis bullous pemphigoid. In one embodiment, a subject having a complement component C5-associated disease has atypical hemolytic uremic syndrome. In another embodiment, a subject having a complement component C5-associated disease has Shiga toxin E. coli-related hemolytic uremic syndrome. In another embodiment, a subject having a complement component C5-associated disease has myasthenia gravis. In yet another embodiment, a subject having a complement component C5-associated disease has neuromyelistis optica. In one embodiment, a subject having a complement component C5-associated disease has dense deposit disease. In one embodiment, a subject having a complement component C5-associated disease has C3 neuropathy. In another embodiment, a subject having a complement component C5-associated disease has age-related macular degeneration. In another embodiment, a subject having a complement component C5-associated disease has cold agglutinin disease. In one embodiment, a subject having a complement component C5-associated disease has anti-neutrophil cytoplasmic antibody-associated vasculitis. In another embodiment, a subject having a complement component C5-associated disease has humoral and vascular transplant rejection. In one embodiment, a subject having a complement component C5-associated disease has graft dysfunction. In one embodiment, a subject having a complement component C5-associated disease has had a myocardial infarction. In another embodiment, a subject having a complement component C5-associated disease is a sensitized recipient of a transplant. In yet another embodiment, a subject having a complement component C5-associated disease has sepsis.

[0463] Treatment of a subject that would benefit from a reduction and/or inhibition of C5 gene expression includes therapeutic and prophylactic (e.g., the subject is to undergo sensitized (or allogenic) transplant surgery) treatment.

[0464] The invention further provides methods and uses of an antisense polynucleotide agent or a pharmaceutical composition thereof (including methods and uses of an antisense polynucleotide agent or a pharmaceutical composition comprising an antisense polynucleotide agent and an anti-complement component C5 antibody, or antigen-binding fragment thereof) for treating a subject that would benefit from reduction and/or inhibition of C5 expression, e.g., a subject having a complement component C5-associated disease, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an antisense polynucleotide agent targeting C5 is administered in combination with, e.g., an agent useful in treating a complement component C5-associated disease as described elsewhere herein.

[0465] For example, additional therapeutics and therapeutic methods suitable for treating a subject that would benefit from reduction in C5 expression, e.g., a subject having a complement component C5-associated disease, include plasmaphoresis, thrombolytic therapy (e.g., streptokinase), antiplatelet agents, folic acid, corticosteroids; immunosuppressive agents; estrogens, methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine, olsalazine, chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate (intramuscular and oral), azathioprine, cochicine, corticosteroids (oral, inhaled and local injection), beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signalling by proinflammatory cytokines, such as TNF-.alpha. or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting enzyme inhibitors, TNF.alpha.converting enzyme (TACE) inhibitors, T-cell signalling inhibitors, such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNF receptors and the derivatives p75TNFRIgG (Enbrel.TM. and p55TNFRIgG (Lenercept)), sIL-RI, sIL-RII, and sIL-6R), antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and TGF.beta.), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib, etanercept, infliximonoclonal antibody, naproxen, valdecoxib, sulfasalazine, methylprednisolone, meloxicam, methylprednisolone acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide, propoxyphene napsylate/apap, folate, nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodone bitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol hcl, salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium, prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin, glucosamine sulf/chondroitin, amitriptyline hcl, sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl, misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximonoclonal antibody, IL-TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485, CDC-801, Mesopram, cyclosporine, cytokine suppressive anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNF.alpha. antibody; Celltech/Bayer); cA2/infliximonoclonal antibody (chimeric anti-TNF.alpha. antibody; Centocor); 75 kdTNFR-IgG/etanercept (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g., (1994) Arthr. Rheum. 37: S295; (1996) J. Invest. Med. 44: 235A); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody; IDEC/SmithKline; see e.g., (1995) Arthr. Rheum. 38: S185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; see e.g., (1993) Arthrit. Rheum. 36: 1223); Anti-Tac (humanized anti-IL-2R.alpha.; Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-RA (IL-receptor antagonist; Synergen/Amgen); anakinra (Kineret.RTM./Amgen); TNF-bp/s-TNF (soluble TNF binding protein; see e.g., (1996) Arthr. Rheum. 39(9 (supplement)): S284; (1995) Amer. J. Physiol.--Heart and Circ. Physiol. 268: 37-42); R973401 (phosphodiesterase Type IV inhibitor; see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S282); MK-966 (COX-2 Inhibitor; see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S81); Iloprost (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S82); methotrexate; thalidomide (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S282) and thalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatory and cytokine inhibitor; see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S131; (1996) Inflamm. Res. 45: 103-107); tranexamic acid (inhibitor of plasminogen activation; see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S284); T-614 (cytokine inhibitor; see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S282); prostaglandin El (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S282); Tenidap (non-steroidal anti-inflammatory drug; see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S280); Naproxen (non-steroidal anti-inflammatory drug; see e.g., (1996) Neuro. Report 7: 1209-1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S281); Azathioprine (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S281); ICE inhibitor (inhibitor of the enzyme interleukin-1.beta. converting enzyme); zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinase zap-70 or lck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitors of vascular endothelial cell growth factor or vascular endothelial cell growth factor receptor; inhibitors of angiogenesis); corticosteroid anti-inflammatory drugs (e.g., SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies; anti-IL-18 antibodies; interleukin-1 (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S296); interleukin-13 (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S308); interleukin-17 inhibitors (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S120); gold; penicillamine; chloroquine; chlorambucil; hydroxychloroquine; cyclosporine; cyclophosphamide; total lymphoid irradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins; orally-administered peptides and collagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligo-deoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulphate; minocycline; anti-IL2R antibodies; marine and botanical lipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21: 759-777); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immune globulin; zileuton; azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see Bruncko, M. et al. (2007) J. Med. Chem. 50(4): 641-662); antivirals and immune-modulating agents, small molecule inhibitor of KDR, small molecule inhibitor of Tie-2; methotrexate; prednisone; celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib; etanercept; infliximonoclonal antibody; leflunomide; naproxen; valdecoxib; sulfasalazine; methylprednisolone; ibuprofen; meloxicam; methylprednisolone acetate; gold sodium thiomalate; aspirin; azathioprine; triamcinolone acetonide; propxyphene napsylate/apap; folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium; oxaprozin; oxycodone hcl; hydrocodone bitartrate/apap; diclofenac sodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol hcl; salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen; alendronate sodium; prednisolone; morphine sulfate; lidocaine hydrochloride; indomethacin; glucosamine sulfate/chondroitin; cyclosporine; amitriptyline hcl; sulfadiazine; oxycodone hcl/acetaminophen; olopatadine hcl; misoprostol; naproxen sodium; omeprazole; mycophenolate mofetil; cyclophosphamide; rituximonoclonal antibody; IL-TRAP; MRA; CTLA4-IG; IL-18 BP; IL-12/23; anti-IL 18; anti-IL 15; BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485; CDC-801; mesopramn, albuterol, salmeterol/fluticasone, montelukast sodium, fluticasone propionate, budesonide, prednisone, salmeterol xinafoate, levalbuterol hcl, albuterol sulfate/ipratropium, prednisolone sodium phosphate, triamcinolone acetonide, beclomethasone dipropionate, ipratropium bromide, azithromycin, pirbuterol acetate, prednisolone, theophylline anhydrous, methylprednisolone sodium succinate, clarithromycin, zafirlukast, formoterol fumarate, influenza virus vaccine, methylprednisolone, amoxicillin trihydrate, flunisolide, allergy injection, cromolyn sodium, fexofenadine hydrochloride, flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhaler assist device, guaifenesin, dexamethasone sodium phosphate, moxifloxacin hcl, doxycycline hyclate, guaifenesin/d-methorphan, p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride, mometasone furoate, salmeterol xinafoate, benzonatate, cephalexin, pe/hydrocodone/chlorphenir, cetirizine hcl/pseudoephed, phenylephrine/cod/promethazine, codeine/promethazine, cefprozil, dexamethasone, guaifenesin/pseudoephedrine, chlorpheniramine/hydrocodone, nedocromil sodium, terbutaline sulfate, epinephrine, methylprednisolone, metaproterenol sulfate, aspirin, nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin sodium, clopidogrel bisulfate, carvedilol, atenolol, morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril, isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril, tenecteplase, enalapril maleate, torsemide, retavase, losartan potassium, quinapril hcl/mag carb, bumetanide, alteplase, enalaprilat, amiodarone hydrochloride, tirofiban hcl m-hydrate, diltiazem hydrochloride, captopril, irbesartan, valsartan, propranolol hydrochloride, fosinopril sodium, lidocaine hydrochloride, eptifibatide, cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone, interferon, sotalol hydrochloride, potassium chloride, docusate sodium, dobutamine hcl, alprazolam, pravastatin sodium, atorvastatin calcium, midazolam hydrochloride, meperidine hydrochloride, isosorbide dinitrate, epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin, ezetimibe/simvastatin, avasimibe, and cariporide.

[0466] The antisense polynucleotide agent (and/or an anti-complement component C5 antibody) and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

[0467] The present invention also provides methods of using an antisense polynucleotide agent of the invention and/or a composition containing an antisense polynucleotide agent of the invention to reduce and/or inhibit complement component C5 expression in a cell. In other aspects, the present invention provides an antisense polynucleotide agent of the invention and/or a composition comprising an antisense polynucleotide agent of the invention for use in reducing and/or inhibiting C5 expression in a cell. In yet other aspects, use of an antisense polynucleotide agent of the invention and/or a composition comprising an antisense polynucleotide agent of the invention for the manufacture of a medicament for reducing and/or inhibiting C5 expression in a cell are provided.

[0468] The methods and uses include contacting the cell with an antisense polynucleotide agent, e.g., a antisense polynucleotide agent, of the invention and maintaining the cell for a time sufficient to obtain antisense inhibition of a C5 gene, thereby inhibiting expression of the C5 gene in the cell.

[0469] Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of C5 may be determined by determining the mRNA expression level of C5 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, by determining the protein level of C5 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques, flow cytometry methods, ELISA, and/or by determining a biological activity of C5, such as CH.sub.50 or AH.sub.50 hemolysis assay, and/or by determining the biological activity of one or more molecules associated with the complement system, e.g., C5 products, such as C5a and C5b (or, in an in vivo setting, e.g., hemolysis).

[0470] In the methods and uses of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject. In embodiments of the invention in which the cell is within a subject, the methods may include further contacting the cell with an anti-complement component C5 antibody, e.g., eculizumab.

[0471] A cell suitable for treatment using the methods of the invention may be any cell that expresses a C5 gene. A cell suitable for use in the methods and uses of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.

[0472] C5 expression may be inhibited in the cell by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

[0473] The in vivo methods and uses of the invention may include administering to a subject a composition containing an antisense polynucleotide agent, where the antisense polynucleotide agent includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the C5 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection.

[0474] In some embodiments, the administration is via a depot injection. A depot injection may release the antisense polynucleotide agent in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of C5, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

[0475] In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the antisense polynucleotide agent to the liver.

[0476] The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

[0477] In one aspect, the present invention also provides methods for inhibiting the expression of a C5 gene in a mammal, e.g., a human. The present invention also provides a composition comprising an antisense polynucleotide agent that targets a C5 gene in a cell of a mammal for use in inhibiting expression of the C5 gene in the mammal. In another aspect, the present invention provides use of an antisense polynucleotide agent that targets a C5 gene in a cell of a mammal in the manufacture of a medicament for inhibiting expression of the C5 gene in the mammal.

[0478] The methods and uses include administering to the mammal, e.g., a human, a composition comprising an antisense polynucleotide agent that targets a C5 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain antisense inhibition of the mRNA transcript of the C5 gene, thereby inhibiting expression of the C5 gene in the mammal. In some embodiment, the methods further comprise administering an anti-complement component C5 antibody, e.g., eculizumab, to the subject.

[0479] Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g., ELISA or Western blotting, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in C5 gene and/or protein expression. In another embodiment, a blood sample serves as the tissue material for monitoring the reduction in C5 gene and/or protein expression. In other embodiments, inhibition of the expression of a C5 gene is monitored indirectly by, for example, determining the expression and/or activity of a gene in a C5 pathway, including, for example, C5a, C5b, and soluble C5b-9 (see, e.g., FIG. 1). For example, the activity of CD59 may be monitored to determine the inhibition of expression of a C5 gene. CH.sub.50, AH.sub.50, clot formation and/or serum lactate dehydrogenase (LDH), in a sample, e.g., a blood or liver sample, may also be measured. Suitable assays are further described in the Examples section below.

[0480] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the antisense polynucleotide agents and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

Example 1. Antisense Synthesis

[0481] The antisense polynucleotides targeting C5 were synthesized using standard synthesis methods well known in the art.

[0482] A detailed list of antisense molecules targeting complement component C5 is shown in Table 3.

Example 2. In Vitro Screening

[0483] In vitro screening of the antisense polynucleotides was performed by transfecting Huh7 cells with a single 5 nM dose of an antisense polynucleotide using methods well known in the art. Table 4 shows the results of single dose transfection screen in cells transfected with the indicated antisense polynucleotide.

TABLE-US-00002 TABLE 2 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds. Abbre- viation Nucleotide(s) A Adenosine-3'-phosphate Af 2'-fluoroadenosine-3'-phosphate Afs 2'-fluoroadenosine-3'-phosphorothioate As adenosine-3'-phosphorothioate a 2'-O-methyladenosine-3'-phosphate as 2'-O-methyladenosine-3'-phosphorothioate C cytidine-3'-phosphate dA 2'-deoxyadenosine-3'-phosphate dAs 2'-deoxyadenosine-3'-phosphorothioate Cf 2'-fluorocytidine-3'-phosphate Cfs 2'-fluorocytidine-3'-phosphorothioate Cs cytidine-3'-phosphorothioate c 2'-O-methylcytidine-3'-phosphate cs 2'-O-methylcytidine-3'-phosphorothioate dC 2'-deoxycytidine-3'-phosphate dCs 2'-deoxycytidine-3'-phosphorothioate G guanosine-3'-phosphate Gf 2'-fluoroguanosine-3'-phosphate Gfs 2'-fluoroguanosine-3'-phosphorothioate Gs guanosine-3'-phosphorothioate g 2'-O-methylguanosine-3'-phosphate gs 2'-O-methylguanosine-3'-phosphorothioate dG 2'-deoxyguanosine-3'-phosphate dGs 2'-deoxyguanosine-3'-phosphorothioate T 5'-methyluridine-3'-phosphate Tf 2'-fluoro-5-methyluridine-3'-phosphate Tfs 2'-fluoro-5-methyluridine-3'-phosphorothioate Ts 5-methyluridine-3'-phosphorothioate t 2'-O-methyl-5-methyluridine-3'-phosphate ts 2'-O-methyl-5-methyluridine-3'-phosphorothioate dT 2'-deoxythymidine-3'-phosphate dTs 2'-deoxythymidine-3'-phosphorothioate U Uridine-3'-phosphate Uf 2'-fluorouridine-3'-phosphate Ufs 2'-fluorouridine-3'-phosphorothioate Us uridine-3'-phosphorothioate u 2'-O-methyluridine-3'-phosphate us 2'-O-methyluridine-3'-phosphorothioate dU 2'-deoxyuridine-3'-phosphate dUs 2'-deoxyuridine-3'-phosphorothioate s phosphorothioate linkage N any nucleotide (G, A, C, T or U) L96 N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3 (dt) deoxy-thymine (5MdC) 5'-methyl-deoxycytidine-3'-phosphate (5MdC)s 5'-methyl-deoxycytidine-3'-phosphorothioate

TABLE-US-00003 TABLE 3 Antisense polynucleotides targeting complement component C5. Start End position position relative to relative to SEQ NM_001735.2 NM_001735.2 Sequence ID (SEQ ID (SEQ ID ID NO: Modified Sequence (5' to 3') NO: 1) NO: 1) A-128563.1 13 asgscsasgsdGsdAsdAsdAs(5MdC)s(5MdC)sdAs(5MdC)sdGsdGsasusasusa 1 20 A-128564.1 14 usgsgsususdGsdGsdAsdGsdGsdTsdAsdGs(5MdC)sdAsgsgsasasa 12 31 A-128565.1 15 csasasasasdGsdGs(5MdC)s(5MdC)s(5MdC)sdAsdTsdGsdGsdTsusgsgsasg 23 42 A-128566.1 16 csasasasgsdTsdAsdTsdTs(5MdC)s(5MdC)s(5MdC)sdAsdAsdAsasgsgscsc 34 53 A-128567.1 17 asgsasususdAsdAsdAsdAsdAsdAs(5MdC)sdAsdAsdAsgsusasusu 45 64 A-128568.1 18 usususcscs(5MdC)s(5MdC)sdAsdGsdGsdAsdAsdGsdAsdTsusasasasa 56 75 A-128569.1 19 usgsuscscs(5MdC)s(5MdC)sdAsdGsdGsdTsdTsdTsdTs(5MdC)scscscsasg 67 86 A-128570.1 20 asusgsususdTsdGs(5MdC)sdTs(5MdC)s(5MdC)sdTsdGsdTs(5MdC)scscscsasg 78 97 A-128571.1 21 usgsasasasdTsdGsdAs(5MdC)sdAsdTsdAsdTsdGsdTsususgscsu 89 108 A-128572.1 22 asususususdTsdGsdGsdTsdGs(5MdC)sdTsdGsdAsdAsasusgsasc 100 119 A-128573.1 23 csasascsas(5MdC)sdGsdGsdAsdAsdTsdAsdTsdTsdTsususgsgsu 111 130 A-128574.1 24 ususcsasgsdAsdTsdGs(5MdC)sdTs(5MdC)s(5MdC)sdAsdAs(5MdC)sascsgsgsa 122 141 A-128575.1 25 asuscsascsdAsdAsdTsdAsdTsdTsdTsdTs(5MdC)sdAsgsasusgsc 133 152 A-128576.1 26 csasusasasdAs(5MdC)sdTsdTsdGsdAsdAsdTs(5MdC)sdAscsasasusa 144 163 A-128577.1 27 ususcsasgsdTsdGsdTsdAsdTs(5MdC)s(5MdC)sdAsdTsdAsasascsusu 155 174 A-128578.1 28 gscsasuscsdAsdAsdAsdTsdGs(5MdC)sdTsdTs(5MdC)sdAsgsusgsusa 166 185 A-128579.1 29 usasgsasgsdAsdTsdTsdGsdTsdTsdGs(5MdC)sdAsdTscsasasasu 177 196 A-128580.1 30 asusasascsdTsdTsdTsdTsdAsdAsdTsdAsdGsdAsgsasususg 188 207 A-128581.1 31 usususususdAsdTs(5MdC)sdAsdGsdGsdAsdTsdAsdAscsusususu 199 218 A-128582.1 32 asgsusasas(5MdC)sdTsdAsdAsdAsdTsdTsdTsdTsdTsusasuscsa 210 229 A-128583.1 33 asusgsgscs(5MdC)sdTsdGsdAsdGsdGsdAsdGsdTsdAsascsusasa 221 240 A-128584.1 34 gsasusasasdAsdTsdGsdAsdAs(5MdC)sdAsdTsdGsdGscscsusgsa 232 251 A-128585.1 35 usasususcsdTs(5MdC)sdTsdGsdAsdGsdGsdAsdTsdAsasasusgsa 243 262 A-128586.1 36 gsususususdGsdGsdAsdAsdTsdTsdTsdAsdTsdTscsuscsusg 254 273 A-128587.1 37 asasgsasusdTsdGs(5MdC)sdAsdGsdAsdGsdTsdTsdTsusgsgsasa 265 284 A-128588.1 38 gsususgsusdAsdTsdTsdGsdTsdTsdAsdAsdGsdAsususgscsa 276 295 A-128589.1 39 csasasususdGsdTsdTsdTsdTsdGsdGsdTsdTsdGsusasususg 287 306 A-128590.1 40 usgsuscscsdTs(5MdC)s(5MdC)sdAsdGsdGs(5MdC)sdAsdAsdTsusgsususu 298 317 A-128591.1 41 asasascsusdGsdGsdGsdTsdTsdTsdTsdGsdTs(5MdC)scsuscscsa 309 328 A-128592.1 42 asusascsas(5MdC)sdAsdTsdAsdAsdGsdAsdAsdAs(5MdC)susgsgsgsu 320 339 A-128593.1 43 ascsasascsdTsdTs(5MdC)s(5MdC)sdAsdAsdAsdTsdAs(5MdC)sascsasusa 331 350 A-128594.1 44 asasusgscsdTsdTsdTsdGsdAsdTsdAs(5MdC)sdAsdAscsususcsc 342 361 A-128595.1 45 usgsasususdTsdTsdGsdAsdAsdAsdAsdAsdTsdGscsusususg 353 372 A-128596.1 46 gsgscsasusdTs(5MdC)sdTsdTsdTsdTsdTsdGsdAsdTsusususgsa 364 383 A-128597.1 47 csasusasgsdGsdTsdTsdAsdTsdTsdGsdGs(5MdC)sdAsususcsusu 375 394 A-128598.1 48 asasasuscs(5MdC)sdAsdTsdTsdGsdTs(5MdC)sdAsdTsdAsgsgsususa 386 405 A-128599.1 49 usgsasasusdGsdAsdAsdGsdAsdGsdAsdAsdAsdTscscsasusu 397 416 A-128600.1 50 gsusususgsdTs(5MdC)sdTsdGsdTsdAsdTsdGsdAsdAsusgsasasg 408 427 A-128601.1 51 asgsusasusdAsdAsdAs(5MdC)sdAsdGsdGsdTsdTsdTsgsuscsusg 419 438 A-128602.1 52 gsascsusgsdGsdTs(5MdC)sdTsdGsdGsdAsdGsdTsdAsusasasasc 430 449 A-128603.1 53 usasascsusdTsdTsdTsdAs(5MdC)sdTsdGsdAs(5MdC)sdTsgsgsuscsu 441 460 A-128604.1 54 csgsasasusdAsdAsdAs(5MdC)sdTs(5MdC)sdTsdAsdAs(5MdC)sususususa 452 471 A-128605.1 55 uscsgsuscsdAsdTsdTs(5MdC)sdAsdAs(5MdC)sdGsdAsdAsusasasasc 463 482 A-128606.1 56 csusgsgscsdTsdTs(5MdC)sdAsdAsdGsdTs(5MdC)sdGsdTscsasususc 474 493 A-128607.1 57 ususcsuscsdTsdTsdTsdTsdGsdGs(5MdC)sdTsdGsdGscsususcsa 485 504 A-128608.1 58 gsususasasdGsdAs(5MdC)sdAsdGsdTsdTsdTs(5MdC)sdTscsusususu 496 515 A-128609.1 59 gsasuscsusdAsdTsdGsdAsdAsdAsdGsdTsdTsdAsasgsascsa 507 526 A-128610.1 60 usgsasuscs(5MdC)sdTsdTs(5MdC)sdAsdGsdGsdAsdTs(5MdC)susasusgsa 518 537 A-128611.1 61 asusgsuscsdAsdAs(5MdC)sdTsdTs(5MdC)sdTsdGsdAsdTscscsususc 529 548 A-128612.1 62 usususcsusdTs(5MdC)sdTsdAs(5MdC)s(5MdC)sdAsdTsdGsdTscsasascsu 540 559 A-128613.1 63 asasusasusdGsdAsdTs(5MdC)sdAsdAsdTsdTsdTs(5MdC)sususcsusa 551 570 A-128614.1 64 gsasgsasusdAsdAsdTsdTs(5MdC)s(5MdC)sdAsdAsdTsdAsusgsasusc 562 581 A-128615.1 65 asgsuscsasdGsdGsdAsdAsdAsdAsdGsdAsdGsdAsusasasusu 573 592 A-128616.1 66 csgsgsasasdTs(5MdC)sdTsdTsdGsdAsdAsdGsdTs(5MdC)sasgsgsasa 584 603 A-128617.1 67 csusasgsgsdAsdTsdTsdAsdGsdAs(5MdC)sdGsdGsdAsasuscsusu 595 614 A-128618.1 68 ascsasusas(5MdC)s(5MdC)sdAsdTsdAsdTs(5MdC)sdTsdAsdGsgsasususa 606 625 A-128619.1 69 csususgsasdTs(5MdC)sdGsdTs(5MdC)s(5MdC)sdAs(5MdC)sdAsdTsascscsasu 617 636 A-128620.1 70 ususasusasdTsdTsdTsdAsdGs(5MdC)s(5MdC)sdTsdTsdGsasuscsgsu 628 647 A-128621.1 71 asasasasgsdTs(5MdC)s(5MdC)sdTs(5MdC)sdTsdTsdTsdAsdTsasusususa 639 658 A-128622.1 72 uscscsasgsdTsdTsdGsdTsdTsdGsdAsdAsdAsdAsgsuscscsu 650 669 A-128623.1 73 asasasusasdTsdGs(5MdC)sdGsdGsdTsdTs(5MdC)s(5MdC)sdAsgsususgsu 661 680 A-128624.1 74 csusususasdAs(5MdC)sdTsdTs(5MdC)sdAsdAsdAsdAsdTsasusgscsg 672 691 A-128625.1 75 csasasgsas(5MdC)sdAsdTsdAsdTsdTs(5MdC)sdTsdTsdTsasascsusu 683 702 A-128626.1 76 gsasasasasdAsdTsdGsdTsdGsdGs(5MdC)sdAsdAsdGsascsasusa 694 713 A-128627.1 77 csgsasususdGsdAsdGsdAs(5MdC)sdAsdGsdAsdAsdAsasasusgsu 705 724 A-128628.1 78 asusasusus(5MdC)sdTsdGsdGs(5MdC)sdTs(5MdC)sdGsdAsdTsusgsasgsa 716 735 A-128629.1 79 cscsasasusdGsdAsdAsdAsdTsdTsdAsdTsdAsdTsuscsusgsg 727 746 A-128630.1 80 asgsususcsdTsdTsdGsdTsdAsdAs(5MdC)s(5MdC)sdAsdAsusgsasasa 738 757 A-128631.1 81 asasasasusdTs(5MdC)sdTsdTsdAsdAsdAsdGsdTsdTscsususgsu 749 768 A-128632.1 82 asusasgsusdAsdAsdTsdTsdTs(5MdC)sdAsdAsdAsdAsususcsusu 760 779 A-128633.1 83 asuscsususdGs(5MdC)sdTsdTsdTsdTsdAsdTsdAsdGsusasasusu 771 790 A-128634.1 84 asususasusdAsdAsdAsdAsdAsdTsdAsdTs(5MdC)sdTsusgscsusu 782 801 A-128635.1 85 gsusgsascsdTsdAs(5MdC)sdTsdTsdTsdAsdTsdTsdAsusasasasa 793 812 A-128636.1 86 csgsuscsasdGs(5MdC)s(5MdC)sdTs(5MdC)sdAsdGsdTsdGsdAscsusascsu 804 823 A-128637.1 87 usgsusgsasdTsdAsdTsdAsdAsdAs(5MdC)sdGsdTs(5MdC)sasgscscsu 815 834 A-128638.1 88 csususasusdTs(5MdC)s(5MdC)sdAsdAsdAsdTsdGsdTsdGsasusasusa 826 845 A-128639.1 89 ususasasgsdTs(5MdC)sdTsdTs(5MdC)sdTs(5MdC)sdTsdTsdAsususcscsa 837 856 A-128640.1 90 ususgsasus(5MdC)sdAsdTs(5MdC)sdTsdTsdTsdTsdAsdAsgsuscsusu 848 867 A-128641.1 91 asuscsasusdTsdTs(5MdC)sdTsdTsdTsdTsdTsdGsdAsuscsasusc 859 878

A-128642.1 92 ususgscsusdGsdTsdTsdTsdGs(5MdC)sdAsdTs(5MdC)sdAsusususcsu 870 889 A-128643.1 93 usgsusgsusdTsdTsdTsdGs(5MdC)sdAsdTsdTsdGs(5MdC)susgsususu 881 900 A-128644.1 94 usususasus(5MdC)sdAsdAs(5MdC)sdAsdTsdTsdGsdTsdGsususususg 892 911 A-128645.1 95 gsasgscsasdAsdTsdTs(5MdC)s(5MdC)sdAsdTsdTsdTsdAsuscsasasc 903 922 A-128646.1 96 asasasusgsdTsdGsdAs(5MdC)sdTsdTsdGsdAsdGs(5MdC)sasasususc 914 933 A-128647.1 97 gsusususcsdAsdGsdAsdAsdTs(5MdC)sdAsdAsdAsdTsgsusgsasc 925 944 A-128648.1 98 csusususgsdAs(5MdC)sdTsdGs(5MdC)sdTsdGsdTsdTsdTscsasgsasa 936 955 A-128649.1 99 gsusasusgsdAs(5MdC)sdAsdGsdTsdTs(5MdC)sdTsdTsdTsgsascsusg 947 966 A-128650.1 100 uscsusasasdAs(5MdC)sdTsdGsdTsdAsdGsdTsdAsdTsgsascsasg 958 977 A-128651.1 101 usgsusususdAsdAsdAsdTs(5MdC)sdTsdTs(5MdC)sdTsdAsasascsusg 969 988 A-128652.1 102 asasgsgsusdAs(5MdC)sdTsdTsdGsdTsdTsdGsdTsdTsusasasasu 980 999 A-128653.1 103 ascsasgscsdAsdAsdTsdAsdTsdAsdAsdAsdGsdGsusascsusu 991 1010 A-128654.1 104 csusasusgsdAs(5MdC)sdTsdGsdTsdTsdAs(5MdC)sdAsdGscsasasusa 1002 1021 A-128655.1 105 ascscsusgsdTsdAsdGsdAs(5MdC)sdTs(5MdC)sdTsdAsdTsgsascsusg 1013 1032 A-128656.1 106 uscsasgsasdAsdAsdAsdTs(5MdC)s(5MdC)sdAs(5MdC)s(5MdC)sdTsgsusasgsa 1024 1043 A-128657.1 107 usususcsusdGs(5MdC)s(5MdC)sdTs(5MdC)sdTsdTs(5MdC)sdAsdGsasasasasu 1035 1054 A-128658.1 108 gsasusgscs(5MdC)sdAsdGsdGsdTsdAsdTsdTsdTs(5MdC)susgscscsu 1046 1065 A-128659.1 109 asgsgsascsdAsdTsdAsdTsdTsdTsdGsdAsdTsdGscscsasgsg 1057 1076 A-128660.1 110 usgsusasgsdGsdGsdAsdGsdAsdGsdAsdGsdGsdAscsasusasu 1068 1087 A-128661.1 111 csasasasusdTs(5MdC)sdAsdGsdTsdTsdTsdGsdTsdAsgsgsgsasg 1079 1098 A-128662.1 112 gsgsasgsusdAsdGs(5MdC)sdAsdAs(5MdC)s(5MdC)sdAsdAsdAsususcsasg 1090 1109 A-128663.1 113 uscsasgsgsdAsdAsdAsdAsdGsdAsdGsdGsdAsdGsusasgscsa 1101 1120 A-128664.1 114 asasuscscs(5MdC)sdAsdGsdGs(5MdC)sdTsdTs(5MdC)sdAsdGsgsasasasa 1112 1131 A-128665.1 115 asusgsgsgsdAsdTsdAsdTsdGsdGsdAsdAsdTs(5MdC)scscsasgsg 1123 1142 A-128666.1 116 cscsusgscsdAs(5MdC)s(5MdC)sdTsdTsdGsdAsdTsdGsdGsgsasusasu 1134 1153 A-128667.1 117 csgsasasus(5MdC)sdTsdTsdTsdAsdAs(5MdC)s(5MdC)sdTsdGscsascscsu 1145 1164 A-128668.1 118 asascsusgsdGsdTs(5MdC)sdAsdAsdGs(5MdC)sdGsdAsdAsuscsususu 1156 1175 A-128669.1 119 csuscscsus(5MdC)s(5MdC)sdTsdAs(5MdC)s(5MdC)sdAsdAs(5MdC)sdTsgsgsu 1167 1186 scsa A-128670.1 120 usgsususas(5MdC)sdTsdGsdGsdGsdAs(5MdC)sdTs(5MdC)s(5MdC)suscscsusa 1178 1197 A-128671.1 121 usgsusgscsdAsdTsdTs(5MdC)sdAsdGsdTsdGsdTsdTsascsusgsg 1189 1208 A-128672.1 122 csasuscsasdAsdTsdTsdGsdTsdTsdTsdGsdTsdGscsasususc 1200 1219 A-128673.1 123 csuscsususdGsdGsdTsdTsdTsdAs(5MdC)sdAsdTs(5MdC)sasasususg 1211 1230 A-128674.1 124 asasgsuscsdAsdGsdAsdTsdGsdTs(5MdC)sdTs(5MdC)sdTsusgsgsusu 1222 1241 A-128675.1 125 usgscsususdGsdGsdAsdTs(5MdC)s(5MdC)sdAsdAsdGsdTscsasgsasu 1233 1252 A-128676.1 126 usgsususas(5MdC)sdAs(5MdC)sdTsdTsdTsdTsdGs(5MdC)sdTsusgsgsasu 1244 1263 A-128677.1 127 uscsasuscsdAsdAs(5MdC)sdAs(5MdC)sdGsdTsdGsdTsdTsascsascsu 1255 1274 A-128678.1 128 asasgscsusdAs(5MdC)sdTs(5MdC)s(5MdC)sdAsdTs(5MdC)sdAsdTscsasascsa 1266 1285 A-128679.1 129 asasgscsas(5MdC)sdAsdAsdAsdGsdGsdAsdAsdGs(5MdC)susascsusc 1277 1296 A-128680.1 130 gsasusgsgsdGsdAsdGsdAsdTsdTsdAsdAsdGs(5MdC)sascsasasa 1288 1307 A-128681.1 131 cscsgsuscsdAs(5MdC)sdTs(5MdC)s(5MdC)sdAsdGsdAsdTsdGsgsgsasgsa 1299 1318 A-128682.1 132 asasascsus(5MdC)s(5MdC)sdAsdGs(5MdC)sdAs(5MdC)s(5MdC)sdGsdTscsasc 1310 1329 susc A-128683.1 133 gsususususdGsdAs(5MdC)sdAsdTsdTsdAsdAsdAs(5MdC)suscscsasg 1321 1340 A-128684.1 134 csusgsgsasdGs(5MdC)sdAsdTs(5MdC)sdAsdGsdTsdTsdTsusgsascsa 1332 1351 A-128685.1 135 ususcsusgsdGsdAsdAsdGsdAsdTs(5MdC)sdTsdGsdGsasgscsasu 1343 1362 A-128686.1 136 gscscsusgsdAsdTsdTsdTsdTs(5MdC)sdTsdTs(5MdC)sdTsgsgsasasg 1354 1373 A-128687.1 137 asascscsusdTs(5MdC)s(5MdC)s(5MdC)sdTsdGsdGs(5MdC)s(5MdC)sdTsgsasu 1365 1384 susu A-128688.1 138 usasususgs(5MdC)sdTs(5MdC)sdGsdGsdTsdAsdAs(5MdC)s(5MdC)sususcscsc 1376 1395 A-128689.1 139 gsasusgsasdGsdTsdAsdTsdGs(5MdC)sdTsdAsdTsdTsgscsuscsg 1387 1406 A-128690.1 140 usususgsgs(5MdC)sdTsdGsdAsdGsdAsdGsdAsdTsdGsasgsusasu 1398 1417 A-128691.1 141 asusasasasdGsdGsdTsdAsdAs(5MdC)sdTsdTsdTsdGsgscsusgsa 1409 1428 A-128692.1 142 gsuscscsasdAsdTs(5MdC)sdAsdAsdTsdAsdTsdAsdAsasgsgsusa 1420 1439 A-128693.1 143 usasusgsgsdTsdTsdAsdTs(5MdC)sdAsdGsdTs(5MdC)s(5MdC)sasasuscsa 1431 1450 A-128694.1 144 usasgscsasdAsdAsdGs(5MdC)s(5MdC)sdTsdTsdAsdTsdGsgsususasu 1442 1461 A-128695.1 145 usgsususcsdTs(5MdC)s(5MdC)s(5MdC)sdAs(5MdC)sdTsdAsdGs(5MdC)sasasa 1453 1472 sgsc A-128696.1 146 usasasusasdTsdTs(5MdC)sdAsdGsdAsdTsdGsdTsdTscsuscscsc 1464 1483 A-128697.1 147 gsgsgsgsgsdTsdAsdAs(5MdC)sdAsdAsdTsdAsdAsdTsasususcsa 1475 1494 A-128698.1 148 usasusgsgsdGs(5MdC)sdTsdTsdTsdTsdGsdGsdGsdGsgsusasasc 1486 1505 A-128699.1 149 ususususgsdTs(5MdC)sdAsdAsdTsdAsdTsdAsdTsdGsgsgscsusu 1497 1516 A-128700.1 150 asusasgsusdGsdAsdGsdTsdTsdAsdTsdTsdTsdTsgsuscsasa 1508 1527 A-128701.1 151 asuscsasasdGsdTsdAsdAsdTsdTsdAsdTsdAsdGsusgsasgsu 1519 1538 A-128702.1 152 cscsususgsdGsdAsdTsdAsdAsdAsdAsdTs(5MdC)sdAsasgsusasa 1530 1549 A-128703.1 153 gsasusasasdTsdTsdTsdTsdGs(5MdC)s(5MdC)s(5MdC)sdTsdTsgsgsasusa 1541 1560 A-128704.1 154 gsusgscscsdAsdAsdAsdGsdTsdGsdGsdAsdTsdAsasusususu 1552 1571 A-128705.1 155 asusususcsdTs(5MdC)s(5MdC)s(5MdC)sdTs(5MdC)sdGsdTsdGs(5MdC)scsasa 1563 1582 sasg A-128706.1 156 usgscsasus(5MdC)sdTsdGsdAsdAsdAsdAsdTsdTsdTscsuscscsc 1574 1593 A-128707.1 157 csusususgsdAsdTsdAsdAsdGsdAsdTsdGs(5MdC)sdAsuscsusgsa 1585 1604 A-128708.1 158 gsasasusgsdTsdTsdTsdAsdTsdAs(5MdC)sdTsdTsdTsgsasusasa 1596 1615 A-128709.1 159 csusgsusgsdTsdTsdAs(5MdC)sdTsdGsdGsdAsdAsdTsgsusususa 1607 1626 A-128710.1 160 gsgsasascs(5MdC)sdAsdTsdGsdTsdTs(5MdC)sdTsdGsdTsgsususasc 1618 1637 A-128711.1 161 gsuscsgsgsdGsdAsdTsdGsdAsdAsdGsdGsdAsdAscscsasusg 1629 1648 A-128712.1 162 asusasgsas(5MdC)s(5MdC)sdAsdGsdAsdAsdGsdTs(5MdC)sdGsgsgsasusg 1640 1659 A-128713.1 163 gsusgsascsdGsdAsdTsdGsdTsdAsdAsdTsdAsdGsascscsasg 1651 1670 A-128714.1 164 uscsusgsusdTs(5MdC)sdTs(5MdC)s(5MdC)sdTsdGsdTsdGsdAscsgsasusg 1662 1681 A-128715.1 165 usasasusus(5MdC)sdTsdGs(5MdC)sdTsdGsdTs(5MdC)sdTsdGsususcsusc 1673 1692 A-128716.1 166 gsasasuscsdAsdGsdAs(5MdC)sdAs(5MdC)sdTsdAsdAsdTsuscsusgsc 1684 1703 A-128717.1 167 ususasascs(5MdC)sdAsdGsdAs(5MdC)sdTsdGsdAsdAsdTscsasgsasc 1695 1714 A-128718.1 168 ususcsusus(5MdC)sdAsdAsdTsdAsdTsdTsdTsdAsdAscscsasgsa 1706 1725 A-128719.1 169 ususgscscsdAs(5MdC)sdAsdTsdTsdTsdTsdTs(5MdC)sdTsuscsasasu 1717 1736 A-128720.1 170 cscsusgsgsdAsdGs(5MdC)sdTsdGsdGsdTsdTsdGs(5MdC)scsascsasu 1728 1747

A-128721.1 171 asgsascsasdGsdAsdTsdGsdAsdAs(5MdC)s(5MdC)sdTsdGsgsasgscsu 1739 1758 A-128722.1 172 uscsusgscsdAsdTs(5MdC)sdAsdGsdGsdAsdGsdAs(5MdC)sasgsasusg 1750 1769 A-128723.1 173 gsasgsasasdTsdAsdTsdGs(5MdC)sdAsdTs(5MdC)sdTsdGscsasuscsa 1761 1780 A-128724.1 174 asgsusususdGsdGs(5MdC)s(5MdC)sdTsdGsdGsdAsdGsdAsasusasusg 1772 1791 A-128725.1 175 ususasasgsdAsdGsdAs(5MdC)sdAs(5MdC)sdAsdGsdTsdTsusgsgscsc 1783 1802 A-128726.1 176 csasgsususdGs(5MdC)s(5MdC)sdAsdTsdAsdTsdTsdAsdAsgsasgsasc 1794 1813 A-128727.1 177 gsgsasasus(5MdC)s(5MdC)sdAsdTsdTs(5MdC)s(5MdC)sdAsdGsdTsusgscscsa 1805 1824 A-128728.1 178 asasusgscs(5MdC)sdAs(5MdC)s(5MdC)s(5MdC)sdAsdGsdGsdAsdAsuscscsasu 1816 1835 A-128729.1 179 cscsascsusdGs(5MdC)sdTsdGs(5MdC)sdTsdAsdAsdTsdGscscsascsc 1827 1846 A-128730.1 180 csascsasgs(5MdC)sdAs(5MdC)sdTsdGsdTs(5MdC)s(5MdC)sdAs(5MdC)susgsc 1838 1857 susg A-128731.1 181 usgsgsascsdTs(5MdC)s(5MdC)sdAsdTsdAs(5MdC)sdAs(5MdC)sdAsgscsascsu 1849 1868 A-128732.1 182 usgsgscsus(5MdC)s(5MdC)sdTs(5MdC)sdTsdTsdTsdGsdGsdAscsuscscsa 1860 1879 A-128733.1 183 csasasgsgsdGs(5MdC)sdTsdTsdTsdTsdTsdGsdGs(5MdC)suscscsusc 1871 1890 A-128734.1 184 asasusascsdTs(5MdC)sdTsdTsdTs(5MdC)s(5MdC)sdAsdAsdGsgsgscsusu 1882 1901 A-128735.1 185 csusasasgsdAsdAsdTsdTsdGsdAsdAsdAsdTsdAscsuscsusu 1893 1912 A-128736.1 186 asuscsascsdTs(5MdC)sdTsdTs(5MdC)sdTs(5MdC)sdTsdAsdAsgsasasusu 1904 1923 A-128737.1 187 cscsascsasdGs(5MdC)s(5MdC)s(5MdC)sdAsdGsdAsdTs(5MdC)sdAscsuscsusu 1915 1934 A-128738.1 188 csascscsas(5MdC)s(5MdC)sdTsdGs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdAs 1926 1945 (5MdC)sasgscscsc A-128739.1 189 asususgsusdTsdGsdAsdGsdGs(5MdC)s(5MdC)sdAs(5MdC)s(5MdC)sascscsusg 1937 1956 A-128740.1 190 asascsascsdAsdTsdTsdGsdGs(5MdC)sdAsdTsdTsdGsususgsasg 1948 1967 A-128741.1 191 csasgscsusdAsdGsdGsdTsdGsdGsdAsdAs(5MdC)sdAscsasususg 1959 1978 A-128742.1 192 gsasasgsgsdTsdAsdAsdGsdTs(5MdC)s(5MdC)sdAsdGs(5MdC)susasgsgsu 1970 1989 A-128743.1 193 gscsasususdAsdGsdTsdGsdAsdGsdGsdAsdAsdGsgsusasasg 1981 2000 A-128744.1 194 csasuscsusdGs(5MdC)sdAsdTsdTsdTsdGs(5MdC)sdAsdTsusasgsusg 1992 2011 A-128745.1 195 ususcsususdGsdGsdGsdAsdGsdTs(5MdC)sdAsdTs(5MdC)susgscsasu 2003 2022 A-128746.1 196 gsgsususcsdAsdTs(5MdC)sdAsdTsdTsdTsdTs(5MdC)sdTsusgsgsgsa 2014 2033 A-128747.1 197 usususcsusdTsdTsdAs(5MdC)sdAsdAsdGsdGsdTsdTscsasuscsa 2025 2044 A-128748.1 198 usgsgscscsdTsdGsdAsdGsdAsdAsdTsdTsdTs(5MdC)susususasc 2036 2055 A-128749.1 199 asgscsgsusdTs(5MdC)sdTsdTs(5MdC)sdTsdTsdGsdGs(5MdC)scsusgsasg 2047 2066 A-128750.1 200 uscsususcsdTsdTsdTsdTsdGs(5MdC)sdAsdGs(5MdC)sdGsususcsusu 2058 2077 A-128751.1 201 usasususus(5MdC)sdTsdTs(5MdC)sdTsdAsdTs(5MdC)sdTsdTscsusususu 2069 2088 A-128752.1 202 usasusususdAsdGs(5MdC)sdAsdGs(5MdC)sdTsdAsdTsdTsuscsususc 2080 2099 A-128753.1 203 csusgsasasdTsdGsdTsdTsdTsdAsdTsdAsdTsdTsusasgscsa 2091 2110 A-128754.1 204 usususcsusdTs(5MdC)sdAs(5MdC)sdTsdAs(5MdC)sdTsdGsdAsasusgsusu 2102 2121 A-128755.1 205 uscsgsusasdAs(5MdC)sdAsdAs(5MdC)sdAsdTsdTsdTs(5MdC)sususcsasc 2113 2132 A-128756.1 206 csgscsasgsdGs(5MdC)sdTs(5MdC)s(5MdC)sdAsdTs(5MdC)sdGsdTsasascsasa 2124 2143 A-128757.1 207 asuscsasusdTsdAsdTsdTsdAsdAs(5MdC)sdGs(5MdC)sdAsgsgscsusc 2135 2154 A-128758.1 208 uscsascsasdGsdGsdTsdTsdTs(5MdC)sdAsdTs(5MdC)sdAsususasusu 2146 2165 A-128759.1 209 csasgscsus(5MdC)sdGs(5MdC)sdTsdGs(5MdC)sdTs(5MdC)sdAs(5MdC)sasgsg 2157 2176 susu A-128760.1 210 ascsusasasdTs(5MdC)s(5MdC)sdGsdTsdGs(5MdC)sdAsdGs(5MdC)suscsgscsu 2168 2187 A-128761.1 211 csususgsgs(5MdC)s(5MdC)s(5MdC)sdTsdAsdAsdAs(5MdC)sdTsdAsasuscscsg 2179 2198 A-128762.1 212 csusususgsdAsdTsdGs(5MdC)sdAsdTs(5MdC)sdTsdTsdGsgscscscsu 2190 2209 A-128763.1 213 ususcsasgsdTsdGsdAsdAsdAsdGs(5MdC)sdTsdTsdTsgsasusgsc 2201 2220 A-128764.1 214 ascsgsascsdAs(5MdC)sdAsdAs(5MdC)sdAsdTsdTs(5MdC)sdAsgsusgsasa 2212 2231 A-128765.1 215 gscsusgsgs(5MdC)sdTsdTsdGs(5MdC)sdGsdAs(5MdC)sdGsdAscsascsasa 2223 2242 A-128766.1 216 asususasgs(5MdC)sdAs(5MdC)sdGsdGsdAsdGs(5MdC)sdTsdGsgscsususg 2234 2253 A-128767.1 217 ususasusgsdAsdGsdAsdGsdAsdTsdAsdTsdTsdAsgscsascsg 2245 2264 A-128768.1 218 asususgscsdAsdTsdGsdTs(5MdC)sdTsdTsdTsdAsdTsgsasgsasg 2256 2275 A-128769.1 219 usasgscscsdTsdTs(5MdC)s(5MdC)s(5MdC)sdAsdAsdTsdTsdGscsasusgsu 2267 2286 A-128770.1 220 gsuscsusus(5MdC)sdAsdTsdGsdTsdGsdTsdAsdGs(5MdC)scsususcsc 2278 2297 A-128771.1 221 csusgsgsusdAsdAs(5MdC)sdAsdGsdGsdGsdTs(5MdC)sdTsuscsasusg 2289 2308 A-128772.1 222 usgsgscsusdTsdGs(5MdC)sdTsdTsdAs(5MdC)sdTsdGsdGsusasascsa 2300 2319 A-128773.1 223 csuscscsgsdAsdAsdTsdTsdTs(5MdC)sdTsdGsdGs(5MdC)sususgscsu 2311 2330 A-128774.1 224 csusgsgsasdAsdAsdAsdTsdAsdAs(5MdC)sdTs(5MdC)s(5MdC)sgsasasusu 2322 2341 A-128775.1 225 csasascscsdAsdGs(5MdC)sdTsdTsdTs(5MdC)sdTsdGsdGsasasasasu 2333 2352 A-128776.1 226 usgsasascsdTsdTs(5MdC)s(5MdC)s(5MdC)sdAs(5MdC)sdAsdAs(5MdC)scsasg 2344 2363 scsu A-128777.1 227 usgsgsgsasdAs(5MdC)sdAsdAsdGsdAsdTsdGsdAsdAscsususcsc 2355 2374 A-128778.1 228 csusgsususdTsdTs(5MdC)sdTsdTs(5MdC)sdTsdGsdGsdGsasascsasa 2366 2385 A-128779.1 229 gscsasasas(5MdC)sdTsdGs(5MdC)sdAsdAs(5MdC)sdTsdGsdTsusususcsu 2377 2396 A-128780.1 230 asasuscsasdGsdGsdTsdAsdGsdGsdGs(5MdC)sdAsdAsascsusgsc 2388 2407 A-128781.1 231 gsgsusgsgsdTsdTsdAsdGsdAsdGsdAsdAsdTs(5MdC)sasgsgsusa 2399 2418 A-128782.1 232 usgsasasusdTsdTs(5MdC)s(5MdC)s(5MdC)sdAsdGsdGsdTsdGsgsususasg 2410 2429 A-128783.1 233 usgscscsasdAs(5MdC)sdGs(5MdC)s(5MdC)sdTsdTsdGsdAsdAsusususcsc 2421 2440 A-128784.1 234 asgsusgsusdTsdTsdGsdAsdAsdAsdTsdGs(5MdC)s(5MdC)sasascsgsc 2432 2451 A-128785.1 235 ascsascsasdTsdAsdTsdAs(5MdC)s(5MdC)sdAsdGsdTsdGsusususgsa 2443 2462 A-128786.1 236 csasgsusasdTs(5MdC)sdAsdGs(5MdC)sdAsdAs(5MdC)sdAs(5MdC)sasusasusa 2454 2473 A-128787.1 237 csusususgs(5MdC)s(5MdC)sdTsdTsdGsdAs(5MdC)sdAsdGsdTsasuscsasg 2465 2484 A-128788.1 238 uscsusususdGsdAsdAs(5MdC)sdAs(5MdC)s(5MdC)sdTsdTsdTsgscscsusu 2476 2495 A-128789.1 239 cscsasgsgsdAsdAsdGsdAs(5MdC)sdAsdTs(5MdC)sdTsdTsusgsasasc 2487 2506 A-128790.1 240 usasusasusdTs(5MdC)sdAsdTsdTsdTs(5MdC)s(5MdC)sdAsdGsgsasasgsa 2498 2517 A-128791.1 241 ascsasgsasdAsdTsdAsdTsdGsdGsdTsdAsdTsdAsususcsasu 2509 2528 A-128792.1 242 csuscscsus(5MdC)sdGsdTsdAs(5MdC)sdAsdAs(5MdC)sdAsdGsasasusasu 2520 2539 A-128793.1 243 ususgsgsasdTs(5MdC)sdTsdGsdTsdTs(5MdC)sdTs(5MdC)s(5MdC)suscsgsusa 2531 2550 A-128794.1 244 gsususcscsdTsdTsdTs(5MdC)sdAsdAsdTsdTsdGsdGsasuscsusg 2542 2561 A-128795.1 245 asgsususgsdTsdAsdAsdAs(5MdC)sdAsdGsdTsdTs(5MdC)scsusususc 2553 2572 A-128796.1 246 asgsasasgsdTs(5MdC)s(5MdC)sdTsdAsdTsdAsdGsdTsdTsgsusasasa 2564 2583 A-128797.1 247 asascsusgs(5MdC)sdAsdTs(5MdC)s(5MdC)s(5MdC)sdAsdGsdAsdAsgsuscscsu 2575 2594 A-128798.1 248

ususususasdAs(5MdC)sdAs(5MdC)sdAsdGsdAsdAs(5MdC)sdTsgscsasusc 2586 2605 A-128799.1 249 csascsasgs(5MdC)sdAsdGsdAs(5MdC)sdAsdTsdTsdTsdTsasascsasc 2597 2616 A-128800.1 250 csasgsasusdTs(5MdC)s(5MdC)s(5MdC)sdTs(5MdC)s(5MdC)sdAs(5MdC)sdAsg 2608 2627 scsasgsa A-128801.1 251 usususcscsdGsdAsdAsdGsdTsdGs(5MdC)sdAsdGsdAsususcscsc 2619 2638 A-128802.1 252 asasusgsas(5MdC)sdTsdGsdGsdGs(5MdC)sdTsdTsdTs(5MdC)scsgsasasg 2630 2649 A-128803.1 253 cscscsusgsdAsdTsdGsdAsdTs(5MdC)sdAsdAsdTsdGsascsusgsg 2641 2660 A-128804.1 254 asgsgsascsdTsdTsdTsdGsdTsdGs(5MdC)s(5MdC)s(5MdC)sdTsgsasusgsa 2652 2671 A-128805.1 255 csascsascsdAsdTsdTsdTsdGsdGsdAsdGsdGsdAscsusususg 2663 2682 A-128806.1 256 ascsususus(5MdC)sdTsdGsdGs(5MdC)sdGs(5MdC)sdAs(5MdC)sdAscsasususu 2674 2693 A-128807.1 257 asgsgsasgs(5MdC)s(5MdC)s(5MdC)sdTs(5MdC)sdTsdAs(5MdC)sdTsdTsuscsu 2685 2704 sgsg A-128808.1 258 csasasgsusdGsdAs(5MdC)sdTsdGsdGsdAsdGsdGsdAsgscscscsu 2696 2715 A-128809.1 259 gsusgsasasdTsdGsdTs(5MdC)sdAs(5MdC)s(5MdC)sdAsdAsdGsusgsascsu 2707 2726 A-128810.1 260 gsasgsgsasdAsdGs(5MdC)sdAs(5MdC)sdAsdGsdTsdGsdAsasusgsusc 2718 2737 A-128811.1 261 gscscsasasdTsdTsdTs(5MdC)s(5MdC)sdAsdGsdAsdGsdGsasasgscsa 2729 2748 A-128812.1 262 asusgsususdGsdTsdGsdAsdAsdGsdGs(5MdC)s(5MdC)sdAsasusususc 2740 2759 A-128813.1 263 gsusgsasasdAsdAsdAsdTsdTsdGsdAsdTsdGsdTsusgsusgsa 2751 2770 A-128814.1 264 cscsasasgsdTs(5MdC)sdTs(5MdC)s(5MdC)sdAsdGsdTsdGsdAsasasasasu 2762 2781 A-128815.1 265 uscsusususdTs(5MdC)s(5MdC)sdAsdAsdAs(5MdC)s(5MdC)sdAsdAsgsuscsusc 2773 2792 A-128816.1 266 ususascsusdAsdAsdGsdAsdTsdTsdTs(5MdC)sdTsdTsususcscsa 2784 2803 A-128817.1 267 uscsgsusasdAsdTsdGsdTsdTsdTsdTsdTsdAs(5MdC)susasasgsa 2795 2814 A-128818.1 268 uscsusgsgs(5MdC)sdAs(5MdC)s(5MdC)sdAs(5MdC)sdTs(5MdC)sdGsdTsasasu 2806 2825 sgsu A-128819.1 269 ususususgsdAs(5MdC)sdAs(5MdC)s(5MdC)sdTsdTs(5MdC)sdTsdGsgscsascsc 2817 2836 A-128820.1 270 asusasgscsdTsdTsdTs(5MdC)s(5MdC)s(5MdC)sdTsdTsdTsdTsgsascsasc 2828 2847 A-128821.1 271 gsusasascsdAs(5MdC)s(5MdC)sdAsdGsdAsdAsdTsdAsdGscsusususc 2839 2858 A-128822.1 272 usasgsgsasdTs(5MdC)s(5MdC)sdAsdAsdAsdGsdTsdAsdAscsascscsa 2850 2869 A-128823.1 273 asusasasasdTsdAs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdTsdAsdGsdGsasuscscsa 2861 2880 A-128824.1 274 csusasasusdGsdGsdTsdAs(5MdC)s(5MdC)sdAsdTsdAsdAsasusascsc 2872 2891 A-128825.1 275 cscsususus(5MdC)sdGsdTs(5MdC)sdTsdGs(5MdC)sdTsdAsdAsusgsgsusa 2883 2902 A-128826.1 276 gsusasusgsdGsdGsdAsdAs(5MdC)sdTs(5MdC)s(5MdC)sdTsdTsuscsgsusc 2894 2913 A-128827.1 277 asasgsgsgsdTsdAsdTs(5MdC)s(5MdC)sdTsdGsdTsdAsdTsgsgsgsasa 2905 2924 A-128828.1 278 gsgsascscsdAsdAsdAsdTs(5MdC)sdTsdAsdAsdGsdGsgsusasusc 2916 2935 A-128829.1 279 ususcsusgsdTsdTsdTsdTsdGsdGsdGsdGsdAs(5MdC)scsasasasu 2927 2946 A-128830.1 280 asuscscsusdTsdTsdTsdGsdAsdTsdTsdTs(5MdC)sdTsgsusususu 2938 2957 A-128831.1 281 ususascsas(5MdC)sdTs(5MdC)sdAsdAsdAsdAsdTs(5MdC)s(5MdC)sususususg 2949 2968 A-128832.1 282 asasgscsasdGsdTs(5MdC)s(5MdC)sdTsdTsdTsdTsdAs(5MdC)sascsuscsa 2960 2979 A-128833.1 283 asuscsuscsdAs(5MdC)s(5MdC)sdTsdAs(5MdC)sdAsdAsdGs(5MdC)sasgsuscsc 2971 2990 A-128834.1 284 csusgscsasdGsdAs(5MdC)sdAsdAsdGsdAsdTs(5MdC)sdTscsascscsu 2982 3001 A-128835.1 285 csusgsascsdTsdTsdAsdGsdAsdAs(5MdC)sdTsdGs(5MdC)sasgsascsa 2993 3012 A-128836.1 286 ususgsasusdGs(5MdC)s(5MdC)sdTsdTs(5MdC)s(5MdC)sdTsdGsdAscsususasg 3004 3023 A-128837.1 287 gsgsgsususdAsdGsdGsdAsdTsdAsdTsdTsdGsdAsusgscscsu 3015 3034 A-128838.1 288 usususgsgsdGsdGsdAsdGsdGsdTsdGsdGsdGsdTsusasgsgsa 3026 3045 A-128839.1 289 uscsusgscsdAs(5MdC)sdTs(5MdC)s(5MdC)s(5MdC)sdTsdTsdTsdGsgsgsgsasg 3037 3056 A-128840.1 290 uscsasgscsdTs(5MdC)s(5MdC)sdGs(5MdC)s(5MdC)sdTs(5MdC)sdTsdGscsasc 3048 3067 susc A-128841.1 291 gsascsasas(5MdC)sdGs(5MdC)sdTs(5MdC)sdAsdTs(5MdC)sdAsdGscsuscscsg 3059 3078 A-128842.1 292 usasgsasasdTsdAs(5MdC)sdTsdGsdGsdGsdAs(5MdC)sdAsascsgscsu 3070 3089 A-128843.1 293 asgsusgsasdAsdAsdAsdAs(5MdC)sdAsdTsdAsdGsdAsasusascsu 3081 3100 A-128844.1 294 usgsususus(5MdC)s(5MdC)sdAsdGsdGsdTsdAsdGsdTsdGsasasasasa 3092 3111 A-128845.1 295 csasasusgsdAsdTsdTsdTs(5MdC)s(5MdC)sdTsdGsdTsdTsuscscsasg 3103 3122 A-128846.1 296 gsasasasasdAsdTsdGsdTsdTs(5MdC)s(5MdC)sdAsdAsdTsgsasususu 3114 3133 A-128847.1 297 usgsgsgsus(5MdC)sdAsdGsdAsdAsdTsdGsdAsdAsdAsasasusgsu 3125 3144 A-128848.1 298 ususususcsdAsdAsdTsdTsdAsdAsdTsdGsdGsdGsuscsasgsa 3136 3155 A-128849.1 299 uscsasgsusdTsdTs(5MdC)sdTsdGs(5MdC)sdTsdTsdTsdTscsasasusu 3147 3166 A-128850.1 300 usasasususdTsdTsdTsdTs(5MdC)sdTsdTs(5MdC)sdAsdGsusususcsu 3158 3177 A-128851.1 301 asuscscscsdTsdTs(5MdC)sdTsdTsdTsdTsdAsdAsdTsususususu 3169 3188 A-128852.1 302 usasasusgs(5MdC)sdTs(5MdC)sdAsdAs(5MdC)sdAsdTs(5MdC)s(5MdC)scsusu 3180 3199 scsu A-128853.1 303 uscsusgsusdAsdGsdGsdAs(5MdC)sdAsdTsdAsdAsdTsgscsuscsa 3191 3210 A-128854.1 304 usasgsuscsdAsdGs(5MdC)sdAsdTsdTsdTs(5MdC)sdTsdGsusasgsgsa 3202 3221 A-128855.1 305 csascsusgsdTsdAsdAsdGsdAsdGsdTsdAsdGsdTscsasgscsa 3213 3232 A-128856.1 306 ascscscsusdTs(5MdC)s(5MdC)sdAs(5MdC)sdAs(5MdC)sdAs(5MdC)sdTsgsusa 3224 3243 sasg A-128857.1 307 csusasgscsdAs(5MdC)sdTsdTs(5MdC)s(5MdC)sdAs(5MdC)s(5MdC)s(5MdC)su 3235 3254 suscscsa A-128858.1 308 ususasascs(5MdC)sdAsdAsdGsdTsdGs(5MdC)sdTsdAsdGscsascsusu 3246 3265 A-128859.1 309 asgscsasasdAsdAsdGs(5MdC)sdTsdGsdTsdTsdAsdAscscsasasg 3257 3276 A-128860.1 310 asgsusascsdTs(5MdC)sdTsdTsdAsdAsdAsdGs(5MdC)sdAsasasasgsc 3268 3287 A-128861.1 311 ususascsusdTsdGsdTs(5MdC)s(5MdC)sdAsdAsdGsdTsdAscsuscsusu 3279 3298 A-128862.1 312 usascsgsusdAsdTsdTsdTsdAsdTsdTsdTsdAs(5MdC)sususgsusc 3290 3309 A-128863.1 313 usgsgsusus(5MdC)sdTsdGs(5MdC)sdTs(5MdC)sdTsdAs(5MdC)sdGsusasususu 3301 3320 A-128864.1 314 asasasususdGsdAsdAsdTsdTsdTsdTsdGsdGsdTsuscsusgsc 3312 3331 A-128865.1 315 usasasasgsdAsdAsdTsdTsdAs(5MdC)sdAsdAsdAsdTsusgsasasu 3323 3342 A-128866.1 316 ascsusasgs(5MdC)s(5MdC)sdAs(5MdC)sdAsdAsdTsdAsdAsdAsgsasasusu 3334 3353 A-128867.1 317 gsasusasasdTsdTs(5MdC)sdTs(5MdC)sdAsdAs(5MdC)sdTsdAsgscscsasc 3345 3364 A-128868.1 318 asususasus(5MdC)sdTsdAsdAsdTsdTsdGsdAsdTsdAsasususcsu 3356 3375 A-128869.1 319 ususgsasasdAsdGsdAsdTs(5MdC)s(5MdC)sdAsdTsdTsdAsuscsusasa 3367 3386 A-128870.1 320 gsusgsasasdTsdTsdTsdTs(5MdC)s(5MdC)sdTsdTsdGsdAsasasgsasu 3378 3397 A-128871.1 321 usgsgsususdGsdAsdTsdAs(5MdC)sdTsdGsdTsdGsdAsasusususu 3389 3408 A-128872.1 322 usgsusasasdTsdTsdTsdTsdAsdTsdTsdGsdGsdTsusgsasusa 3400 3419 A-128873.1 323 gscsasasgsdGsdTsdAs(5MdC)s(5MdC)s(5MdC)sdTsdGsdTsdAsasusususu 3411 3430

A-128874.1 324 gsgscsusus(5MdC)sdAsdAs(5MdC)sdAsdGsdGs(5MdC)sdAsdAsgsgsusasc 3422 3441 A-128875.1 325 csusgsusus(5MdC)sdTs(5MdC)sdTs(5MdC)sdGsdGsdGs(5MdC)sdTsuscsasasc 3433 3452 A-128876.1 326 usasasgsasdTsdAsdTsdAsdAsdGs(5MdC)sdTsdGsdTsuscsuscsu 3444 3463 A-128877.1 327 asgsusasasdAsdGsdGs(5MdC)sdTsdGsdTsdAsdAsdGsasusasusa 3455 3474 A-128878.1 328 asususcscsdAsdAsdTs(5MdC)sdAs(5MdC)sdAsdGsdTsdAsasasgsgsc 3466 3485 A-128879.1 329 asasgscscsdTsdTsdTs(5MdC)sdTsdAsdAsdTsdTs(5MdC)scsasasusc 3477 3496 A-128880.1 330 gscsasusasdTsdAsdTs(5MdC)sdGsdAsdAsdAsdGs(5MdC)scsusususc 3488 3507 A-128881.1 331 ususcsascs(5MdC)sdAsdGsdGsdGsdGsdGs(5MdC)sdAsdTsasusasusc 3499 3518 A-128882.1 332 csusgsusgsdTs(5MdC)sdGsdAsdTsdTsdTsdTs(5MdC)sdAscscsasgsg 3510 3529 A-128883.1 333 usususasasdTsdTsdAsdGsdAsdGs(5MdC)sdTsdGsdTsgsuscsgsa 3521 3540 A-128884.1 334 asasgsususdGsdTs(5MdC)sdAsdGs(5MdC)sdTsdTsdTsdAsasususasg 3532 3551 A-128885.1 335 usususcsasdAsdGs(5MdC)sdAsdGsdAsdAsdAsdGsdTsusgsuscsa 3543 3562 A-128886.1 336 usgsgscsasdGsdTsdGsdTsdAsdTsdTsdTsdTs(5MdC)sasasgscsa 3554 3573 A-128887.1 337 gsusgscsus(5MdC)sdTsdGsdGsdGs(5MdC)sdTsdGsdGs(5MdC)sasgsusgsu 3565 3584 A-128888.1 338 cscsasasusdGsdTsdAsdAsdAsdGsdGsdTsdGs(5MdC)suscsusgsg 3576 3595 A-128889.1 339 csgscsasgsdAsdAsdAsdTsdGsdGs(5MdC)s(5MdC)sdAsdAsusgsusasa 3587 3606 A-128890.1 340 gsasasasgsdAsdGs(5MdC)sdAsdTsdAs(5MdC)sdGs(5MdC)sdAsgsasasasu 3598 3617 A-128891.1 341 usasuscsus(5MdC)s(5MdC)s(5MdC)sdAsdGsdGsdGsdAsdAsdAsgsasgscsa 3609 3628 A-128892.1 342 usgsgsgsusdGsdAsdGsdTsdTsdTsdTsdAsdTs(5MdC)suscscscsa 3620 3639 A-128893.1 343 gsasascsgsdAsdAsdAs(5MdC)sdTsdGsdTsdGsdGsdGsusgsasgsu 3631 3650 A-128894.1 344 csusgsasasdAs(5MdC)sdAsdAsdTsdTsdGsdAsdAs(5MdC)sgsasasasc 3642 3661 A-128895.1 345 uscsuscsusdTs(5MdC)sdAsdAsdAsdGs(5MdC)sdTsdGsdAsasascsasa 3653 3672 A-128896.1 346 ascscsasasdAsdGs(5MdC)sdTsdTs(5MdC)sdTs(5MdC)sdTs(5MdC)sususcsasa 3664 3683 A-128897.1 347 gsasususas(5MdC)s(5MdC)sdTsdTsdTsdAsdAs(5MdC)s(5MdC)sdAsasasgscsu 3675 3694 A-128898.1 348 asusasasasdTsdGsdGsdGsdTsdGsdGsdAsdTsdTsascscsusu 3686 3705 A-128899.1 349 ususcscsasdAsdAsdAsdAs(5MdC)sdGsdAsdTsdAsdAsasusgsgsg 3697 3716 A-128900.1 350 gsasasgsasdTsdTsdGsdTs(5MdC)sdTsdTsdTs(5MdC)s(5MdC)sasasasasa 3708 3727 A-128901.1 351 gsuscsususdTsdAsdTsdGs(5MdC)sdTsdGsdAsdAsdGsasususgsu 3719 3738 A-128902.1 352 gsgsusascsdAsdGsdAsdGs(5MdC)sdTsdGsdTs(5MdC)sdTsususasusg 3730 3749 A-128903.1 353 usascscsasdGsdTsdGsdTsdTsdAsdGsdGsdTsdAscsasgsasg 3741 3760 A-128904.1 354 csasusascsdGsdTsdGs(5MdC)s(5MdC)sdGsdTsdAs(5MdC)s(5MdC)sasgsusgsu 3752 3771 A-128905.1 355 gsususgsusdTsdTs(5MdC)sdTsdAs(5MdC)s(5MdC)sdAsdTsdAscsgsusgsc 3763 3782 A-128906.1 356 asasgscsasdTsdAsdGsdGs(5MdC)sdAsdGsdTsdTsdGsusususcsu 3774 3793 A-128907.1 357 ascsusgsgsdTsdGsdAsdGsdTsdAsdAsdAsdGs(5MdC)sasusasgsg 3785 3804 A-128908.1 358 ususcsasasdGsdTsdTs(5MdC)sdAsdGsdAs(5MdC)sdTsdGsgsusgsasg 3796 3815 A-128909.1 359 asasusususdAsdTsdAsdTs(5MdC)sdTsdTsdTs(5MdC)sdAsasgsususc 3807 3826 A-128910.1 360 usgsgsgsusdTsdAsdAs(5MdC)sdAsdTsdAsdAsdTsdTsusasusasu 3818 3837 A-128911.1 361 csasusususdGsdAsdTsdGsdAs(5MdC)sdTsdGsdGsdGsususasasc 3829 3848 A-128912.1 362 csususcsusdGsdAsdTsdAsdGs(5MdC)s(5MdC)sdAsdTsdTsusgsasusg 3840 3859 A-128913.1 363 asusascscsdTs(5MdC)sdTsdGs(5MdC)sdTs(5MdC)sdTsdTs(5MdC)susgsasusa 3851 3870 A-128914.1 364 asasgscscsdAs(5MdC)s(5MdC)sdTs(5MdC)s(5MdC)sdAsdTsdAs(5MdC)scsusc 3862 3881 susg A-128915.1 365 gsgsgsususdGsdAsdAsdTsdAsdAsdAsdAsdGs(5MdC)scsascscsu 3873 3892 A-128916.1 366 gsasususgsdTsdGsdTs(5MdC)s(5MdC)sdTsdGsdGsdGsdTsusgsasasu 3884 3903 A-128917.1 367 uscsasasusdGsdGs(5MdC)sdAsdTsdTsdGsdAsdTsdTsgsusgsusc 3895 3914 A-128918.1 368 cscsgsuscsdAsdGsdGs(5MdC)s(5MdC)s(5MdC)sdTs(5MdC)sdAsdAsusgsgscsa 3906 3925 A-128919.1 369 gsasgsusgsdAsdAsdTsdAsdTsdTs(5MdC)s(5MdC)sdGsdTscsasgsgsc 3917 3936 A-128920.1 370 usgsusususdAsdAs(5MdC)s(5MdC)sdAsdGsdGsdAsdGsdTsgsasasusa 3928 3947 A-128921.1 371 uscsasasgs(5MdC)sdGsdGsdAsdGsdTsdTsdGsdTsdTsusasascsc 3939 3958 A-128922.1 372 gsasusgsus(5MdC)s(5MdC)sdAsdTsdAs(5MdC)sdTs(5MdC)sdAsdAsgscsgsgsa 3950 3969 A-128923.1 373 usasasgsasdAsdAs(5MdC)sdAsdTs(5MdC)sdGsdAsdTsdGsuscscsasu 3961 3980 A-128924.1 374 csusususasdTsdGs(5MdC)sdTsdTsdGsdTsdAsdAsdGsasasascsa 3972 3991 A-128925.1 375 asusgsusasdAsdGsdGs(5MdC)sdAs(5MdC)s(5MdC)sdTsdTsdTsasusgscsu 3983 4002 A-128926.1 376 asususususdAsdTsdAsdAsdTsdTsdAsdTsdGsdTsasasgsgsc 3994 4013 A-128927.1 377 uscsususgsdTs(5MdC)sdTsdGsdTs(5MdC)sdAsdTsdTsdTsusasusasa 4005 4024 A-128928.1 378 cscscsasasdGsdGsdAsdAsdAsdTsdTs(5MdC)sdTsdTsgsuscsusg 4016 4035 A-128929.1 379 uscsusascsdTsdGsdGs(5MdC)s(5MdC)sdTs(5MdC)s(5MdC)s(5MdC)sdAsasgsg 4027 4046 sasa A-128930.1 380 usgsasgsasdAsdGs(5MdC)sdAs(5MdC)s(5MdC)sdTs(5MdC)sdTsdAscsusgsgsc 4038 4057 A-128931.1 381 gsasgsgsus(5MdC)sdAsdTs(5MdC)sdAsdTsdTsdGsdAsdGsasasgscsa 4049 4068 A-128932.1 382 gsusascsusdGsdAs(5MdC)sdAsdAsdTsdGsdAsdGsdGsuscsasusc 4060 4079 A-128933.1 383 usgscscsasdAsdAsdTs(5MdC)s(5MdC)sdTsdGsdTsdAs(5MdC)susgsascsa 4071 4090 A-128934.1 384 asgscscsasdAsdGs(5MdC)s(5MdC)sdAs(5MdC)sdTsdGs(5MdC)s(5MdC)sasasa 4082 4101 susc A-128935.1 385 ascsasusgsdTsdAs(5MdC)sdTsdGsdTsdAsdGs(5MdC)s(5MdC)sasasgscsc 4093 4112 A-128936.1 386 csusascsasdGsdTsdTsdGsdTsdTsdAs(5MdC)sdAsdTsgsusascsu 4104 4123 A-128937.1 387 gsgsusususdTsdGsdTsdGsdAsdAs(5MdC)sdTsdAs(5MdC)sasgsususg 4115 4134 A-128938.1 388 uscsasgsasdGsdGsdTsdAs(5MdC)sdTsdGsdGsdTsdTsususgsusg 4126 4145 A-128939.1 389 usgscsasasdAs(5MdC)sdTsdTs(5MdC)s(5MdC)sdTs(5MdC)sdAsdGsasgsgsusa 4137 4156 A-128940.1 390 csasasasusdAsdAsdAsdAsdGs(5MdC)sdTsdGs(5MdC)sdAsasascsusu 4148 4167 A-128941.1 391 gsusasuscsdGsdAsdTsdTsdTsdTs(5MdC)sdAsdAsdAsusasasasa 4159 4178 A-128942.1 392 csasasusasdTs(5MdC)s(5MdC)sdTsdGsdAsdGsdTsdAsdTscsgsasusu 4170 4189 A-128943.1 393 gsusgsgsgsdAsdTsdGs(5MdC)sdTsdTs(5MdC)sdAsdAsdTsasuscscsu 4181 4200 A-128944.1 394 usasgscscsdTs(5MdC)sdTsdGsdTsdAsdGsdTsdGsdGsgsasusgsc 4192 4211 A-128945.1 395 csasgsasgsdTsdTsdTs(5MdC)s(5MdC)sdGsdTsdAsdGs(5MdC)scsuscsusg 4203 4222 A-128946.1 396 gscsgsususdTsdGsdTsdAsdAsdTs(5MdC)sdAsdGsdAsgsusususc 4214 4233 A-128947.1 397 csasusgscsdTsdAs(5MdC)sdTsdAsdTsdGs(5MdC)sdGsdTsususgsusa 4225 4244 A-128948.1 398 usgsusasgs(5MdC)sdTsdGsdGs(5MdC)sdAs(5MdC)sdAsdTsdGscsusascsu 4236 4255 A-128949.1 399 cscsusgscsdTsdGsdGsdGs(5MdC)sdTsdTsdGsdTsdAsgscsusgsg 4247 4266 A-128950.1 400 gsasusgsasdTsdTs(5MdC)sdTsdTs(5MdC)s(5MdC)s(5MdC)sdTsdGscsusgsgsg 4258 4277 A-128951.1 401 asgsgsasus(5MdC)s(5MdC)sdAsdGsdAsdTsdGsdAsdTsdGsasususcsu 4269 4288 A-128952.1 402 csascscsgs(5MdC)sdAsdTsdGsdAsdGsdAsdGsdGsdAsuscscsasg 4280

4299 A-128953.1 403 gsasgsasusdGsdTs(5MdC)s(5MdC)sdAsdTs(5MdC)sdAs(5MdC)s(5MdC)sgscsa 4291 4310 susg A-128954.1 404 csasgsusasdGsdGs(5MdC)sdAsdAsdGsdGsdAsdGsdAsusgsuscsc 4302 4321 A-128955.1 405 usgscsascsdTsdGsdAsdTsdTs(5MdC)s(5MdC)sdAsdGsdTsasgsgscsa 4313 4332 A-128956.1 406 uscsususcsdTsdTs(5MdC)sdAsdTsdTsdTsdGs(5MdC)sdAscsusgsasu 4324 4343 A-128957.1 407 gsgsgscsusdTsdTsdTsdAsdAsdGsdTs(5MdC)sdTsdTscsususcsa 4335 4354 A-128958.1 408 cscscsusus(5MdC)s(5MdC)sdAs(5MdC)sdAsdAsdGsdGsdGs(5MdC)sususususa 4346 4365 A-128959.1 409 asgsususgsdAsdTs(5MdC)s(5MdC)sdAs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdTsu 4357 4376 scscsasc A-128960.1 410 asasuscsasdGsdTsdGsdAsdAsdTsdAsdGsdTsdTsgsasuscsc 4368 4387 A-128961.1 411 usususgsasdTsdTsdTsdGsdGsdTsdAsdAsdTs(5MdC)sasgsusgsa 4379 4398 A-128962.1 412 ascsasusgsdTs(5MdC)s(5MdC)sdAsdTs(5MdC)sdTsdTsdTsdGsasusususg 4390 4409 A-128963.1 413 gsususgscsdAsdGsdAsdAsdTsdAsdAs(5MdC)sdAsdTsgsuscscsa 4401 4420 A-128964.1 414 asasuscsgsdAsdAsdTsdTs(5MdC)sdAsdGsdTsdTsdGscsasgsasa 4412 4431 A-128965.1 415 uscsascsusdGsdGsdAsdGsdGsdGsdAsdAsdTs(5MdC)sgsasasusu 4423 4442 A-128966.1 416 csascsasasdAsdGsdGsdAsdAsdAsdTs(5MdC)sdAs(5MdC)susgsgsasg 4434 4453 A-128967.1 417 cscsgsgsasdAsdTs(5MdC)sdGsdTsdAs(5MdC)sdAs(5MdC)sdAsasasgsgsa 4445 4464 A-128968.1 418 asgsususcsdAsdAsdAsdTsdAsdTs(5MdC)s(5MdC)sdGsdGsasasuscsg 4456 4475 A-128969.1 419 csasascsusdTs(5MdC)sdAsdAsdAsdGsdAsdGsdTsdTscsasasasu 4467 4486 A-128970.1 420 ascsusgsasdGsdAsdAsdAs(5MdC)s(5MdC)s(5MdC)sdAsdAs(5MdC)sususcsasa 4478 4497 A-128971.1 421 asasasgsusdGsdGs(5MdC)sdAsdGsdGsdAs(5MdC)sdTsdGsasgsasasa 4489 4508 A-128972.1 422 csgsusascsdAs(5MdC)sdTsdGsdTsdGsdAsdAsdAsdGsusgsgscsa 4500 4519 A-128973.1 423 uscsusgsusdGsdGsdTsdAsdTsdTs(5MdC)sdGsdTsdAscsascsusg 4511 4530 A-128974.1 424 usgsusususdAsdTs(5MdC)sdTsdGsdGsdTs(5MdC)sdTsdGsusgsgsusa 4522 4541 A-128975.1 425 ascsasusgsdGsdTsdAs(5MdC)sdAs(5MdC)sdTsdGsdTsdTsusasuscsu 4533 4552 A-128976.1 426 asgsusgscsdTsdAsdTsdAsdAsdAsdAs(5MdC)sdAsdTsgsgsusasc 4544 4563 A-128977.1 427 ususgsasusdAsdTsdTsdGsdGsdAsdAsdGsdTsdGscsusasusa 4555 4574 A-128978.1 428 csusususcsdTsdGsdAsdAsdTsdTsdTsdTsdGsdAsusasususg 4566 4585 A-128979.1 429 uscscsusus(5MdC)sdAs(5MdC)sdAsdGsdAs(5MdC)sdTsdTsdTscsusgsasa 4577 4596 A-128980.1 430 ususgscsas(5MdC)sdGs(5MdC)sdGsdGs(5MdC)sdTs(5MdC)s(5MdC)sdTsuscsa 4588 4607 scsa A-128981.1 431 csususcsusdAs(5MdC)sdAs(5MdC)sdAs(5MdC)sdTsdTsdGs(5MdC)sascsgscsg 4599 4618 A-128982.1 432 cscscsascsdAsdAsdTs(5MdC)sdAsdGs(5MdC)sdTsdTs(5MdC)susascsasc 4610 4629 A-128983.1 433 uscscsusgs(5MdC)sdAsdTsdTsdTsdGs(5MdC)s(5MdC)s(5MdC)sdAscsasasusc 4621 4640 A-128984.1 434 gsasuscscsdAsdAsdTsdTs(5MdC)sdTsdTs(5MdC)s(5MdC)sdTsgscsasusu 4632 4651 A-128985.1 435 asgsasgsasdTsdTsdGsdTs(5MdC)sdAsdGsdAsdTs(5MdC)scsasasusu 4643 4662 A-128986.1 436 csususgsus(5MdC)sdTs(5MdC)sdTsdGs(5MdC)sdAsdGsdAsdGsasususgsu 4654 4673 A-128987.1 437 csusgsususdTsdGsdTsdTsdTsdTs(5MdC)sdTsdTsdGsuscsuscsu 4665 4684 A-128988.1 438 usgsgsususdTsdAs(5MdC)sdAsdTsdGs(5MdC)sdTsdGsdTsususgsusu 4676 4695 A-128989.1 439 usasusgscsdAsdAsdTs(5MdC)sdTs(5MdC)sdTsdGsdGsdTsususascsa 4687 4706 A-128990.1 440 csusususasdTsdAsdAsdGs(5MdC)sdAsdTsdAsdTsdGscsasasusc 4698 4717 A-128991.1 441 usgsusgsasdTsdGs(5MdC)sdTsdAsdAs(5MdC)sdTsdTsdTsasusasasg 4709 4728 A-128992.1 442 ascsasgsusdGsdAsdTsdGsdGsdAsdTsdGsdTsdGsasusgscsu 4720 4739 A-128993.1 443 asasascsasdTsdTsdTsdTs(5MdC)sdTsdAs(5MdC)sdAsdGsusgsasusg 4731 4750 A-128994.1 444 gsusascsusdTsdGsdAs(5MdC)sdAsdAsdAsdAsdAs(5MdC)sasusususu 4742 4761 A-128995.1 445 asgsgsgsusdTsdGs(5MdC)s(5MdC)sdTsdTsdGsdTsdAs(5MdC)sususgsasc 4753 4772 A-128996.1 446 asgsasusasdTs(5MdC)s(5MdC)sdAsdGsdAsdAsdGsdGsdGsususgscsc 4764 4783 A-128997.1 447 cscscsasgsdTsdTsdTsdTsdGsdTsdAsdGsdAsdTsasuscscsa 4775 4794 A-128998.1 448 gscsasascsdAsdGs(5MdC)sdTsdTs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdAsgsusu 4786 4805 susu A-128999.1 449 asgsuscsusdTsdTs(5MdC)sdTs(5MdC)sdAsdGs(5MdC)sdAsdAscsasgscsu 4797 4816 A-129000.1 450 gsgsusasasdTs(5MdC)sdTs(5MdC)sdAsdGsdAsdGsdTs(5MdC)susususcsu 4808 4827 A-129001.1 451 usususususdAsdAsdTsdGsdAsdAsdGsdGsdTsdAsasuscsusc 4819 4838 A-129002.1 452 usascsasgsdGsdTsdTsdAs(5MdC)s(5MdC)sdTsdTsdTsdTsusasasusg 4830 4849 A-129003.1 453 csuscsasgs(5MdC)sdGsdTsdTsdAsdGsdTsdAs(5MdC)sdAsgsgsususa 4841 4860 A-129004.1 454 cscsusususdTsdAs(5MdC)s(5MdC)sdAsdGs(5MdC)sdTs(5MdC)sdAsgscsgsusu 4852 4871 A-129005.1 455 asgsusascsdTsdGsdTs(5MdC)sdTsdTs(5MdC)s(5MdC)sdTsdTsususascsc 4863 4882 A-129006.1 456 ascscscsasdTsdAsdAsdTsdTsdAsdAsdGsdTsdAscsusgsusc 4874 4893 A-129007.1 457 asgsgsgscsdTsdTs(5MdC)sdTsdTsdTsdAs(5MdC)s(5MdC)s(5MdC)sasusasasu 4885 4904 A-129008.1 458 asususususdAsdTs(5MdC)sdTsdGsdGsdAsdGsdGsdGscsususcsu 4896 4915 A-129009.1 459 ascsusgsasdAsdAsdTsdTsdGsdTsdAsdTsdTsdTsusasuscsu 4907 4926 A-129010.1 460 asusgsusas(5MdC)s(5MdC)sdTsdGsdAsdAsdAs(5MdC)sdTsdGsasasasusu 4918 4937 A-129011.1 461 csusasasasdGsdGsdGsdTsdAsdGsdAsdTsdGsdTsascscsusg 4929 4948 A-129012.1 462 gsgsuscsasdAsdGsdGsdAsdAsdTs(5MdC)sdTsdAsdAsasgsgsgsu 4940 4959 A-129013.1 463 usasususcsdAsdAsdTs(5MdC)s(5MdC)sdAsdGsdGsdTs(5MdC)sasasgsgsa 4951 4970 A-129014.1 464 csuscsusasdGsdGs(5MdC)s(5MdC)sdAsdGsdTsdAsdTsdTscsasasusc 4962 4981 A-129015.1 465 ascsasusgsdTsdTsdGsdTsdGsdTs(5MdC)sdTs(5MdC)sdTsasgsgscsc 4973 4992 A-129016.1 466 usgsascsas(5MdC)sdGsdAsdTsdGsdAsdAs(5MdC)sdAsdTsgsususgsu 4984 5003 A-129017.1 467 csusasasasdAsdAsdTsdGs(5MdC)sdTsdTsdGsdAs(5MdC)sascsgsasu 4995 5014 A-129018.1 468 asuscsusasdAsdAsdTsdTsdAsdGs(5MdC)sdTsdAsdAsasasasusg 5006 5025 A-129019.1 469 uscsgsgscsdAsdAsdAsdTsdTs(5MdC)sdAsdTs(5MdC)sdTsasasasusu 5017 5036 A-129020.1 470 asasasasgsdAsdTsdAsdTs(5MdC)sdTsdTs(5MdC)sdGsdGscsasasasu 5028 5047 A-129021.1 471 gscsasuscs(5MdC)sdAsdTsdTsdTsdAsdAsdAsdAsdAsgsasusasu 5039 5058 A-129022.1 472 csasgsgsasdAsdTsdTsdTsdTsdAsdGs(5MdC)sdAsdTscscsasusu 5050 5069 A-129023.1 473 csasgscsusdGsdAsdAs(5MdC)sdTsdTs(5MdC)sdAsdGsdGsasasususu 5061 5080 A-129024.1 474 csasasascsdTsdGsdTsdAsdTsdGs(5MdC)sdAsdGs(5MdC)susgsasasc 5072 5091 A-129025.1 475 gsuscscsasdTsdAsdAsdGsdTsdGs(5MdC)sdAsdAsdAscsusgsusa 5083 5102 A-129026.1 476 csasascsasdAs(5MdC)sdAsdGsdGsdAsdGsdTs(5MdC)s(5MdC)sasusasasg 5094 5113 A-129027.1 477 asasasascsdGsdAsdAs(5MdC)sdTsdTs(5MdC)sdAsdAs(5MdC)sasascsasg 5105 5124 A-129028.1 478 asasgsasasdAsdAs(5MdC)sdAsdAsdAsdAsdAsdAsdAscsgsasasc 5116 5135 A-129029.1 479 usususasasdAsdAsdAsdAsdAsdGsdAsdAsdGsdAsasasascsa 5127 5146 A-129030.1 480 asgscsusasdTsdGsdAsdAsdTsdGsdTsdTsdTsdAsasasasasa 5138 5157 A-129031.1 481

csasasasusdAsdAsdGsdAs(5MdC)s(5MdC)sdAsdGs(5MdC)sdTsasusgsasa 5149 5168 A-129032.1 482 asgsusgsasdGs(5MdC)sdTsdTsdTsdAs(5MdC)sdAsdAsdAsusasasgsa 5160 5179 A-129033.1 483 asususcsusdAsdAsdGsdTsdAsdAsdAsdGsdTsdGsasgscsusu 5171 5190 A-129034.1 484 asasgsusgs(5MdC)s(5MdC)sdAs(5MdC)sdTsdAsdAsdTsdTs(5MdC)susasasgsu 5182 5201 A-129035.1 485 csusasasusdAsdAsdAsdAsdGs(5MdC)sdAsdAsdGsdTsgscscsasc 5193 5212 A-129036.1 486 gsasasasus(5MdC)sdAsdTsdTs(5MdC)sdTs(5MdC)sdTsdAsdAsusasasasa 5204 5223 A-129037.1 487 ususascsasdGs(5MdC)sdAsdTsdTsdTsdGsdAsdAsdAsuscsasusu 5215 5234 A-129038.1 488 asusususcsdAsdGsdAsdAsdAsdGsdTsdTsdAs(5MdC)sasgscsasu 5226 5245 A-129039.1 489 asasgsgscs(5MdC)sdAsdTsdGsdTsdTsdAsdTsdTsdTscsasgsasa 5237 5256 A-129040.1 490 uscsasusgs(5MdC)s(5MdC)s(5MdC)sdTs(5MdC)s(5MdC)sdAsdAsdGsdGscscsa 5248 5267 susg A-129041.1 491 asgsusasus(5MdC)sdTsdGsdTs(5MdC)sdTsdTs(5MdC)sdAsdTsgscscscsu 5259 5278 A-129042.1 492 asascscsusdTsdGsdGsdAsdGsdGsdAsdGsdTsdAsuscsusgsu 5270 5289 A-129043.1 493 csgsgsusgsdTs(5MdC)s(5MdC)sdAsdAsdTsdAsdAs(5MdC)s(5MdC)sususgsgsa 5281 5300 A-129044.1 494 usususasusdTsdGsdTsdTsdTs(5MdC)s(5MdC)sdGsdGsdTsgsuscscsa 5292 5311 A-129045.1 495 asgsgsusgsdTsdTs(5MdC)s(5MdC)sdAsdAsdTsdTsdTsdAsususgsusu 5303 5322 A-129046.1 496 usasgsgsusdTsdTsdGsdAsdGsdGsdAsdGsdGsdTsgsususcsc 5314 5333 A-129047.1 497 ususcscsusdGsdAsdGsdTsdGsdGsdTsdAsdGsdGsusususgsa 5325 5344 A-129048.1 498 cscscsasgs(5MdC)sdAsdAsdAs(5MdC)sdAsdTsdTs(5MdC)s(5MdC)susgsasgsu 5336 5355 A-129049.1 499 gsususcsusdTsdTs(5MdC)sdGsdGs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdAsgscsa 5347 5366 sasa A-129050.1 500 ususcsasasdTsdGsdGsdAs(5MdC)sdTsdGsdTsdTs(5MdC)susususcsg 5358 5377 A-129051.1 501 usasasusas(5MdC)sdTs(5MdC)s(5MdC)s(5MdC)sdTsdTsdTs(5MdC)sdAsasusg 5369 5388 sgsa A-129052.1 502 cscsasusgsdTsdTsdTsdTsdTsdGsdTsdAsdAsdTsascsuscsc 5380 5399 A-129053.1 503 uscsasasgs(5MdC)sdAsdAsdAsdGsdGs(5MdC)s(5MdC)sdAsdTsgsusususu 5391 5410 A-129054.1 504 gsgsusasusdTsdTsdTs(5MdC)sdTsdTsdTs(5MdC)sdAsdAsgscsasasa 5402 5421 A-129055.1 505 uscscsusgsdTsdTs(5MdC)s(5MdC)sdTsdTsdGsdGsdTsdAsususususc 5413 5432 A-129056.1 506 asasusgsasdTs(5MdC)sdAsdGsdTsdTsdTs(5MdC)s(5MdC)sdTsgsususcsc 5424 5443 A-129057.1 507 ascsuscsasdGsdGs(5MdC)sdTsdTsdTsdAsdAsdTsdGsasuscsasg 5435 5454 A-129058.1 508 usususgsasdAsdAsdGs(5MdC)sdAsdAsdAs(5MdC)sdTs(5MdC)sasgsgscsu 5446 5465

TABLE-US-00004 TABLE 4 meanval % Corrected Transfection (w/o correction) sd % Efficiency (tfe) A-128636.1 16 4 6 A-128659.1 23 5 19 A-128693.1 23 7 19 A-128637.1 30 5 20 A-128671.1 28 6 23 A-128655.1 28 7 24 A-128594.1 29 10 24 A-128689.1 29 4 25 A-128720.1 29 6 26 A-128591.1 31 8 27 A-128686.1 31 4 27 A-128597.1 31 9 27 A-128606.1 32 6 27 A-128574.1 30 3 27 A-128773.1 38 4 28 A-128570.1 31 9 29 A-128713.1 32 5 29 A-128669.1 34 8 29 A-128619.1 34 6 29 A-128780.1 40 7 30 A-128688.1 34 4 30 A-128678.1 36 5 31 A-128645.1 35 6 31 A-128658.1 35 5 31 A-128603.1 36 4 31 A-128711.1 34 7 31 A-128623.1 42 3 32 A-128630.1 42 5 32 A-128598.1 36 6 32 A-128583.1 36 9 32 A-128714.1 35 7 32 A-128756.1 42 7 33 A-128577.1 36 8 33 A-128709.1 36 8 33 A-128710.1 36 5 33 A-128729.1 41 8 33 A-128648.1 38 7 34 A-128696.1 38 8 34 A-128566.1 37 8 34 A-128596.1 39 9 34 A-129058.1 35 7 34 A-128643.1 39 8 35 A-128703.1 38 6 35 A-128674.1 40 7 35 A-128687.1 39 4 35 A-128668.1 40 6 35 A-128639.1 47 6 36 A-128677.1 41 6 36 A-128787.1 47 5 37 A-128622.1 42 2 38 A-128734.1 46 8 38 A-128584.1 42 8 38 A-128759.1 47 7 38 A-128706.1 41 10 38 A-128676.1 43 5 39 A-129044.1 39 4 39 A-128763.1 49 7 39 A-128642.1 49 1 39 A-128679.1 44 8 39 A-128765.1 50 8 39 A-128667.1 44 7 39 A-128649.1 43 8 39 A-128694.1 43 12 39 A-128695.1 43 17 39 A-128672.1 44 5 40 A-128705.1 42 8 40 A-128646.1 44 10 40 A-128776.1 51 11 40 A-128749.1 49 12 40 A-128592.1 44 6 40 A-128579.1 43 11 40 A-128565.1 43 5 40 A-128975.1 41 4 41 A-128593.1 45 9 41 A-128738.1 49 9 41 A-128793.1 51 6 41 A-128717.1 44 9 41 A-128607.1 46 6 41 A-128867.1 52 5 41 A-128602.1 46 8 42 A-128600.1 46 8 42 A-128760.1 51 7 42 A-128660.1 46 6 42 A-128973.1 43 10 43 A-128682.1 48 2 43 A-128933.1 45 9 43 A-128675.1 48 11 43 A-128628.1 54 8 44 A-128684.1 48 7 44 A-128692.1 48 2 44 A-128611.1 49 4 44 A-128725.1 52 7 44 A-128563.1 47 6 44 A-128727.1 53 8 44 A-128620.1 49 5 44 A-128712.1 48 4 45 A-128770.1 56 7 46 A-128762.1 55 2 46 A-128728.1 54 8 46 A-128716.1 49 9 46 A-128626.1 57 8 46 A-128617.1 51 11 46 A-128721.1 49 8 47 A-128575.1 49 3 47 A-128690.1 51 2 47 A-128967.1 47 2 47 A-128769.1 58 7 47 A-128666.1 52 5 47 A-128612.1 53 5 48 A-128707.1 51 7 48 A-128698.1 52 5 48 A-128578.1 51 7 48 A-128761.1 57 4 48 A-128766.1 59 6 49 A-128588.1 53 7 49 A-128652.1 53 9 49 A-128618.1 54 5 49 A-129056.1 49 16 49 A-128590.1 53 7 49 A-128571.1 52 9 49 A-128625.1 60 7 49 A-128925.1 51 2 49 A-128654.1 53 8 49 A-128640.1 60 4 49 A-128656.1 54 6 50 A-128875.1 60 14 50 A-128893.1 51 16 50 A-128673.1 55 6 50 A-128573.1 53 7 50 A-128779.1 61 6 50 A-128853.1 61 3 50 A-128624.1 61 8 50 A-128856.1 61 4 50 A-128662.1 54 6 50 A-128670.1 55 8 51 A-128585.1 55 9 51 A-128976.1 51 6 51 A-128715.1 53 11 51 A-128968.1 51 7 51 A-128748.1 60 12 51 A-128841.1 60 3 51 A-128730.1 59 8 51 A-129025.1 51 8 51 A-128680.1 56 5 51 A-128732.1 59 7 51 A-129015.1 51 10 51 A-128691.1 55 3 51 A-129046.1 52 7 51 A-128788.1 62 10 51 A-129014.1 52 7 52 A-128934.1 53 1 52 A-128845.1 62 8 52 A-128792.1 62 6 52 A-128638.1 62 8 52 A-128897.1 53 23 52 A-128699.1 56 12 52 A-128737.1 60 9 52 A-129055.1 53 22 52 A-128764.1 63 8 53 A-128569.1 55 8 53 A-128633.1 63 7 53 A-128826.1 62 6 53 A-128742.1 61 3 53 A-128878.1 64 6 53 A-129031.1 53 8 53 A-129022.1 53 4 53 A-128685.1 58 3 54 A-128775.1 64 7 54 A-128783.1 64 7 54 A-128774.1 65 9 54 A-129024.1 54 8 54 A-128888.1 55 9 54 A-128977.1 54 9 54 A-128993.1 54 15 54 A-128966.1 54 6 54 A-128927.1 56 16 55 A-128863.1 65 7 55 A-128789.1 65 10 55 A-128825.1 64 4 55 A-128757.1 64 14 55 A-128908.1 56 12 55 A-128861.1 66 4 55 A-128874.1 66 9 56 A-128681.1 60 3 56 A-128700.1 60 7 56 A-128854.1 66 3 56 A-128842.1 66 2 56 A-128797.1 66 8 56 A-128650.1 60 9 56 A-128859.1 67 3 56 A-128733.1 64 6 56 A-129043.1 57 10 56 A-128922.1 57 11 56 A-128731.1 65 5 57 A-128882.1 67 2 57 A-128855.1 67 5 57 A-128610.1 62 5 57 A-128857.1 68 9 57 A-129045.1 58 5 57 A-128852.1 68 6 57 A-128726.1 65 7 57 A-128723.1 66 12 58 A-128884.1 59 10 58 A-128784.1 68 5 58 A-129013.1 58 7 58 A-128974.1 58 17 58 A-128647.1 62 6 58 A-128866.1 68 7 58 A-129035.1 58 18 58 A-128837.1 67 5 58 A-128772.1 69 5 58 A-128635.1 69 9 58 A-128586.1 63 9 58 A-128777.1 69 14 58 A-128794.1 69 25 58 A-128755.1 68 13 58 A-128807.1 68 10 59 A-128657.1 63 12 59 A-128785.1 69 5 59 A-128663.1 64 6 59 A-128804.1 68 13 59 A-129034.1 59 7 59 A-129004.1 59 12 59 A-128970.1 59 16 59 A-128786.1 69 4 59 A-128704.1 62 8 59 A-128876.1 70 7 59 A-128605.1 64 2 59 A-128939.1 61 8 59 A-128815.1 68 6 59 A-128819.1 68 4 59 A-128864.1 70 9 59 A-128858.1 70 3 59 A-128781.1 70 4 59 A-128814.1 69 5 60 A-128747.1 69 10 60 A-128896.1 61 7 60 A-128894.1 61 8 60 A-128964.1 60 8 60 A-128634.1 71 4 60 A-128833.1 70 5 60 A-128840.1 70 3 60 A-128641.1 71 1 60 A-128771.1 71 8 60

A-128744.1 70 11 61 A-128587.1 65 6 61 A-128938.1 63 7 61 A-128915.1 62 16 61 A-128829.1 70 7 61 A-128741.1 69 6 61 A-128778.1 72 9 61 A-128801.1 71 6 61 A-128665.1 66 7 61 A-128979.1 61 15 61 A-128883.1 63 13 61 A-128899.1 63 20 62 A-128719.1 64 12 62 A-128621.1 67 5 62 A-128805.1 71 10 62 A-129005.1 62 13 62 A-128629.1 73 7 62 A-128836.1 72 5 62 A-128782.1 73 5 62 A-128564.1 65 3 62 A-128918.1 63 20 62 A-128963.1 63 10 62 A-128661.1 66 5 62 A-128601.1 67 6 63 A-128768.1 73 4 63 A-128608.1 67 3 63 A-128935.1 64 14 63 A-128616.1 67 10 63 A-128999.1 63 7 63 A-128898.1 64 13 63 A-128887.1 64 18 63 A-128572.1 66 8 63 A-128902.1 65 7 63 A-129054.1 64 18 63 A-128834.1 73 9 63 A-128724.1 72 8 64 A-128767.1 74 7 64 A-128847.1 74 4 64 A-128937.1 66 13 64 A-128984.1 64 23 64 A-128627.1 74 5 64 A-128980.1 64 14 64 A-128822.1 73 7 64 A-128905.1 65 22 64 A-129047.1 65 16 64 A-128949.1 66 19 65 A-128895.1 66 7 65 A-128802.1 75 6 65 A-128953.1 66 11 65 A-129008.1 65 16 65 A-128972.1 65 12 65 A-128800.1 76 7 66 A-129040.1 66 7 66 A-128820.1 75 2 66 A-128983.1 66 18 66 A-128992.1 66 21 66 A-128885.1 67 31 66 A-128886.1 67 13 66 A-128809.1 75 9 66 A-128921.1 67 6 66 A-128930.1 68 18 66 A-128936.1 68 4 67 A-128982.1 67 17 67 A-128806.1 76 9 67 A-128892.1 68 19 67 A-128739.1 75 11 67 A-128917.1 68 8 67 A-128881.1 77 2 67 A-128873.1 78 10 67 A-128743.1 76 4 67 A-128736.1 75 17 67 A-128848.1 78 6 67 A-128745.1 77 10 68 A-128683.1 72 5 68 A-129006.1 68 18 68 A-128889.1 69 6 68 A-128901.1 69 18 68 A-128589.1 73 6 68 A-128860.1 79 5 68 A-129057.1 69 27 68 A-128827.1 78 6 68 A-129049.1 69 16 68 A-129017.1 68 13 68 A-128609.1 73 7 68 A-128811.1 78 5 69 A-128879.1 79 7 69 A-128986.1 69 19 69 A-128697.1 73 11 69 A-128821.1 78 6 69 A-129016.1 69 15 69 A-128818.1 78 3 69 A-128945.1 70 15 69 A-128995.1 69 20 69 A-128798.1 79 4 69 A-128946.1 70 17 69 A-128947.1 70 8 69 A-128735.1 77 16 69 A-128631.1 80 6 69 A-129019.1 69 7 69 A-128790.1 80 8 69 A-128808.1 78 8 69 A-128614.1 74 10 69 A-128653.1 74 9 69 A-128832.1 79 5 70 A-129020.1 70 15 70 A-128865.1 81 6 70 A-128828.1 80 5 70 A-129003.1 70 27 70 A-128846.1 81 5 70 A-128928.1 72 10 70 A-129050.1 71 7 70 A-128994.1 71 7 70 A-128758.1 80 3 71 A-128944.1 72 24 71 A-128746.1 80 13 71 A-128969.1 71 6 71 A-128877.1 81 7 71 A-128862.1 81 3 71 A-128954.1 72 12 71 A-128595.1 75 18 71 A-129026.1 71 10 71 A-129038.1 71 15 71 A-128923.1 73 6 71 A-129011.1 71 20 71 A-128722.1 74 6 71 A-128576.1 74 11 71 A-128998.1 71 9 71 A-128959.1 72 18 71 A-128843.1 82 3 71 A-128740.1 79 5 71 A-128803.1 80 6 71 A-128965.1 72 18 71 A-128942.1 73 8 71 A-128924.1 74 10 72 A-128971.1 72 18 72 A-128997.1 72 29 72 A-129041.1 72 7 72 A-129000.1 72 22 72 A-128987.1 72 8 72 A-128985.1 73 11 72 A-128880.1 83 3 73 A-128823.1 82 8 73 A-129048.1 73 19 73 A-128839.1 82 4 73 A-128664.1 78 6 73 A-128871.1 84 3 73 A-128835.1 83 8 73 A-128849.1 84 5 73 A-128791.1 84 7 73 A-128914.1 74 20 73 A-128613.1 78 4 74 A-128750.1 83 9 74 A-128996.1 74 21 74 A-128799.1 84 11 74 A-128599.1 78 10 74 A-128916.1 75 18 74 A-128991.1 74 10 74 A-129018.1 74 9 74 A-128844.1 85 4 74 A-129042.1 74 5 74 A-128988.1 74 24 74 A-128568.1 77 6 74 A-128989.1 75 14 74 A-128754.1 84 19 75 A-128870.1 85 3 75 A-128900.1 76 14 75 A-128869.1 85 2 75 A-128581.1 78 11 75 A-128931.1 77 8 75 A-128955.1 76 4 75 A-128956.1 77 17 75 A-128868.1 86 2 75 A-128701.1 79 4 75 A-128812.1 85 10 76 A-128752.1 85 6 76 A-128567.1 78 7 76 A-128978.1 76 22 76 A-129032.1 76 11 76 A-128824.1 85 6 76 A-128615.1 81 14 76 A-128957.1 78 19 76 A-129012.1 77 11 77 A-128932.1 78 10 77 A-128943.1 78 21 77 A-128951.1 78 19 77 A-129051.1 77 24 77 A-128948.1 78 17 77 A-129023.1 77 17 77 A-128810.1 86 7 77 A-129021.1 77 12 77 A-128751.1 87 3 77 A-129033.1 78 19 78 A-129039.1 78 15 78 A-128632.1 89 4 78 A-128950.1 79 12 78 A-128644.1 83 6 79 A-128962.1 80 17 79 A-128906.1 80 21 79 A-128911.1 80 14 79 A-128831.1 89 5 79 A-128891.1 81 16 80 A-128813.1 89 5 80 A-128850.1 91 7 80 A-128851.1 91 4 80 A-128940.1 82 9 80 A-129027.1 81 9 81 A-128958.1 82 15 81 A-128838.1 91 4 81 A-128604.1 86 8 81 A-129037.1 82 16 82 A-128816.1 91 3 82 A-128907.1 83 30 82 A-128753.1 91 23 82 A-128817.1 91 3 82 A-129007.1 82 18 82 A-128651.1 86 5 82 A-128981.1 83 13 82 A-128960.1 85 11 83 A-128582.1 86 6 84 A-128872.1 94 2 84 A-128580.1 86 9 84 A-128990.1 84 17 84 A-128830.1 94 5 84 A-128718.1 87 9 85 A-128919.1 86 38 85 A-128909.1 86 13 85 A-128913.1 87 31 86 A-128702.1 90 8 86 A-129036.1 87 17 87 A-129052.1 87 23 87 A-128929.1 88 9 87 A-128926.1 89 17 87 A-128941.1 89 22 87 A-128796.1 98 9 88 A-128890.1 89 39 88 A-128708.1 91 5 88 A-128903.1 89 21 88 A-128912.1 89 16 89 A-128910.1 91 18 90 A-128795.1 101 12 90 A-128952.1 92 10 91 A-129010.1 91 10 91 A-128920.1 93 22 92 A-129001.1 93 16 93 A-129009.1 95 10 95 A-128904.1 99 24 98 A-129053.1 101 6 101 A-129029.1 102 19 101 A-128961.1 107 32 105 A-129028.1 108 30 108 A-129030.1 117 11 117

A-129002.1 129 27 129

Sequence CWU 1

1

50815480DNAHomo sapiens 1tatatccgtg gtttcctgct acctccaacc atgggccttt tgggaatact ttgtttttta 60atcttcctgg ggaaaacctg gggacaggag caaacatatg tcatttcagc accaaaaata 120ttccgtgttg gagcatctga aaatattgtg attcaagttt atggatacac tgaagcattt 180gatgcaacaa tctctattaa aagttatcct gataaaaaat ttagttactc ctcaggccat 240gttcatttat cctcagagaa taaattccaa aactctgcaa tcttaacaat acaaccaaaa 300caattgcctg gaggacaaaa cccagtttct tatgtgtatt tggaagttgt atcaaagcat 360ttttcaaaat caaaaagaat gccaataacc tatgacaatg gatttctctt cattcataca 420gacaaacctg tttatactcc agaccagtca gtaaaagtta gagtttattc gttgaatgac 480gacttgaagc cagccaaaag agaaactgtc ttaactttca tagatcctga aggatcagaa 540gttgacatgg tagaagaaat tgatcatatt ggaattatct cttttcctga cttcaagatt 600ccgtctaatc ctagatatgg tatgtggacg atcaaggcta aatataaaga ggacttttca 660acaactggaa ccgcatattt tgaagttaaa gaatatgtct tgccacattt ttctgtctca 720atcgagccag aatataattt cattggttac aagaacttta agaattttga aattactata 780aaagcaagat atttttataa taaagtagtc actgaggctg acgtttatat cacatttgga 840ataagagaag acttaaaaga tgatcaaaaa gaaatgatgc aaacagcaat gcaaaacaca 900atgttgataa atggaattgc tcaagtcaca tttgattctg aaacagcagt caaagaactg 960tcatactaca gtttagaaga tttaaacaac aagtaccttt atattgctgt aacagtcata 1020gagtctacag gtggattttc tgaagaggca gaaatacctg gcatcaaata tgtcctctct 1080ccctacaaac tgaatttggt tgctactcct cttttcctga agcctgggat tccatatccc 1140atcaaggtgc aggttaaaga ttcgcttgac cagttggtag gaggagtccc agtaacactg 1200aatgcacaaa caattgatgt aaaccaagag acatctgact tggatccaag caaaagtgta 1260acacgtgttg atgatggagt agcttccttt gtgcttaatc tcccatctgg agtgacggtg 1320ctggagttta atgtcaaaac tgatgctcca gatcttccag aagaaaatca ggccagggaa 1380ggttaccgag caatagcata ctcatctctc agccaaagtt acctttatat tgattggact 1440gataaccata aggctttgct agtgggagaa catctgaata ttattgttac ccccaaaagc 1500ccatatattg acaaaataac tcactataat tacttgattt tatccaaggg caaaattatc 1560cactttggca cgagggagaa attttcagat gcatcttatc aaagtataaa cattccagta 1620acacagaaca tggttccttc atcccgactt ctggtctatt acatcgtcac aggagaacag 1680acagcagaat tagtgtctga ttcagtctgg ttaaatattg aagaaaaatg tggcaaccag 1740ctccaggttc atctgtctcc tgatgcagat gcatattctc caggccaaac tgtgtctctt 1800aatatggcaa ctggaatgga ttcctgggtg gcattagcag cagtggacag tgctgtgtat 1860ggagtccaaa gaggagccaa aaagcccttg gaaagagtat ttcaattctt agagaagagt 1920gatctgggct gtggggcagg tggtggcctc aacaatgcca atgtgttcca cctagctgga 1980cttaccttcc tcactaatgc aaatgcagat gactcccaag aaaatgatga accttgtaaa 2040gaaattctca ggccaagaag aacgctgcaa aagaagatag aagaaatagc tgctaaatat 2100aaacattcag tagtgaagaa atgttgttac gatggagcct gcgttaataa tgatgaaacc 2160tgtgagcagc gagctgcacg gattagttta gggccaagat gcatcaaagc tttcactgaa 2220tgttgtgtcg tcgcaagcca gctccgtgct aatatctctc ataaagacat gcaattggga 2280aggctacaca tgaagaccct gttaccagta agcaagccag aaattcggag ttattttcca 2340gaaagctggt tgtgggaagt tcatcttgtt cccagaagaa aacagttgca gtttgcccta 2400cctgattctc taaccacctg ggaaattcaa ggcgttggca tttcaaacac tggtatatgt 2460gttgctgata ctgtcaaggc aaaggtgttc aaagatgtct tcctggaaat gaatatacca 2520tattctgttg tacgaggaga acagatccaa ttgaaaggaa ctgtttacaa ctataggact 2580tctgggatgc agttctgtgt taaaatgtct gctgtggagg gaatctgcac ttcggaaagc 2640ccagtcattg atcatcaggg cacaaagtcc tccaaatgtg tgcgccagaa agtagagggc 2700tcctccagtc acttggtgac attcactgtg cttcctctgg aaattggcct tcacaacatc 2760aatttttcac tggagacttg gtttggaaaa gaaatcttag taaaaacatt acgagtggtg 2820ccagaaggtg tcaaaaggga aagctattct ggtgttactt tggatcctag gggtatttat 2880ggtaccatta gcagacgaaa ggagttccca tacaggatac ccttagattt ggtccccaaa 2940acagaaatca aaaggatttt gagtgtaaaa ggactgcttg taggtgagat cttgtctgca 3000gttctaagtc aggaaggcat caatatccta acccacctcc ccaaagggag tgcagaggcg 3060gagctgatga gcgttgtccc agtattctat gtttttcact acctggaaac aggaaatcat 3120tggaacattt ttcattctga cccattaatt gaaaagcaga aactgaagaa aaaattaaaa 3180gaagggatgt tgagcattat gtcctacaga aatgctgact actcttacag tgtgtggaag 3240ggtggaagtg ctagcacttg gttaacagct tttgctttaa gagtacttgg acaagtaaat 3300aaatacgtag agcagaacca aaattcaatt tgtaattctt tattgtggct agttgagaat 3360tatcaattag ataatggatc tttcaaggaa aattcacagt atcaaccaat aaaattacag 3420ggtaccttgc ctgttgaagc ccgagagaac agcttatatc ttacagcctt tactgtgatt 3480ggaattagaa aggctttcga tatatgcccc ctggtgaaaa tcgacacagc tctaattaaa 3540gctgacaact ttctgcttga aaatacactg ccagcccaga gcacctttac attggccatt 3600tctgcgtatg ctctttccct gggagataaa actcacccac agtttcgttc aattgtttca 3660gctttgaaga gagaagcttt ggttaaaggt aatccaccca tttatcgttt ttggaaagac 3720aatcttcagc ataaagacag ctctgtacct aacactggta cggcacgtat ggtagaaaca 3780actgcctatg ctttactcac cagtctgaac ttgaaagata taaattatgt taacccagtc 3840atcaaatggc tatcagaaga gcagaggtat ggaggtggct tttattcaac ccaggacaca 3900atcaatgcca ttgagggcct gacggaatat tcactcctgg ttaaacaact ccgcttgagt 3960atggacatcg atgtttctta caagcataaa ggtgccttac ataattataa aatgacagac 4020aagaatttcc ttgggaggcc agtagaggtg cttctcaatg atgacctcat tgtcagtaca 4080ggatttggca gtggcttggc tacagtacat gtaacaactg tagttcacaa aaccagtacc 4140tctgaggaag tttgcagctt ttatttgaaa atcgatactc aggatattga agcatcccac 4200tacagaggct acggaaactc tgattacaaa cgcatagtag catgtgccag ctacaagccc 4260agcagggaag aatcatcatc tggatcctct catgcggtga tggacatctc cttgcctact 4320ggaatcagtg caaatgaaga agacttaaaa gcccttgtgg aaggggtgga tcaactattc 4380actgattacc aaatcaaaga tggacatgtt attctgcaac tgaattcgat tccctccagt 4440gatttccttt gtgtacgatt ccggatattt gaactctttg aagttgggtt tctcagtcct 4500gccactttca cagtgtacga ataccacaga ccagataaac agtgtaccat gttttatagc 4560acttccaata tcaaaattca gaaagtctgt gaaggagccg cgtgcaagtg tgtagaagct 4620gattgtgggc aaatgcagga agaattggat ctgacaatct ctgcagagac aagaaaacaa 4680acagcatgta aaccagagat tgcatatgct tataaagtta gcatcacatc catcactgta 4740gaaaatgttt ttgtcaagta caaggcaacc cttctggata tctacaaaac tggggaagct 4800gttgctgaga aagactctga gattaccttc attaaaaagg taacctgtac taacgctgag 4860ctggtaaaag gaagacagta cttaattatg ggtaaagaag ccctccagat aaaatacaat 4920ttcagtttca ggtacatcta ccctttagat tccttgacct ggattgaata ctggcctaga 4980gacacaacat gttcatcgtg tcaagcattt ttagctaatt tagatgaatt tgccgaagat 5040atctttttaa atggatgcta aaattcctga agttcagctg catacagttt gcacttatgg 5100actcctgttg ttgaagttcg tttttttgtt ttcttctttt tttaaacatt catagctggt 5160cttatttgta aagctcactt tacttagaat tagtggcact tgcttttatt agagaatgat 5220ttcaaatgct gtaactttct gaaataacat ggccttggag ggcatgaaga cagatactcc 5280tccaaggtta ttggacaccg gaaacaataa attggaacac ctcctcaaac ctaccactca 5340ggaatgtttg ctggggccga aagaacagtc cattgaaagg gagtattaca aaaacatggc 5400ctttgcttga aagaaaatac caaggaacag gaaactgatc attaaagcct gagtttgctt 5460tcaaaaaaaa aaaaaaaaaa 548025384DNAMacaca mulattamodified_base(2876)..(2895)a, c, t, g, unknown or other 2catgatttcc tgctacctcc aaccatgggc cttttgggaa tactttgttt tttaatcttc 60ctgggaaaaa cttggggaca ggagcaaaca tatgtcattt cagcaccaaa aatattccgt 120gttggagcat ctgaaaacat tgtgattcaa gtttatggat acactgaagc atttgatgca 180acaatctcta ttaaaagtta tcctgataaa aaatttagtt actcctcagg ccatgttcat 240ttatcctcag agaataaatt ccaaaactcg gcagtcttaa caatacaacc aaaacaatta 300cctggaggac aaaaccaagt ttcttatgtg tatttggaag ttgtatcaaa gcatttttca 360aaatcaaaaa aaattccaat aacctatgac aatggatttc tcttcattca tacagacaaa 420cctgtttata ctccagacca atcagtaaag gttagagttt attcgttgaa tgatgacttg 480aagccagcca aaagagaaac tgtcttaact ttcatagatc ctgaaggatc agaaattgac 540atggtagaag aaattgatca tattggaatt atctcttttc ctgacttcaa gattccgtct 600aatcctagat atggtatgtg gatgatccag gctaaatata aagaggactt ttcaacaact 660ggaactgcat tttttgaagt taaagaatat gtcttgccac atttttctgt ctcagtagaa 720ccagaaagta atttcattgg ttataagaac tttaagaatt ttgaaattac tataaaagca 780agatattttt ataataaagt agtcactgag gctgatgttt atatcacatt tggaataaga 840gaagacttaa aagatgatca aaaagaaatg atgcaaacag caatgcaaaa cacaatgttg 900ataaatggaa ttgctcaagt cacatttgat tctgaaacag cagtcaaaga actgtcatac 960tacagtttag aagatttaaa caacaagtac ctttatattg ctgtaacagt catagagtct 1020acaggtggat tttctgaaga ggcagaaata cctggcatca aatatgtcct ctctccctac 1080aaactgaatt tggttgctac tcctcttttc ctgaagcctg ggattccata ttccatcaag 1140gtgcaggtta aagatgcgct tgaccagttg gtaggagggg tcccagtaac actgaatgca 1200caaacaattg atgtcaacca agagacatct gacttggagc caaggaaaag tgtaacacgt 1260gttgatgatg gagtagcttc gtttgtggtt aatctcccat ctggagtgac ggtgctggag 1320tttaatgtca aaactgatgc tccagatctt ccagacgaaa atcaggccag ggaaggttac 1380cgagcaatag catactcatc tctcagccaa agttaccttt atatcgattg gactgataac 1440cacaaggctt tgctagtggg agaatatttg aatattattg ttacccccaa aagcccatat 1500attgacaaaa taactcacta taattacttg attttatcca agggcaaaat tatccacttt 1560ggcacaaggg agaaactttc agatgcatct tatcaaagta taaacattcc agtaacgcag 1620aacatggttc cttcatcccg actcctggtc tattacatcg tcacaggaga gcagacagca 1680gaattagtgt ctgattcagt ctggttaaat attgaagaaa aatgtggcaa ccagctccag 1740gttcatctgt ctcctgatgc agatacatat tctccaggcc aaactgtgtc tcttaatatg 1800gtaactggga tggattcctg ggtggcatta acagcagtgg acagcgctgt gtatggagtc 1860caaagaagag ccaaaaagcc cttggaaaga gtatttcaat tcttagagaa gagtgatctg 1920ggctgtgggg caggtggtgg cctcaacaat gccaatgtgt tccacctagc tggacttacc 1980ttcctcacta atgcaaatgc agatgactcc caagaaaatg atgaaccttg taaagaaatt 2040atcaggccaa gaagaatgct acaagagaag atagaagaaa tagctgctaa atataaacat 2100ttagtagtga agaaatgttg ttacgatgga gtccgtatta atcatgatga aacctgtgag 2160cagcgagctg cacggattag tgtagggccg agatgcgtca aagctttcac tgaatgttgt 2220gtcgtcgcaa gccagctccg tgctaataac tctcataaag acttgcaatt gggaaggcta 2280cacatgaaga ccctgttacc agtaagcaag ccagaaattc ggagttattt tccagaaagc 2340tggttatggg aagttcatct tgttcccaga agaaaacagt tgcagtttgc cctacctgat 2400tctgtaacta cctgggaaat tcaaggtgtt ggcatttcaa acagtggtat atgtgttgct 2460gatactatta aggcaaaggt gttcaaagat gtcttcctgg aaatgaatat accatattct 2520gttgtacgag gagaacaggt ccagttgaaa ggaactgttt acaactatag gacttctggg 2580atgcagttct gtgttaaaat gtctgctgtg gagggaatct gcacttcaga aagcccagtc 2640attgatcatc agggcacaaa gtcctccaaa tgtgtgcgac agaaagtaga gggctcctct 2700aatcacttgg tgacctttac tgtgcttcct ctggaaattg gccttcagaa catcaatttc 2760tcactggaga cttcgtttgg aaaagaaatc ttagtaaaat cgttacgagt ggtgccagaa 2820ggtgtcaaaa gggaaagcta ttctggtatt actttggatc ctaggggtat ttatgnnnnn 2880nnnnnnnnnn nnnnncgaaa ggagttccca tacaggatac cattagattt ggtccccaaa 2940acagaaatca aaaggatttt gagtgtaaaa ggactgcttg taggtgagat cttgtctgca 3000gttctaagtc gggaaggcat caatatccta acccacctcc ccaaagggag tgcagaggcg 3060gagctgatga gcgttgtccc agtattctat gtttttcact acctggaaac aggaaatcat 3120tggaacattt ttcattccga cccattaatt gaaaagcgga acctggagaa aaaattaaaa 3180gaagggatgg tgagcattat gtcctacaga aatgctgact attcttacag cgtgtggaag 3240ggtggcagtg ctagcacttg gttaacagct tttgctttaa gagtacttgg acaagtacat 3300aaatatgtag agcagaacca aaattcaata tgtaattctt tattgtggct ggttgagaat 3360tatcagttag ataatggatc cttcaaggaa aattcacagt atcaaccaat aaaattacag 3420aaaatcaaca cagctctaat taaagctgac acctttctgc ttgaaaatac actgccagcc 3480cagagcacct ttacattggc catttctgcc tatgctcttt ccctgggaga taaaactcac 3540ccacagtttt gttcaattgt ttcagctttg aagagagaag ctttggttaa aggtaatcca 3600cccatttatc gtttttggaa agacagtctt caacataaag acagctctgt acctaacact 3660ggtacagcac gtatggtaga aacaactgcc tatgctttac tcaccagtct gaacttgaaa 3720gacataaatt atgttaaccc aatcatcaaa tggctatcag aagagcagag gtatggaggt 3780ggcttttatt caacccagga cacaatcaat gccatcgagg gcctgacaga atattcactc 3840ctggttaaac agctccgctt gaatatggac atcgatgttg cttacaagca taaaggtccc 3900ttacataatt ataaaatgac agacaagaat ttccttggga ggccagtaga ggtgcttctc 3960aatgatgacc tcgttgtcag tacaggattt ggcagtggct tggctacggt acatgtaaca 4020actgtagttc acaaaaccag tacctctgag gaagtttgca gcttttattt gaaaattgat 4080actcaggata ttgaagcatc ccactacaga ggctacggaa actctgatta caaacgcata 4140gtagcatgtg ccagctacaa gcccagcaag gaagaatcat cttctggatc ctctcatgca 4200gtgatggaca tctccttgcc tactggaatc aatgcaaatg aagaagactt aaaagctctt 4260gtggaagggg tggatcagct attcactgat taccaaataa aagatggaca tgttattctg 4320caactgaatt cgatcccctc cagtgatttc ctttgtgtac gattccggat ttttgaactc 4380tttgaagttg ggtttcttag tcctgccact ttcacagtgt atgaatacca cagaccagat 4440aaacagtgta ccatgtttta tagcacttcc aatatcaaaa ttcagaaagt ctgtgaagga 4500gccacgtgca agtgtataga agctgattgt gggcaaatgc agaaagaatt ggatctgaca 4560atctctgcag agactagaaa acaaacagca tgtaacccag agattgcata tgcttataaa 4620gttatcatca catccatcac tacagaaaat gtttttgtca agtacaaggc aacccttctg 4680gatatctaca aaactgggga agctgttgct gaaaaagact ctgaaatcac cttcattaaa 4740aaggtaacct gcactaacgc tgagctggtg aaaggaagac agtacttaat tatggggaaa 4800gaagctctcc agataaaata caatttcact ttcaggtaca tctacccttt agattccttg 4860acctggattg aatactggcc tagagacaca acatgttcat cgtgtcaagc atttttagct 4920aatttagatg aatttgctga agacatcttt ttaaatggat gctaaaattc ctgaagttca 4980gctgcataca gtttgcactt atggactcct gttgttgaag tttgtttttt tttctcgttt 5040ttttgtcttt aaacattcac agctggtctt atttgtaaag ctcactttac ttagaattag 5100tggcacttgc ttttattaga gaatgatttt aaacgctgta actttctgaa ataacatggc 5160cttggagggc atgaagacag atactcctcc aaggttattg gacaccggaa acaataaatt 5220agaacacctc ctcaaaccta ccacttagga atgtttgctg gagccgaaag aacagtccat 5280tgaaatggag tattacaaaa acatggcctt tgcttgaaag aaaataccag gggacaggaa 5340actgatcatt aaagcctgag tttgctttca aactgtgcta aaaa 538435448DNAMus musculus 3tttaaaagga aagtggttac agggaggcca tgcccatggg tttatgccgc taccagccat 60gggtctttgg ggaatacttt gtcttttaat tttcctggac aaaacttggg gacaggaaca 120aacctacgtc atttcagcac ccaaaatcct ccgggtcggc tcgtctgaaa atgtggtaat 180tcaagtccat ggctacactg aagcatttga tgcaactctt tctctaaaaa gctatcctga 240caaaaaagtc accttctctt caggctatgt taatttgtcc ccggaaaaca aattccaaaa 300cgcggcactg ttgacactac agcccaatca agttcctaga gaagaaagcc cagtctctca 360cgtgtatctg gaagttgtgt caaaacactt ttcaaaatca aagaaaatac caattaccta 420taacaatgga attctcttca tccatacaga caaacctgtt tacacgccgg accagtcagt 480aaagatcaga gtctattctc tgggtgacga cttgaagcca gccaaacggg agactgtctt 540aactttcata gaccccgaag gatcagaagt tgacattgta gaagaaaatg attacaccgg 600aattatctct tttcctgact tcaagattcc atctaatccc aagtatggtg tttggacaat 660taaagctaac tataagaagg attttacaac aactggaact gcatactttg aaattaaaga 720atatgtcttg ccacgattct ctgtttcaat agaactagaa agaaccttca ttggctataa 780aaactttaag aactttgaaa tcactgtgaa agcaagatat ttttataata aagtggtacc 840tgatgctgaa gtgtatgcct tttttggatt gagagaggac ataaaagatg aggagaagca 900gatgatgcac aaagccacac aagccgcaaa gttggttgac ggagttgctc agatctcttt 960tgattctgaa acagcagtta aagagctgtc ctacaacagt ctagaagact taaacaacaa 1020gtacctttat attgcagtaa cagtcacaga atcttcaggt ggattttcag aagaggcaga 1080aatccctgga gtcaaatatg tcctctctcc ctacacactg aatttggtcg ctactcctct 1140tttcgtgaag cccgggattc cattttccat caaggcacag gttaaagatt cactcgagca 1200ggcggtagga ggggtcccag taactctgat ggcacaaaca gtcgatgtga atcaagagac 1260atctgacttg gaaacaaaga ggagcatcac tcatgacact gatggagtag ctgtgtttgt 1320gctgaacctc ccatcaaatg tgacggtgct aaagtttgag atcagaactg atgacccaga 1380acttcccgaa gaaaatcaag ccagcaaaga gtacgaagca gttgcgtact cgtctctcag 1440ccaaagttac atttacatcg cttggactga aaactacaag cccatgcttg tgggagaata 1500cctgaatatt atggttaccc ccaagagccc atatatcgac aaaataactc actataatta 1560cttgatttta tccaaaggca aaattgtaca gtacggcaca agagagaaac ttttctcctc 1620aacttatcaa aatataaata ttccagtgac acagaacatg gttccttcag cacgactcct 1680ggtctattac atagtcacag gggagcaaac agcagaatta gtggctgacg cagtctggat 1740aaatattgag gagaagtgtg gcaaccagct ccaggtccat ctgtctccag atgaatatgt 1800gtattctcca ggccaaactg tgtcccttga catggtgact gaagcagact catgggtagc 1860actatcagca gtggacagag ctgtgtataa agtccaggga aacgccaaaa gggccatgca 1920aagagtcttt caagctttgg atgaaaagag tgacctgggc tgtggggcag gtggtggcca 1980tgacaatgca gatgtattcc atctagctgg gctcaccttc ctcaccaacg caaacgcaga 2040tgactcccat tatcgtgatg actcttgtaa agaaattctc aggtcaaaga gaaacctgca 2100tctcctaagg cagaaaatag aagaacaagc tgctaagtac aaacatagtg tgccaaagaa 2160atgctgctat gacggagccc gagtgaactt ctacgaaacc tgtgaggagc gagtggcccg 2220ggttaccata ggccctctct gcatcagggc cttcaacgag tgctgtacta ttgcgaacaa 2280gatccgaaaa gaaagccccc ataaacctgt ccaactggga aggatccaca ttaagaccct 2340gttaccagtg atgaaggcag atatccgaag ctactttcca gagagctggc tatgggaaat 2400tcatcgcgtt cccaaaagaa aacagctgca ggtcacgctg cctgactcac taacgacttg 2460ggaaattcaa ggcattggca tttcagacaa tggtatatgt gttgctgata cactcaaggc 2520aaaggtgttc aaagaagtct tcctggagat gaacatacca tattctgttg tgcgaggaga 2580acagatccaa ttgaaaggaa ctgtttacaa ctatatgacc tcagggacaa agttctgtgt 2640taaaatgtct gctgtggagg ggatctgcac ttcaggaagc tcagctgcta gccttcacac 2700ctccaggccc tccagatgtg tgttccagag gatagagggc tcgtccagtc acttggtgac 2760cttcaccctg cttcctctgg aaattggcct tcactccata aacttctcac tagagacctc 2820atttgggaaa gacatcttag taaagacatt acgggtagtg ccagaaggag tcaagaggga 2880aagctatgcc ggcgtgattc tggaccctaa gggaattcgt ggtattgtta acagacgaaa 2940ggaattccca tacaggatcc cattagattt ggtccccaag accaaagttg aaaggatttt 3000gagtgtcaaa ggactgcttg taggggagtt cttgtccacg gttctgagta aggaaggcat 3060caacatccta acccacctcc ccaagggcag tgcagaggca gagctcatga gcatagctcc 3120ggtgttctat gttttccact acctggaagc aggaaaccat tggaatattt tctatcctga 3180tacactgagt aaaagacaga gcctggagaa aaaaataaaa caaggggtgg tgagcgtcat 3240gtcctacaga aacgctgact attcctacag catgtggaag ggggcgagcg ctagtacctg 3300gctgacagct tttgctctga gagtgcttgg acaggtggcc aagtatgtaa aacaggatga 3360aaactcaatt tgtaactctt tgctatggct ggttgagaag tgtcagctgg aaaacggctc 3420tttcaaggaa aattcccaat atctaccaat aaaattacag ggtactttgc ctgctgaagc 3480ccaagagaaa actttgtatc ttacagcctt ttctgtgatt ggaattagaa aggcagttga 3540catatgcccc accatgaaaa tccacacagc gctagataaa gccgactcct tcctgcttga 3600aaacaccctg ccatccaaga gcaccttcac actggccatt gtagcctatg ctctttccct 3660aggagacaga acccacccga ggtttcgtct aattgtgtcg gccctgagga aggaagcttt 3720tgttaaaggt gatccgccca tttaccgtta ctggagagat accctcaaac gtccagacag 3780ctctgtgccc agcagcggca cagcaggtat ggttgaaacc acagcctatg ctttgctcgc 3840cagcctgaaa ctgaaggata tgaattacgc caaccccatc atcaagtggc tatctgaaga 3900gcagaggtat ggaggcggct tttattccac ccaggatacg attaatgcca tcgagggcct 3960gacagaatat tcactcctgt taaaacaaat tcatttggat

atggacatca atgtcgccta 4020caaacacgaa ggtgacttcc acaagtataa ggtgacagag aagcatttcc tggggaggcc 4080agtggaggta tctctcaatg atgaccttgt tgtcagcaca ggctacagca gtggcttggc 4140cacagtatat gtaaaaactg tggttcacaa aattagtgtc tctgaggaat tttgcagctt 4200ttacttgaaa attgataccc aagatattga agcatccagc cacttcaggc tcagtgactc 4260tggattcaag cgcataatag catgtgccag ctacaagccc agcaaggagg agtcaacatc 4320cgggtcctcc catgcagtaa tggatatatc actgccgact ggaatcggag caaacgagga 4380agatttacgg gctcttgtgg aaggagtgga tcaactacta actgattacc agatcaaaga 4440tggccatgtc attctgcaac tgaattcgat cccctccaga gatttcctct gtgtccggtt 4500ccggatattt gaacttttcc aagttgggtt tctgaatcct gctaccttca cggtgtacga 4560gtatcacaga ccagataagc agtgcaccat gatttatagc atttctgaca ccaggcttca 4620gaaagtctgt gaaggagcag cttgcacatg tgtggaagct gactgtgcgc aactgcaggc 4680agaagtagac ctagccatct ctgcagactc cagaaaagag aaagcctgta aaccagagac 4740tgcatatgct tataaagtca ggatcacatc agccactgaa gaaaatgttt ttgtcaagta 4800cactgcgact cttctggtca cttacaaaac aggggaagct gctgatgaga attcggaggt 4860caccttcatt aaaaagatga gctgtaccaa tgccaacctg gtgaaaggga agcagtattt 4920aatcatgggc aaagaggttc tgcagatcaa acacaatttc agtttcaagt atatataccc 4980tctagattcc tccacctgga ttgaatattg gcccacagac acaacgtgtc catcctgtca 5040agcatttgta gagaatttga ataactttgc tgaagacctc tttttaaaca gctgtgaatg 5100aaaagttctg ctgcacgaag attcctcctg cggcgggggg attgctcctc ctctggcttg 5160gaaacctagc ctagaatcag atacactttc tttagagtaa agcacaagct gatgagttac 5220gactttgtga aatggatagc cttgagggga ggcgaaaaca ggtcccccaa ggctatcaga 5280tgtcagtgcc aatagactga aacaagtctg taaagttagc agtcaggggt gttggttggg 5340gccggaagaa gagacccact gaaactgtag ccccttatca aaacatatcc ttgcttgaaa 5400gaaaaatacc aaggacagaa aatgccataa aatcttgact ttgcactc 544844497DNARattus norvegicus 4atggatagca cagagaccga cagatgtcct acagcccgcc atcatctttc cggaaacatt 60aactcagtgc ttgctgccct tgtaggtggg ttttcggaag aggcagaaat tcctggcatc 120aaatacgtcc tctctcccta tacactgaat ttggtcgcta cccctctttt cctgaagcct 180gggattccat tttccatcaa ggtacaggtt aaggattcac tcgagcagtt ggtaggaggg 240gtcccagtaa ctctgatggc acaaacagtc aatgtgaatc aagagacatc tgacttggaa 300ccaaagagga gcatcacaca ctctgctgat ggagtggctt catttgtggt gaacctccca 360tcagaagtga catcactgaa gtttgaggtc aaaactgatg ccccggaact tcccgaagaa 420aatcaagcca gcaaagaata tgaagcagtt acatactcat ccctcagcca gagttacatt 480tacattggct ggactgaaaa ctacaagccc atgcttgtgg gagaatatct gaatattatc 540gtcaccccca agagtccata tattgacaaa ataactcact ataattactt gattttatcc 600aaaggcaaaa ttgtacagta tggcacaaag gagaaacttc tctattcatc ttatcaaaat 660ataaacatcc cagtgacaca ggacatggtt ccttcagcgc ggctcctggt ctattacata 720gtcacggggg agcagacagc agaattggtg gctgacgcag tctggataaa cattgaggag 780aagtgtggca accagctcca ggtccatctg tctccagata aagacgtgta ttctccaggc 840caaactgtgt cccttgacat ggtgactgaa gcagactcat gggtggcact atctgcggtg 900gacagcgctg tgtatggagt ccggggaaaa gccaaaaggg ccatgcaaag agtgttccaa 960gcttttgatg acaagagtga cctgggctgt ggggcaggtg gtggccgtga caatgtagat 1020gtattccatc tagctgggct caccttcctc accaatgcaa acgcagatga ctcccaatac 1080cacgatgact cttgtaagga aattctcagg ccaaagagag acctgcagct cctgcatcag 1140aaagtggaag aacaagctgc taaatacaaa caccgtgtgc ccaagaaatg ctgttatgat 1200ggagcccgag aaaacaaata cgaaacctgt gagcagcgag ttgcccgggt gaccataggc 1260ccacactgca tcagggcctt caacgagtgt tgtactattg cggataagat ccgaaaagaa 1320agccaccaca aaggcatgct gttgggaagg atccaaataa aggccctgtt accagtgatg 1380aaggcagaaa tccgaagcta ctttccagag agctggctat gggaagttca tcgtgttccc 1440aaaagaaacc agctgcaggt tgcactgcct gactcactga cgacctggga aattcaaggc 1500atcggcatct cagacaatgg tatatgtgtt gctgacacac tcaaggcaaa ggtgttcaaa 1560gatgtcttcc tggagatgaa cataccatat tctgttgtac gaggggagca gatccaattg 1620aagggaaccg tttacaatta taggacctct gggacaatgt tctgtgttaa aatgtctgcc 1680gtggagggaa tctgcactcc aggaagctcg gctgctagcc ctcagacctc taggtcctcc 1740agatgtgtgc gccagagaat agagggctcc tccagtcact tggtgacctt cagcctgctt 1800cctctggaaa ttggccttca ctccataaac ttctcactag agacttcatt tgggaaagaa 1860atcttagtga agacattacg ggtagtgcca gaagggatca aaagggaaag ctatgctggt 1920gtgactctgg accccagggg agtttatggt attgttaaca gacgaaagga attcccatac 1980aggataccat tagatttggt ccccaaaacc aacgtcaaaa ggattttgag tgtaaaagga 2040ctgcttatag gggaattctt gtccacggtt ctgagtaaag aaggcatcga catcctaacc 2100cacctcccca agggcagcgc cgaggcagaa ctcatgagca tagtcccggt gttctacgtt 2160ttccactacc tggaagcagg aaaccattgg aatattttcc accctgatac gttagctaga 2220aaacagagcc tgcagaaaaa aataaaagaa gggctggtga gcgtcatgtc ctacagaaac 2280gctgactatt cctacagcat gtggaaggga gcaagctcta gtgcctggct gacagctttt 2340gctctgagag tgcttggaca ggtgaacaag tatgtgaaac aagaccaata ctcgatctgt 2400aactccttgt tatggctgat tgagaagtgt cagctggaaa acggatcttt caaggaaaat 2460tcccaatatc taccaataaa attacagggt actttgcctg ctgaagccca agagaacact 2520ttatatctta cagccttttc tgtgattgga attagaaagg ctattggcat atgccccacg 2580gagaaaatct acacagcgct ggctaaagct gactccttcc tacttgaaag gaccctgcct 2640tccaagagca ccttcaccct ggccattgtg gcctatgctc tctccctggg agacagaacc 2700cacccgaagt ttcgttctat tgtgtcagcc ctgaagaggg aagctttggt taaaggagac 2760ccgcccattt accgtttctg gagagacact ctccaacgtc cagacagctc agcacccaac 2820agcggcacag caggtatggt agaaaccacg gcctatgctt tgctcaccag cctgaacctg 2880aaggagacga gttatgtcaa cccgatcatc aagtggctat ctgaggagca gaggtatgga 2940ggcggctttt attccaccca ggataccatt aacgccatcg agggcctgac agagtattca 3000ctcctggtta aacaacttca tttggatatg gatatcaatg tctcctacaa acacaaaggg 3060gatttctacc agtataaagt gacagagaag aacttcctcg ggaggccagt ggaggtaccc 3120ctcaatgatg acctcatcgt caccacaggc tatagcagtg gcttggctac agtatatgta 3180aaaactgtgg ttcacaaaac tagtgtcgct gaggaatttt gcagctttta cttgaaaatt 3240gatacccaag aagttgaagc ctccagctac ctcagctaca gtgactcggg acacaagcgc 3300ataatagcct gtgccagcta caagcccagc aaggaggagt cagcatctgg gtcctcccat 3360gcagtaatgg atatactgct gccgaccgga atcggagcaa accaagaaga tttacgagct 3420cttgtggaag gagtagatca actcctaact gattaccaga tcaaagacag tcatgttatt 3480ctgcaattga attcgattcc ctccagagat ttcctttgtg ttcggttccg gatatttgaa 3540cttttccaag ttgggtttct gaatcctgct acgttcacgg tgtacgagta tcacagacca 3600gataagcagt gtaccatgat ttacagcact tctgacacca accttcagag agtctgtgaa 3660ggagcggcat gcaaatgcgt tgaagctgat tgtgggcaac tgcaggcaga actggacctg 3720gccatctctg cagacaccag gaaagaaaca gcatgtaaac cagagattgc atatgcttat 3780aaggtcagga tcacgtcggc cacggaagaa aacatttttg tcaagtacac tgcgacgctt 3840ctggatattt acaaaacagg ggaagccgct gctgagaagg actctgagat caccttcatt 3900aaaaagataa gctgtaccaa cgccaacctg gtgaaaggaa agcaatattt aatcatgggc 3960aaagaggctc tgcagatcaa acacaatttc agtttcaagt atatataccc tctagattcc 4020tccacctgga ttgaatattg gcccacagac acaacgtgtc catcctgcca agcgtttgta 4080gctaatttgg acgagttcgc tgaagacatc tttctaaatg gctgtgaaaa tgcctgagga 4140agttctgctg cgtggccttc ccgggtactc ctgttggtgg ctcctaggag ccaggatcgc 4200ttggaaactt agcctagaat cggatacatt ttctttatag taaagcgtaa gttgaagagt 4260tactttgtga aacaaaatag ccttgtggag agccgaaggc aggtccccca aggctattgg 4320acatcagcac caataagctg gaacaagtct gtaacgttag cagccagggg tgtttgttgg 4380ggccggaaga agagactcac tgaaattgta gccccttagg aaaacatggt cttgcttgaa 4440aaaaaaaata ccaaggacag aaaatgccat aaaagcttga ctttgcactc aactgta 449755480DNAHomo sapiens 5tttttttttt ttttttttga aagcaaactc aggctttaat gatcagtttc ctgttccttg 60gtattttctt tcaagcaaag gccatgtttt tgtaatactc cctttcaatg gactgttctt 120tcggccccag caaacattcc tgagtggtag gtttgaggag gtgttccaat ttattgtttc 180cggtgtccaa taaccttgga ggagtatctg tcttcatgcc ctccaaggcc atgttatttc 240agaaagttac agcatttgaa atcattctct aataaaagca agtgccacta attctaagta 300aagtgagctt tacaaataag accagctatg aatgtttaaa aaaagaagaa aacaaaaaaa 360cgaacttcaa caacaggagt ccataagtgc aaactgtatg cagctgaact tcaggaattt 420tagcatccat ttaaaaagat atcttcggca aattcatcta aattagctaa aaatgcttga 480cacgatgaac atgttgtgtc tctaggccag tattcaatcc aggtcaagga atctaaaggg 540tagatgtacc tgaaactgaa attgtatttt atctggaggg cttctttacc cataattaag 600tactgtcttc cttttaccag ctcagcgtta gtacaggtta cctttttaat gaaggtaatc 660tcagagtctt tctcagcaac agcttcccca gttttgtaga tatccagaag ggttgccttg 720tacttgacaa aaacattttc tacagtgatg gatgtgatgc taactttata agcatatgca 780atctctggtt tacatgctgt ttgttttctt gtctctgcag agattgtcag atccaattct 840tcctgcattt gcccacaatc agcttctaca cacttgcacg cggctccttc acagactttc 900tgaattttga tattggaagt gctataaaac atggtacact gtttatctgg tctgtggtat 960tcgtacactg tgaaagtggc aggactgaga aacccaactt caaagagttc aaatatccgg 1020aatcgtacac aaaggaaatc actggaggga atcgaattca gttgcagaat aacatgtcca 1080tctttgattt ggtaatcagt gaatagttga tccacccctt ccacaagggc ttttaagtct 1140tcttcatttg cactgattcc agtaggcaag gagatgtcca tcaccgcatg agaggatcca 1200gatgatgatt cttccctgct gggcttgtag ctggcacatg ctactatgcg tttgtaatca 1260gagtttccgt agcctctgta gtgggatgct tcaatatcct gagtatcgat tttcaaataa 1320aagctgcaaa cttcctcaga ggtactggtt ttgtgaacta cagttgttac atgtactgta 1380gccaagccac tgccaaatcc tgtactgaca atgaggtcat cattgagaag cacctctact 1440ggcctcccaa ggaaattctt gtctgtcatt ttataattat gtaaggcacc tttatgcttg 1500taagaaacat cgatgtccat actcaagcgg agttgtttaa ccaggagtga atattccgtc 1560aggccctcaa tggcattgat tgtgtcctgg gttgaataaa agccacctcc atacctctgc 1620tcttctgata gccatttgat gactgggtta acataattta tatctttcaa gttcagactg 1680gtgagtaaag cataggcagt tgtttctacc atacgtgccg taccagtgtt aggtacagag 1740ctgtctttat gctgaagatt gtctttccaa aaacgataaa tgggtggatt acctttaacc 1800aaagcttctc tcttcaaagc tgaaacaatt gaacgaaact gtgggtgagt tttatctccc 1860agggaaagag catacgcaga aatggccaat gtaaaggtgc tctgggctgg cagtgtattt 1920tcaagcagaa agttgtcagc tttaattaga gctgtgtcga ttttcaccag ggggcatata 1980tcgaaagcct ttctaattcc aatcacagta aaggctgtaa gatataagct gttctctcgg 2040gcttcaacag gcaaggtacc ctgtaatttt attggttgat actgtgaatt ttccttgaaa 2100gatccattat ctaattgata attctcaact agccacaata aagaattaca aattgaattt 2160tggttctgct ctacgtattt atttacttgt ccaagtactc ttaaagcaaa agctgttaac 2220caagtgctag cacttccacc cttccacaca ctgtaagagt agtcagcatt tctgtaggac 2280ataatgctca acatcccttc ttttaatttt ttcttcagtt tctgcttttc aattaatggg 2340tcagaatgaa aaatgttcca atgatttcct gtttccaggt agtgaaaaac atagaatact 2400gggacaacgc tcatcagctc cgcctctgca ctccctttgg ggaggtgggt taggatattg 2460atgccttcct gacttagaac tgcagacaag atctcaccta caagcagtcc ttttacactc 2520aaaatccttt tgatttctgt tttggggacc aaatctaagg gtatcctgta tgggaactcc 2580tttcgtctgc taatggtacc ataaataccc ctaggatcca aagtaacacc agaatagctt 2640tcccttttga caccttctgg caccactcgt aatgttttta ctaagatttc ttttccaaac 2700caagtctcca gtgaaaaatt gatgttgtga aggccaattt ccagaggaag cacagtgaat 2760gtcaccaagt gactggagga gccctctact ttctggcgca cacatttgga ggactttgtg 2820ccctgatgat caatgactgg gctttccgaa gtgcagattc cctccacagc agacatttta 2880acacagaact gcatcccaga agtcctatag ttgtaaacag ttcctttcaa ttggatctgt 2940tctcctcgta caacagaata tggtatattc atttccagga agacatcttt gaacaccttt 3000gccttgacag tatcagcaac acatatacca gtgtttgaaa tgccaacgcc ttgaatttcc 3060caggtggtta gagaatcagg tagggcaaac tgcaactgtt ttcttctggg aacaagatga 3120acttcccaca accagctttc tggaaaataa ctccgaattt ctggcttgct tactggtaac 3180agggtcttca tgtgtagcct tcccaattgc atgtctttat gagagatatt agcacggagc 3240tggcttgcga cgacacaaca ttcagtgaaa gctttgatgc atcttggccc taaactaatc 3300cgtgcagctc gctgctcaca ggtttcatca ttattaacgc aggctccatc gtaacaacat 3360ttcttcacta ctgaatgttt atatttagca gctatttctt ctatcttctt ttgcagcgtt 3420cttcttggcc tgagaatttc tttacaaggt tcatcatttt cttgggagtc atctgcattt 3480gcattagtga ggaaggtaag tccagctagg tggaacacat tggcattgtt gaggccacca 3540cctgccccac agcccagatc actcttctct aagaattgaa atactctttc caagggcttt 3600ttggctcctc tttggactcc atacacagca ctgtccactg ctgctaatgc cacccaggaa 3660tccattccag ttgccatatt aagagacaca gtttggcctg gagaatatgc atctgcatca 3720ggagacagat gaacctggag ctggttgcca catttttctt caatatttaa ccagactgaa 3780tcagacacta attctgctgt ctgttctcct gtgacgatgt aatagaccag aagtcgggat 3840gaaggaacca tgttctgtgt tactggaatg tttatacttt gataagatgc atctgaaaat 3900ttctccctcg tgccaaagtg gataattttg cccttggata aaatcaagta attatagtga 3960gttattttgt caatatatgg gcttttgggg gtaacaataa tattcagatg ttctcccact 4020agcaaagcct tatggttatc agtccaatca atataaaggt aactttggct gagagatgag 4080tatgctattg ctcggtaacc ttccctggcc tgattttctt ctggaagatc tggagcatca 4140gttttgacat taaactccag caccgtcact ccagatggga gattaagcac aaaggaagct 4200actccatcat caacacgtgt tacacttttg cttggatcca agtcagatgt ctcttggttt 4260acatcaattg tttgtgcatt cagtgttact gggactcctc ctaccaactg gtcaagcgaa 4320tctttaacct gcaccttgat gggatatgga atcccaggct tcaggaaaag aggagtagca 4380accaaattca gtttgtaggg agagaggaca tatttgatgc caggtatttc tgcctcttca 4440gaaaatccac ctgtagactc tatgactgtt acagcaatat aaaggtactt gttgtttaaa 4500tcttctaaac tgtagtatga cagttctttg actgctgttt cagaatcaaa tgtgacttga 4560gcaattccat ttatcaacat tgtgttttgc attgctgttt gcatcatttc tttttgatca 4620tcttttaagt cttctcttat tccaaatgtg atataaacgt cagcctcagt gactacttta 4680ttataaaaat atcttgcttt tatagtaatt tcaaaattct taaagttctt gtaaccaatg 4740aaattatatt ctggctcgat tgagacagaa aaatgtggca agacatattc tttaacttca 4800aaatatgcgg ttccagttgt tgaaaagtcc tctttatatt tagccttgat cgtccacata 4860ccatatctag gattagacgg aatcttgaag tcaggaaaag agataattcc aatatgatca 4920atttcttcta ccatgtcaac ttctgatcct tcaggatcta tgaaagttaa gacagtttct 4980cttttggctg gcttcaagtc gtcattcaac gaataaactc taacttttac tgactggtct 5040ggagtataaa caggtttgtc tgtatgaatg aagagaaatc cattgtcata ggttattggc 5100attctttttg attttgaaaa atgctttgat acaacttcca aatacacata agaaactggg 5160ttttgtcctc caggcaattg ttttggttgt attgttaaga ttgcagagtt ttggaattta 5220ttctctgagg ataaatgaac atggcctgag gagtaactaa attttttatc aggataactt 5280ttaatagaga ttgttgcatc aaatgcttca gtgtatccat aaacttgaat cacaatattt 5340tcagatgctc caacacggaa tatttttggt gctgaaatga catatgtttg ctcctgtccc 5400caggttttcc ccaggaagat taaaaaacaa agtattccca aaaggcccat ggttggaggt 5460agcaggaaac cacggatata 548065384DNAMacaca mulattamodified_base(2490)..(2509)a, c, t, g, unknown or other 6tttttagcac agtttgaaag caaactcagg ctttaatgat cagtttcctg tcccctggta 60ttttctttca agcaaaggcc atgtttttgt aatactccat ttcaatggac tgttctttcg 120gctccagcaa acattcctaa gtggtaggtt tgaggaggtg ttctaattta ttgtttccgg 180tgtccaataa ccttggagga gtatctgtct tcatgccctc caaggccatg ttatttcaga 240aagttacagc gtttaaaatc attctctaat aaaagcaagt gccactaatt ctaagtaaag 300tgagctttac aaataagacc agctgtgaat gtttaaagac aaaaaaacga gaaaaaaaaa 360caaacttcaa caacaggagt ccataagtgc aaactgtatg cagctgaact tcaggaattt 420tagcatccat ttaaaaagat gtcttcagca aattcatcta aattagctaa aaatgcttga 480cacgatgaac atgttgtgtc tctaggccag tattcaatcc aggtcaagga atctaaaggg 540tagatgtacc tgaaagtgaa attgtatttt atctggagag cttctttccc cataattaag 600tactgtcttc ctttcaccag ctcagcgtta gtgcaggtta cctttttaat gaaggtgatt 660tcagagtctt tttcagcaac agcttcccca gttttgtaga tatccagaag ggttgccttg 720tacttgacaa aaacattttc tgtagtgatg gatgtgatga taactttata agcatatgca 780atctctgggt tacatgctgt ttgttttcta gtctctgcag agattgtcag atccaattct 840ttctgcattt gcccacaatc agcttctata cacttgcacg tggctccttc acagactttc 900tgaattttga tattggaagt gctataaaac atggtacact gtttatctgg tctgtggtat 960tcatacactg tgaaagtggc aggactaaga aacccaactt caaagagttc aaaaatccgg 1020aatcgtacac aaaggaaatc actggagggg atcgaattca gttgcagaat aacatgtcca 1080tcttttattt ggtaatcagt gaatagctga tccacccctt ccacaagagc ttttaagtct 1140tcttcatttg cattgattcc agtaggcaag gagatgtcca tcactgcatg agaggatcca 1200gaagatgatt cttccttgct gggcttgtag ctggcacatg ctactatgcg tttgtaatca 1260gagtttccgt agcctctgta gtgggatgct tcaatatcct gagtatcaat tttcaaataa 1320aagctgcaaa cttcctcaga ggtactggtt ttgtgaacta cagttgttac atgtaccgta 1380gccaagccac tgccaaatcc tgtactgaca acgaggtcat cattgagaag cacctctact 1440ggcctcccaa ggaaattctt gtctgtcatt ttataattat gtaagggacc tttatgcttg 1500taagcaacat cgatgtccat attcaagcgg agctgtttaa ccaggagtga atattctgtc 1560aggccctcga tggcattgat tgtgtcctgg gttgaataaa agccacctcc atacctctgc 1620tcttctgata gccatttgat gattgggtta acataattta tgtctttcaa gttcagactg 1680gtgagtaaag cataggcagt tgtttctacc atacgtgctg taccagtgtt aggtacagag 1740ctgtctttat gttgaagact gtctttccaa aaacgataaa tgggtggatt acctttaacc 1800aaagcttctc tcttcaaagc tgaaacaatt gaacaaaact gtgggtgagt tttatctccc 1860agggaaagag cataggcaga aatggccaat gtaaaggtgc tctgggctgg cagtgtattt 1920tcaagcagaa aggtgtcagc tttaattaga gctgtgttga ttttctgtaa ttttattggt 1980tgatactgtg aattttcctt gaaggatcca ttatctaact gataattctc aaccagccac 2040aataaagaat tacatattga attttggttc tgctctacat atttatgtac ttgtccaagt 2100actcttaaag caaaagctgt taaccaagtg ctagcactgc cacccttcca cacgctgtaa 2160gaatagtcag catttctgta ggacataatg ctcaccatcc cttcttttaa ttttttctcc 2220aggttccgct tttcaattaa tgggtcggaa tgaaaaatgt tccaatgatt tcctgtttcc 2280aggtagtgaa aaacatagaa tactgggaca acgctcatca gctccgcctc tgcactccct 2340ttggggaggt gggttaggat attgatgcct tcccgactta gaactgcaga caagatctca 2400cctacaagca gtccttttac actcaaaatc cttttgattt ctgttttggg gaccaaatct 2460aatggtatcc tgtatgggaa ctcctttcgn nnnnnnnnnn nnnnnnnnnc ataaataccc 2520ctaggatcca aagtaatacc agaatagctt tcccttttga caccttctgg caccactcgt 2580aacgatttta ctaagatttc ttttccaaac gaagtctcca gtgagaaatt gatgttctga 2640aggccaattt ccagaggaag cacagtaaag gtcaccaagt gattagagga gccctctact 2700ttctgtcgca cacatttgga ggactttgtg ccctgatgat caatgactgg gctttctgaa 2760gtgcagattc cctccacagc agacatttta acacagaact gcatcccaga agtcctatag 2820ttgtaaacag ttcctttcaa ctggacctgt tctcctcgta caacagaata tggtatattc 2880atttccagga agacatcttt gaacaccttt gccttaatag tatcagcaac acatatacca 2940ctgtttgaaa tgccaacacc ttgaatttcc caggtagtta cagaatcagg tagggcaaac 3000tgcaactgtt ttcttctggg aacaagatga acttcccata accagctttc tggaaaataa 3060ctccgaattt ctggcttgct tactggtaac agggtcttca tgtgtagcct tcccaattgc 3120aagtctttat gagagttatt agcacggagc tggcttgcga cgacacaaca ttcagtgaaa 3180gctttgacgc atctcggccc tacactaatc cgtgcagctc gctgctcaca ggtttcatca 3240tgattaatac ggactccatc gtaacaacat ttcttcacta ctaaatgttt atatttagca 3300gctatttctt ctatcttctc ttgtagcatt cttcttggcc tgataatttc tttacaaggt 3360tcatcatttt cttgggagtc atctgcattt gcattagtga ggaaggtaag tccagctagg 3420tggaacacat tggcattgtt gaggccacca cctgccccac

agcccagatc actcttctct 3480aagaattgaa atactctttc caagggcttt ttggctcttc tttggactcc atacacagcg 3540ctgtccactg ctgttaatgc cacccaggaa tccatcccag ttaccatatt aagagacaca 3600gtttggcctg gagaatatgt atctgcatca ggagacagat gaacctggag ctggttgcca 3660catttttctt caatatttaa ccagactgaa tcagacacta attctgctgt ctgctctcct 3720gtgacgatgt aatagaccag gagtcgggat gaaggaacca tgttctgcgt tactggaatg 3780tttatacttt gataagatgc atctgaaagt ttctcccttg tgccaaagtg gataattttg 3840cccttggata aaatcaagta attatagtga gttattttgt caatatatgg gcttttgggg 3900gtaacaataa tattcaaata ttctcccact agcaaagcct tgtggttatc agtccaatcg 3960atataaaggt aactttggct gagagatgag tatgctattg ctcggtaacc ttccctggcc 4020tgattttcgt ctggaagatc tggagcatca gttttgacat taaactccag caccgtcact 4080ccagatggga gattaaccac aaacgaagct actccatcat caacacgtgt tacacttttc 4140cttggctcca agtcagatgt ctcttggttg acatcaattg tttgtgcatt cagtgttact 4200gggacccctc ctaccaactg gtcaagcgca tctttaacct gcaccttgat ggaatatgga 4260atcccaggct tcaggaaaag aggagtagca accaaattca gtttgtaggg agagaggaca 4320tatttgatgc caggtatttc tgcctcttca gaaaatccac ctgtagactc tatgactgtt 4380acagcaatat aaaggtactt gttgtttaaa tcttctaaac tgtagtatga cagttctttg 4440actgctgttt cagaatcaaa tgtgacttga gcaattccat ttatcaacat tgtgttttgc 4500attgctgttt gcatcatttc tttttgatca tcttttaagt cttctcttat tccaaatgtg 4560atataaacat cagcctcagt gactacttta ttataaaaat atcttgcttt tatagtaatt 4620tcaaaattct taaagttctt ataaccaatg aaattacttt ctggttctac tgagacagaa 4680aaatgtggca agacatattc tttaacttca aaaaatgcag ttccagttgt tgaaaagtcc 4740tctttatatt tagcctggat catccacata ccatatctag gattagacgg aatcttgaag 4800tcaggaaaag agataattcc aatatgatca atttcttcta ccatgtcaat ttctgatcct 4860tcaggatcta tgaaagttaa gacagtttct cttttggctg gcttcaagtc atcattcaac 4920gaataaactc taacctttac tgattggtct ggagtataaa caggtttgtc tgtatgaatg 4980aagagaaatc cattgtcata ggttattgga attttttttg attttgaaaa atgctttgat 5040acaacttcca aatacacata agaaacttgg ttttgtcctc caggtaattg ttttggttgt 5100attgttaaga ctgccgagtt ttggaattta ttctctgagg ataaatgaac atggcctgag 5160gagtaactaa attttttatc aggataactt ttaatagaga ttgttgcatc aaatgcttca 5220gtgtatccat aaacttgaat cacaatgttt tcagatgctc caacacggaa tatttttggt 5280gctgaaatga catatgtttg ctcctgtccc caagtttttc ccaggaagat taaaaaacaa 5340agtattccca aaaggcccat ggttggaggt agcaggaaat catg 538475448DNAMus musculus 7gagtgcaaag tcaagatttt atggcatttt ctgtccttgg tatttttctt tcaagcaagg 60atatgttttg ataaggggct acagtttcag tgggtctctt cttccggccc caaccaacac 120ccctgactgc taactttaca gacttgtttc agtctattgg cactgacatc tgatagcctt 180gggggacctg ttttcgcctc ccctcaaggc tatccatttc acaaagtcgt aactcatcag 240cttgtgcttt actctaaaga aagtgtatct gattctaggc taggtttcca agccagagga 300ggagcaatcc ccccgccgca ggaggaatct tcgtgcagca gaacttttca ttcacagctg 360tttaaaaaga ggtcttcagc aaagttattc aaattctcta caaatgcttg acaggatgga 420cacgttgtgt ctgtgggcca atattcaatc caggtggagg aatctagagg gtatatatac 480ttgaaactga aattgtgttt gatctgcaga acctctttgc ccatgattaa atactgcttc 540cctttcacca ggttggcatt ggtacagctc atctttttaa tgaaggtgac ctccgaattc 600tcatcagcag cttcccctgt tttgtaagtg accagaagag tcgcagtgta cttgacaaaa 660acattttctt cagtggctga tgtgatcctg actttataag catatgcagt ctctggttta 720caggctttct cttttctgga gtctgcagag atggctaggt ctacttctgc ctgcagttgc 780gcacagtcag cttccacaca tgtgcaagct gctccttcac agactttctg aagcctggtg 840tcagaaatgc tataaatcat ggtgcactgc ttatctggtc tgtgatactc gtacaccgtg 900aaggtagcag gattcagaaa cccaacttgg aaaagttcaa atatccggaa ccggacacag 960aggaaatctc tggaggggat cgaattcagt tgcagaatga catggccatc tttgatctgg 1020taatcagtta gtagttgatc cactccttcc acaagagccc gtaaatcttc ctcgtttgct 1080ccgattccag tcggcagtga tatatccatt actgcatggg aggacccgga tgttgactcc 1140tccttgctgg gcttgtagct ggcacatgct attatgcgct tgaatccaga gtcactgagc 1200ctgaagtggc tggatgcttc aatatcttgg gtatcaattt tcaagtaaaa gctgcaaaat 1260tcctcagaga cactaatttt gtgaaccaca gtttttacat atactgtggc caagccactg 1320ctgtagcctg tgctgacaac aaggtcatca ttgagagata cctccactgg cctccccagg 1380aaatgcttct ctgtcacctt atacttgtgg aagtcacctt cgtgtttgta ggcgacattg 1440atgtccatat ccaaatgaat ttgttttaac aggagtgaat attctgtcag gccctcgatg 1500gcattaatcg tatcctgggt ggaataaaag ccgcctccat acctctgctc ttcagatagc 1560cacttgatga tggggttggc gtaattcata tccttcagtt tcaggctggc gagcaaagca 1620taggctgtgg tttcaaccat acctgctgtg ccgctgctgg gcacagagct gtctggacgt 1680ttgagggtat ctctccagta acggtaaatg ggcggatcac ctttaacaaa agcttccttc 1740ctcagggccg acacaattag acgaaacctc gggtgggttc tgtctcctag ggaaagagca 1800taggctacaa tggccagtgt gaaggtgctc ttggatggca gggtgttttc aagcaggaag 1860gagtcggctt tatctagcgc tgtgtggatt ttcatggtgg ggcatatgtc aactgccttt 1920ctaattccaa tcacagaaaa ggctgtaaga tacaaagttt tctcttgggc ttcagcaggc 1980aaagtaccct gtaattttat tggtagatat tgggaatttt ccttgaaaga gccgttttcc 2040agctgacact tctcaaccag ccatagcaaa gagttacaaa ttgagttttc atcctgtttt 2100acatacttgg ccacctgtcc aagcactctc agagcaaaag ctgtcagcca ggtactagcg 2160ctcgccccct tccacatgct gtaggaatag tcagcgtttc tgtaggacat gacgctcacc 2220accccttgtt ttattttttt ctccaggctc tgtcttttac tcagtgtatc aggatagaaa 2280atattccaat ggtttcctgc ttccaggtag tggaaaacat agaacaccgg agctatgctc 2340atgagctctg cctctgcact gcccttgggg aggtgggtta ggatgttgat gccttcctta 2400ctcagaaccg tggacaagaa ctcccctaca agcagtcctt tgacactcaa aatcctttca 2460actttggtct tggggaccaa atctaatggg atcctgtatg ggaattcctt tcgtctgtta 2520acaataccac gaattccctt agggtccaga atcacgccgg catagctttc cctcttgact 2580ccttctggca ctacccgtaa tgtctttact aagatgtctt tcccaaatga ggtctctagt 2640gagaagttta tggagtgaag gccaatttcc agaggaagca gggtgaaggt caccaagtga 2700ctggacgagc cctctatcct ctggaacaca catctggagg gcctggaggt gtgaaggcta 2760gcagctgagc ttcctgaagt gcagatcccc tccacagcag acattttaac acagaacttt 2820gtccctgagg tcatatagtt gtaaacagtt cctttcaatt ggatctgttc tcctcgcaca 2880acagaatatg gtatgttcat ctccaggaag acttctttga acacctttgc cttgagtgta 2940tcagcaacac atataccatt gtctgaaatg ccaatgcctt gaatttccca agtcgttagt 3000gagtcaggca gcgtgacctg cagctgtttt cttttgggaa cgcgatgaat ttcccatagc 3060cagctctctg gaaagtagct tcggatatct gccttcatca ctggtaacag ggtcttaatg 3120tggatccttc ccagttggac aggtttatgg gggctttctt ttcggatctt gttcgcaata 3180gtacagcact cgttgaaggc cctgatgcag agagggccta tggtaacccg ggccactcgc 3240tcctcacagg tttcgtagaa gttcactcgg gctccgtcat agcagcattt ctttggcaca 3300ctatgtttgt acttagcagc ttgttcttct attttctgcc ttaggagatg caggtttctc 3360tttgacctga gaatttcttt acaagagtca tcacgataat gggagtcatc tgcgtttgcg 3420ttggtgagga aggtgagccc agctagatgg aatacatctg cattgtcatg gccaccacct 3480gccccacagc ccaggtcact cttttcatcc aaagcttgaa agactctttg catggccctt 3540ttggcgtttc cctggacttt atacacagct ctgtccactg ctgatagtgc tacccatgag 3600tctgcttcag tcaccatgtc aagggacaca gtttggcctg gagaatacac atattcatct 3660ggagacagat ggacctggag ctggttgcca cacttctcct caatatttat ccagactgcg 3720tcagccacta attctgctgt ttgctcccct gtgactatgt aatagaccag gagtcgtgct 3780gaaggaacca tgttctgtgt cactggaata tttatatttt gataagttga ggagaaaagt 3840ttctctcttg tgccgtactg tacaattttg cctttggata aaatcaagta attatagtga 3900gttattttgt cgatatatgg gctcttgggg gtaaccataa tattcaggta ttctcccaca 3960agcatgggct tgtagttttc agtccaagcg atgtaaatgt aactttggct gagagacgag 4020tacgcaactg cttcgtactc tttgctggct tgattttctt cgggaagttc tgggtcatca 4080gttctgatct caaactttag caccgtcaca tttgatggga ggttcagcac aaacacagct 4140actccatcag tgtcatgagt gatgctcctc tttgtttcca agtcagatgt ctcttgattc 4200acatcgactg tttgtgccat cagagttact gggacccctc ctaccgcctg ctcgagtgaa 4260tctttaacct gtgccttgat ggaaaatgga atcccgggct tcacgaaaag aggagtagcg 4320accaaattca gtgtgtaggg agagaggaca tatttgactc cagggatttc tgcctcttct 4380gaaaatccac ctgaagattc tgtgactgtt actgcaatat aaaggtactt gttgtttaag 4440tcttctagac tgttgtagga cagctcttta actgctgttt cagaatcaaa agagatctga 4500gcaactccgt caaccaactt tgcggcttgt gtggctttgt gcatcatctg cttctcctca 4560tcttttatgt cctctctcaa tccaaaaaag gcatacactt cagcatcagg taccacttta 4620ttataaaaat atcttgcttt cacagtgatt tcaaagttct taaagttttt atagccaatg 4680aaggttcttt ctagttctat tgaaacagag aatcgtggca agacatattc tttaatttca 4740aagtatgcag ttccagttgt tgtaaaatcc ttcttatagt tagctttaat tgtccaaaca 4800ccatacttgg gattagatgg aatcttgaag tcaggaaaag agataattcc ggtgtaatca 4860ttttcttcta caatgtcaac ttctgatcct tcggggtcta tgaaagttaa gacagtctcc 4920cgtttggctg gcttcaagtc gtcacccaga gaatagactc tgatctttac tgactggtcc 4980ggcgtgtaaa caggtttgtc tgtatggatg aagagaattc cattgttata ggtaattggt 5040attttctttg attttgaaaa gtgttttgac acaacttcca gatacacgtg agagactggg 5100ctttcttctc taggaacttg attgggctgt agtgtcaaca gtgccgcgtt ttggaatttg 5160ttttccgggg acaaattaac atagcctgaa gagaaggtga cttttttgtc aggatagctt 5220tttagagaaa gagttgcatc aaatgcttca gtgtagccat ggacttgaat taccacattt 5280tcagacgagc cgacccggag gattttgggt gctgaaatga cgtaggtttg ttcctgtccc 5340caagttttgt ccaggaaaat taaaagacaa agtattcccc aaagacccat ggctggtagc 5400ggcataaacc catgggcatg gcctccctgt aaccactttc cttttaaa 544884497DNARattus norvegicus 8tacagttgag tgcaaagtca agcttttatg gcattttctg tccttggtat tttttttttc 60aagcaagacc atgttttcct aaggggctac aatttcagtg agtctcttct tccggcccca 120acaaacaccc ctggctgcta acgttacaga cttgttccag cttattggtg ctgatgtcca 180atagccttgg gggacctgcc ttcggctctc cacaaggcta ttttgtttca caaagtaact 240cttcaactta cgctttacta taaagaaaat gtatccgatt ctaggctaag tttccaagcg 300atcctggctc ctaggagcca ccaacaggag tacccgggaa ggccacgcag cagaacttcc 360tcaggcattt tcacagccat ttagaaagat gtcttcagcg aactcgtcca aattagctac 420aaacgcttgg caggatggac acgttgtgtc tgtgggccaa tattcaatcc aggtggagga 480atctagaggg tatatatact tgaaactgaa attgtgtttg atctgcagag cctctttgcc 540catgattaaa tattgctttc ctttcaccag gttggcgttg gtacagctta tctttttaat 600gaaggtgatc tcagagtcct tctcagcagc ggcttcccct gttttgtaaa tatccagaag 660cgtcgcagtg tacttgacaa aaatgttttc ttccgtggcc gacgtgatcc tgaccttata 720agcatatgca atctctggtt tacatgctgt ttctttcctg gtgtctgcag agatggccag 780gtccagttct gcctgcagtt gcccacaatc agcttcaacg catttgcatg ccgctccttc 840acagactctc tgaaggttgg tgtcagaagt gctgtaaatc atggtacact gcttatctgg 900tctgtgatac tcgtacaccg tgaacgtagc aggattcaga aacccaactt ggaaaagttc 960aaatatccgg aaccgaacac aaaggaaatc tctggaggga atcgaattca attgcagaat 1020aacatgactg tctttgatct ggtaatcagt taggagttga tctactcctt ccacaagagc 1080tcgtaaatct tcttggtttg ctccgattcc ggtcggcagc agtatatcca ttactgcatg 1140ggaggaccca gatgctgact cctccttgct gggcttgtag ctggcacagg ctattatgcg 1200cttgtgtccc gagtcactgt agctgaggta gctggaggct tcaacttctt gggtatcaat 1260tttcaagtaa aagctgcaaa attcctcagc gacactagtt ttgtgaacca cagtttttac 1320atatactgta gccaagccac tgctatagcc tgtggtgacg atgaggtcat cattgagggg 1380tacctccact ggcctcccga ggaagttctt ctctgtcact ttatactggt agaaatcccc 1440tttgtgtttg taggagacat tgatatccat atccaaatga agttgtttaa ccaggagtga 1500atactctgtc aggccctcga tggcgttaat ggtatcctgg gtggaataaa agccgcctcc 1560atacctctgc tcctcagata gccacttgat gatcgggttg acataactcg tctccttcag 1620gttcaggctg gtgagcaaag cataggccgt ggtttctacc atacctgctg tgccgctgtt 1680gggtgctgag ctgtctggac gttggagagt gtctctccag aaacggtaaa tgggcgggtc 1740tcctttaacc aaagcttccc tcttcagggc tgacacaata gaacgaaact tcgggtgggt 1800tctgtctccc agggagagag cataggccac aatggccagg gtgaaggtgc tcttggaagg 1860cagggtcctt tcaagtagga aggagtcagc tttagccagc gctgtgtaga ttttctccgt 1920ggggcatatg ccaatagcct ttctaattcc aatcacagaa aaggctgtaa gatataaagt 1980gttctcttgg gcttcagcag gcaaagtacc ctgtaatttt attggtagat attgggaatt 2040ttccttgaaa gatccgtttt ccagctgaca cttctcaatc agccataaca aggagttaca 2100gatcgagtat tggtcttgtt tcacatactt gttcacctgt ccaagcactc tcagagcaaa 2160agctgtcagc caggcactag agcttgctcc cttccacatg ctgtaggaat agtcagcgtt 2220tctgtaggac atgacgctca ccagcccttc ttttattttt ttctgcaggc tctgttttct 2280agctaacgta tcagggtgga aaatattcca atggtttcct gcttccaggt agtggaaaac 2340gtagaacacc gggactatgc tcatgagttc tgcctcggcg ctgcccttgg ggaggtgggt 2400taggatgtcg atgccttctt tactcagaac cgtggacaag aattccccta taagcagtcc 2460ttttacactc aaaatccttt tgacgttggt tttggggacc aaatctaatg gtatcctgta 2520tgggaattcc tttcgtctgt taacaatacc ataaactccc ctggggtcca gagtcacacc 2580agcatagctt tcccttttga tcccttctgg cactacccgt aatgtcttca ctaagatttc 2640tttcccaaat gaagtctcta gtgagaagtt tatggagtga aggccaattt ccagaggaag 2700caggctgaag gtcaccaagt gactggagga gccctctatt ctctggcgca cacatctgga 2760ggacctagag gtctgagggc tagcagccga gcttcctgga gtgcagattc cctccacggc 2820agacatttta acacagaaca ttgtcccaga ggtcctataa ttgtaaacgg ttcccttcaa 2880ttggatctgc tcccctcgta caacagaata tggtatgttc atctccagga agacatcttt 2940gaacaccttt gccttgagtg tgtcagcaac acatatacca ttgtctgaga tgccgatgcc 3000ttgaatttcc caggtcgtca gtgagtcagg cagtgcaacc tgcagctggt ttcttttggg 3060aacacgatga acttcccata gccagctctc tggaaagtag cttcggattt ctgccttcat 3120cactggtaac agggccttta tttggatcct tcccaacagc atgcctttgt ggtggctttc 3180ttttcggatc ttatccgcaa tagtacaaca ctcgttgaag gccctgatgc agtgtgggcc 3240tatggtcacc cgggcaactc gctgctcaca ggtttcgtat ttgttttctc gggctccatc 3300ataacagcat ttcttgggca cacggtgttt gtatttagca gcttgttctt ccactttctg 3360atgcaggagc tgcaggtctc tctttggcct gagaatttcc ttacaagagt catcgtggta 3420ttgggagtca tctgcgtttg cattggtgag gaaggtgagc ccagctagat ggaatacatc 3480tacattgtca cggccaccac ctgccccaca gcccaggtca ctcttgtcat caaaagcttg 3540gaacactctt tgcatggccc ttttggcttt tccccggact ccatacacag cgctgtccac 3600cgcagatagt gccacccatg agtctgcttc agtcaccatg tcaagggaca cagtttggcc 3660tggagaatac acgtctttat ctggagacag atggacctgg agctggttgc cacacttctc 3720ctcaatgttt atccagactg cgtcagccac caattctgct gtctgctccc ccgtgactat 3780gtaatagacc aggagccgcg ctgaaggaac catgtcctgt gtcactggga tgtttatatt 3840ttgataagat gaatagagaa gtttctcctt tgtgccatac tgtacaattt tgcctttgga 3900taaaatcaag taattatagt gagttatttt gtcaatatat ggactcttgg gggtgacgat 3960aatattcaga tattctccca caagcatggg cttgtagttt tcagtccagc caatgtaaat 4020gtaactctgg ctgagggatg agtatgtaac tgcttcatat tctttgctgg cttgattttc 4080ttcgggaagt tccggggcat cagttttgac ctcaaacttc agtgatgtca cttctgatgg 4140gaggttcacc acaaatgaag ccactccatc agcagagtgt gtgatgctcc tctttggttc 4200caagtcagat gtctcttgat tcacattgac tgtttgtgcc atcagagtta ctgggacccc 4260tcctaccaac tgctcgagtg aatccttaac ctgtaccttg atggaaaatg gaatcccagg 4320cttcaggaaa agaggggtag cgaccaaatt cagtgtatag ggagagagga cgtatttgat 4380gccaggaatt tctgcctctt ccgaaaaccc acctacaagg gcagcaagca ctgagttaat 4440gtttccggaa agatgatggc gggctgtagg acatctgtcg gtctctgtgc tatccat 4497916PRTUnknownsource/note="Description of Unknown RFGF peptide" 9Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 151011PRTUnknownsource/note="Description of Unknown RFGF analogue peptide" 10Ala Ala Leu Leu Pro Val Leu Leu Ala Ala Pro1 5 101113PRTHuman immunodeficiency virus 11Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln1 5 101216PRTDrosophila sp. 12Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10 151320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 13agcaggaaac cacggauaua 201420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 14ugguuggagg tagcaggaaa 201520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 15caaaaggccc atggtuggag 201620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 16caaagtattc ccaaaaggcc 201720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 17agauuaaaaa acaaaguauu 201820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 18uuuccccagg aagatuaaaa 201920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 19uguccccagg ttttccccag 202020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 20auguutgctc ctgtccccag 202120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 21ugaaatgaca tatgtuugcu 202220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 22auuuutggtg ctgaaaugac 202320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 23caacacggaa

tatttuuggu 202420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 24uucagatgct ccaacacgga 202520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 25aucacaatat tttcagaugc 202620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 26cauaaacttg aatcacaaua 202720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 27uucagtgtat ccataaacuu 202820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 28gcaucaaatg cttcagugua 202920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 29uagagattgt tgcatcaaau 203020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 30auaactttta atagagauug 203120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 31uuuuuatcag gataacuuuu 203220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 32aguaactaaa tttttuauca 203320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 33auggcctgag gagtaacuaa 203420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 34gauaaatgaa catggccuga 203520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 35uauuctctga ggataaauga 203620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 36guuuuggaat ttattcucug 203720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 37aagautgcag agtttuggaa 203820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 38guuguattgt taagauugca 203920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 39caauugtttt ggttguauug 204020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 40ugucctccag gcaatuguuu 204120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 41aaacugggtt ttgtccucca 204220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 42auacacataa gaaacugggu 204320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 43acaacttcca aatacacaua 204420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 44aaugctttga tacaacuucc 204520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 45ugauuttgaa aaatgcuuug 204620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 46ggcautcttt ttgatuuuga 204720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 47cauaggttat tggcauucuu 204820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 48aaauccattg tcatagguua 204920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 49ugaaugaaga gaaatccauu 205020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 50guuugtctgt atgaaugaag 205120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 51aguauaaaca ggtttgucug 205220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 52gacuggtctg gagtauaaac 205320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 53uaacutttac tgactggucu 205420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 54cgaauaaact ctaacuuuua 205520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 55ucgucattca acgaauaaac 205620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 56cuggcttcaa gtcgtcauuc 205720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 57uucucttttg gctggcuuca 205820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 58guuaagacag tttctcuuuu 205920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 59gaucuatgaa agttaagaca 206020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 60ugauccttca ggatcuauga 206120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 61augucaactt ctgatccuuc 206220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 62uuucutctac catgtcaacu 206320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 63aauaugatca atttcuucua 206420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 64gagauaattc caataugauc 206520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 65agucaggaaa agagauaauu 206620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 66cggaatcttg aagtcaggaa 206720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 67cuaggattag acggaaucuu 206820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 68acauaccata tctaggauua 206920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 69cuugatcgtc cacataccau 207020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 70uuauatttag ccttgaucgu 207120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 71aaaagtcctc tttatauuua 207220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 72uccagttgtt gaaaaguccu 207320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 73aaauatgcgg ttccaguugu 207420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 74cuuuaacttc aaaataugcg 207520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 75caagacatat tctttaacuu 207620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 76gaaaaatgtg gcaagacaua 207720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 77cgauugagac agaaaaaugu 207820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 78auauuctggc tcgatugaga 207920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 79ccaaugaaat tatatucugg 208020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 80aguucttgta accaaugaaa 208120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 81aaaautctta aagttcuugu 208220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 82auaguaattt caaaauucuu 208320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 83aucuugcttt tataguaauu 208420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 84auuauaaaaa tatctugcuu 208520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 85gugactactt tattauaaaa 208620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of

Combined DNA/RNA Molecule Synthetic oligonucleotide" 86cgucagcctc agtgacuacu 208720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 87ugugatataa acgtcagccu 208820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 88cuuautccaa atgtgauaua 208920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 89uuaagtcttc tcttauucca 209020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 90uugaucatct tttaagucuu 209120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 91aucauttctt tttgaucauc 209220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 92uugcugtttg catcauuucu 209320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 93ugugutttgc attgcuguuu 209420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 94uuuaucaaca ttgtguuuug 209520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 95gagcaattcc atttaucaac 209620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 96aaaugtgact tgagcaauuc 209720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 97guuucagaat caaatgugac 209820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 98cuuugactgc tgtttcagaa 209920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 99guaugacagt tctttgacug 2010020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 100ucuaaactgt agtatgacag 2010120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 101uguuuaaatc ttctaaacug 2010220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 102aagguacttg ttgttuaaau 2010320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 103acagcaatat aaagguacuu 2010420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 104cuaugactgt tacagcaaua 2010520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 105accugtagac tctatgacug 2010620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 106ucagaaaatc cacctguaga 2010720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 107uuucugcctc ttcagaaaau 2010820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 108gaugccaggt atttcugccu 2010920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 109aggacatatt tgatgccagg 2011020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 110uguagggaga gaggacauau 2011120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 111caaautcagt ttgtagggag 2011220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 112ggaguagcaa ccaaauucag 2011320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 113ucaggaaaag aggaguagca 2011420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 114aaucccaggc ttcaggaaaa 2011520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 115augggatatg gaatcccagg 2011620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 116ccugcacctt gatgggauau 2011720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 117cgaaucttta acctgcaccu 2011820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 118aacuggtcaa gcgaaucuuu 2011920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 119cuccucctac caactgguca 2012020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 120uguuactggg actccuccua 2012120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 121ugugcattca gtgttacugg 2012220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 122caucaattgt ttgtgcauuc 2012320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 123cucuuggttt acatcaauug 2012420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 124aagucagatg tctctugguu 2012520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 125ugcuuggatc caagtcagau 2012620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 126uguuacactt ttgctuggau 2012720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 127ucaucaacac gtgttacacu 2012820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 128aagcuactcc atcatcaaca 2012920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 129aagcacaaag gaagcuacuc 2013020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 130gaugggagat taagcacaaa 2013120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 131ccgucactcc agatgggaga 2013220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 132aaacuccagc accgtcacuc 2013320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 133guuuugacat taaacuccag 2013420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 134cuggagcatc agtttugaca 2013520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 135uucuggaaga tctggagcau 2013620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 136gccugatttt cttctggaag 2013720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 137aaccutccct ggcctgauuu 2013820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 138uauugctcgg taaccuuccc 2013920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 139gaugagtatg ctattgcucg 2014020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 140uuuggctgag agatgaguau 2014120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 141auaaaggtaa ctttggcuga 2014220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 142guccaatcaa tataaaggua 2014320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 143uauggttatc agtccaauca 2014420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 144uagcaaagcc ttatgguuau 2014520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 145uguuctccca ctagcaaagc 2014620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 146uaauattcag atgttcuccc 2014720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 147gggggtaaca ataatauuca 2014820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 148uaugggcttt tggggguaac 2014920DNAArtificial Sequencesource/note="Description of Artificial

Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 149uuuugtcaat atatgggcuu 2015020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 150auagugagtt attttgucaa 2015120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 151aucaagtaat tatagugagu 2015220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 152ccuuggataa aatcaaguaa 2015320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 153gauaattttg cccttggaua 2015420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 154gugccaaagt ggataauuuu 2015520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 155auuuctccct cgtgccaaag 2015620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 156ugcauctgaa aatttcuccc 2015720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 157cuuugataag atgcaucuga 2015820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 158gaaugtttat actttgauaa 2015920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 159cugugttact ggaatguuua 2016020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 160ggaaccatgt tctgtguuac 2016120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 161gucgggatga aggaaccaug 2016220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 162auagaccaga agtcgggaug 2016320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 163gugacgatgt aatagaccag 2016420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 164ucugutctcc tgtgacgaug 2016520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 165uaauuctgct gtctguucuc 2016620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 166gaaucagaca ctaatucugc 2016720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 167uuaaccagac tgaatcagac 2016820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 168uucuucaata tttaaccaga 2016920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 169uugccacatt tttctucaau 2017020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 170ccuggagctg gttgccacau 2017120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 171agacagatga acctggagcu 2017220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 172ucugcatcag gagacagaug 2017320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 173gagaatatgc atctgcauca 2017420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 174aguuuggcct ggagaauaug 2017520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 175uuaagagaca cagttuggcc 2017620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 176caguugccat attaagagac 2017720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 177ggaauccatt ccagtugcca 2017820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 178aaugccaccc aggaauccau 2017920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 179ccacugctgc taatgccacc 2018020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 180cacagcactg tccacugcug 2018120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 181uggactccat acacagcacu 2018220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 182uggcucctct ttggacucca 2018320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 183caagggcttt ttggcuccuc 2018420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 184aauactcttt ccaagggcuu 2018520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 185cuaagaattg aaatacucuu 2018620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 186aucactcttc tctaagaauu 2018720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 187ccacagccca gatcacucuu 2018820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 188caccacctgc cccacagccc 2018920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 189auugutgagg ccaccaccug 2019020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 190aacacattgg cattguugag 2019120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 191cagcuaggtg gaacacauug 2019220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 192gaaggtaagt ccagcuaggu 2019320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 193gcauuagtga ggaagguaag 2019420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 194caucugcatt tgcatuagug 2019520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 195uucuugggag tcatcugcau 2019620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 196gguucatcat tttctuggga 2019720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 197uuucuttaca aggttcauca 2019820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 198uggcctgaga atttcuuuac 2019920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 199agcgutcttc ttggccugag 2020020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 200ucuucttttg cagcguucuu 2020120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 201uauuucttct atcttcuuuu 2020220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 202uauuuagcag ctattucuuc 2020320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 203cugaatgttt atattuagca 2020420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 204uuucutcact actgaauguu 2020520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 205ucguaacaac atttcuucac 2020620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 206cgcaggctcc atcgtaacaa 2020720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 207aucautatta acgcaggcuc 2020820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 208ucacaggttt catcauuauu 2020920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 209cagcucgctg ctcacagguu 2021020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 210acuaatccgt gcagcucgcu 2021120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 211cuuggcccta aactaauccg

2021220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 212cuuugatgca tcttggcccu 2021320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 213uucagtgaaa gctttgaugc 2021420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 214acgacacaac attcagugaa 2021520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 215gcuggcttgc gacgacacaa 2021620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 216auuagcacgg agctggcuug 2021720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 217uuaugagaga tattagcacg 2021820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 218auugcatgtc tttatgagag 2021920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 219uagccttccc aattgcaugu 2022020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 220gucuucatgt gtagccuucc 2022120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 221cugguaacag ggtctucaug 2022220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 222uggcutgctt actgguaaca 2022320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 223cuccgaattt ctggcuugcu 2022420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 224cuggaaaata actccgaauu 2022520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 225caaccagctt tctggaaaau 2022620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 226ugaacttccc acaaccagcu 2022720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 227ugggaacaag atgaacuucc 2022820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 228cuguuttctt ctgggaacaa 2022920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 229gcaaactgca actgtuuucu 2023020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 230aaucaggtag ggcaaacugc 2023120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 231gguggttaga gaatcaggua 2023220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 232ugaauttccc aggtgguuag 2023320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 233ugccaacgcc ttgaauuucc 2023420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 234aguguttgaa atgccaacgc 2023520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 235acacatatac cagtguuuga 2023620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 236caguatcagc aacacauaua 2023720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 237cuuugccttg acagtaucag 2023820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 238ucuuugaaca cctttgccuu 2023920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 239ccaggaagac atcttugaac 2024020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 240uauautcatt tccaggaaga 2024120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 241acagaatatg gtatauucau 2024220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 242cuccucgtac aacagaauau 2024320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 243uuggatctgt tctccucgua 2024420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 244guucctttca attggaucug 2024520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 245aguugtaaac agttccuuuc 2024620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 246agaagtccta tagttguaaa 2024720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 247aacugcatcc cagaaguccu 2024820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 248uuuuaacaca gaactgcauc 2024920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 249cacagcagac attttaacac 2025020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 250cagautccct ccacagcaga 2025120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 251uuuccgaagt gcagauuccc 2025220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 252aaugactggg ctttccgaag 2025320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 253cccugatgat caatgacugg 2025420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 254aggactttgt gccctgauga 2025520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 255cacacatttg gaggacuuug 2025620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 256acuuuctggc gcacacauuu 2025720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 257aggagccctc tacttucugg 2025820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 258caagugactg gaggagcccu 2025920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 259gugaatgtca ccaagugacu 2026020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 260gaggaagcac agtgaauguc 2026120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 261gccaatttcc agaggaagca 2026220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 262auguugtgaa ggccaauuuc 2026320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 263gugaaaaatt gatgtuguga 2026420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 264ccaagtctcc agtgaaaaau 2026520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 265ucuuutccaa accaagucuc 2026620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 266uuacuaagat ttcttuucca 2026720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 267ucguaatgtt tttacuaaga 2026820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 268ucuggcacca ctcgtaaugu 2026920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 269uuuugacacc ttctggcacc 2027020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 270auagctttcc cttttgacac 2027120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 271guaacaccag aatagcuuuc 2027220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 272uaggatccaa agtaacacca 2027320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 273auaaataccc ctaggaucca 2027420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 274cuaauggtac

cataaauacc 2027520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 275ccuuucgtct gctaauggua 2027620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 276guaugggaac tccttucguc 2027720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 277aagggtatcc tgtatgggaa 2027820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 278ggaccaaatc taaggguauc 2027920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 279uucugttttg gggaccaaau 2028020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 280auccutttga tttctguuuu 2028120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 281uuacactcaa aatccuuuug 2028220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 282aagcagtcct tttacacuca 2028320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 283aucucaccta caagcagucc 2028420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 284cugcagacaa gatctcaccu 2028520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 285cugacttaga actgcagaca 2028620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 286uugaugcctt cctgacuuag 2028720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 287ggguuaggat attgaugccu 2028820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 288uuuggggagg tgggtuagga 2028920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 289ucugcactcc ctttggggag 2029020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 290ucagctccgc ctctgcacuc 2029120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 291gacaacgctc atcagcuccg 2029220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 292uagaatactg ggacaacgcu 2029320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 293agugaaaaac atagaauacu 2029420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 294uguuuccagg tagtgaaaaa 2029520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 295caaugatttc ctgttuccag 2029620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 296gaaaaatgtt ccaatgauuu 2029720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 297ugggucagaa tgaaaaaugu 2029820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 298uuuucaatta atgggucaga 2029920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 299ucaguttctg cttttcaauu 2030020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 300uaauuttttc ttcaguuucu 2030120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 301aucccttctt ttaatuuuuu 2030220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 302uaaugctcaa catcccuucu 2030320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 303ucuguaggac ataatgcuca 2030420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 304uagucagcat ttctguagga 2030520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 305cacugtaaga gtagtcagca 2030620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 306acccutccac acactguaag 2030720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 307cuagcacttc cacccuucca 2030820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 308uuaaccaagt gctagcacuu 2030920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 309agcaaaagct gttaaccaag 2031020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 310aguactctta aagcaaaagc 2031120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 311uuacutgtcc aagtacucuu 2031220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 312uacguattta tttacuuguc 2031320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 313ugguuctgct ctacguauuu 2031420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 314aaauugaatt ttggtucugc 2031520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 315uaaagaatta caaatugaau 2031620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 316acuagccaca ataaagaauu 2031720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 317gauaattctc aactagccac 2031820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 318auuauctaat tgataauucu 2031920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 319uugaaagatc cattaucuaa 2032020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 320gugaattttc cttgaaagau 2032120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 321ugguugatac tgtgaauuuu 2032220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 322uguaatttta ttggtugaua 2032320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 323gcaaggtacc ctgtaauuuu 2032420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 324ggcuucaaca ggcaagguac 2032520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 325cuguuctctc gggctucaac 2032620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 326uaagatataa gctgtucucu 2032720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 327aguaaaggct gtaagauaua 2032820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 328auuccaatca cagtaaaggc 2032920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 329aagcctttct aattccaauc 2033020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 330gcauatatcg aaagccuuuc 2033120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 331uucaccaggg ggcatauauc 2033220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 332cugugtcgat tttcaccagg 2033320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 333uuuaattaga gctgtgucga 2033420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 334aaguugtcag ctttaauuag 2033520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 335uuucaagcag aaagtuguca 2033620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 336uggcagtgta ttttcaagca 2033720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of

Combined DNA/RNA Molecule Synthetic oligonucleotide" 337gugcuctggg ctggcagugu 2033820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 338ccaaugtaaa ggtgcucugg 2033920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 339cgcagaaatg gccaauguaa 2034020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 340gaaagagcat acgcagaaau 2034120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 341uaucucccag ggaaagagca 2034220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 342ugggugagtt ttatcuccca 2034320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 343gaacgaaact gtgggugagu 2034420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 344cugaaacaat tgaacgaaac 2034520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 345ucucutcaaa gctgaaacaa 2034620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 346accaaagctt ctctcuucaa 2034720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 347gauuaccttt aaccaaagcu 2034820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 348auaaatgggt ggattaccuu 2034920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 349uuccaaaaac gataaauggg 2035020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 350gaagattgtc tttccaaaaa 2035120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 351gucuutatgc tgaagauugu 2035220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 352gguacagagc tgtctuuaug 2035320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 353uaccagtgtt aggtacagag 2035420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 354cauacgtgcc gtaccagugu 2035520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 355guuguttcta ccatacgugc 2035620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 356aagcataggc agttguuucu 2035720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 357acuggtgagt aaagcauagg 2035820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 358uucaagttca gactggugag 2035920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 359aauuuatatc tttcaaguuc 2036020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 360ugggutaaca taattuauau 2036120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 361cauuugatga ctggguuaac 2036220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 362cuucugatag ccattugaug 2036320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 363auacctctgc tcttcugaua 2036420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 364aagccacctc cataccucug 2036520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 365ggguugaata aaagccaccu 2036620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 366gauugtgtcc tgggtugaau 2036720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 367ucaauggcat tgattguguc 2036820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 368ccgucaggcc ctcaauggca 2036920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 369gagugaatat tccgtcaggc 2037020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 370uguuuaacca ggagtgaaua 2037120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 371ucaagcggag ttgttuaacc 2037220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 372gauguccata ctcaagcgga 2037320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 373uaagaaacat cgatguccau 2037420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 374cuuuatgctt gtaagaaaca 2037520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 375auguaaggca cctttaugcu 2037620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 376auuuuataat tatgtaaggc 2037720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 377ucuugtctgt catttuauaa 2037820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 378cccaaggaaa ttcttgucug 2037920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 379ucuactggcc tcccaaggaa 2038020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 380ugagaagcac ctctacuggc 2038120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 381gaggucatca ttgagaagca 2038220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 382guacugacaa tgaggucauc 2038320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 383ugccaaatcc tgtacugaca 2038420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 384agccaagcca ctgccaaauc 2038520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 385acaugtactg tagccaagcc 2038620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 386cuacagttgt tacatguacu 2038720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 387gguuutgtga actacaguug 2038820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 388ucagaggtac tggttuugug 2038920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 389ugcaaacttc ctcagaggua 2039020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 390caaauaaaag ctgcaaacuu 2039120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 391guaucgattt tcaaauaaaa 2039220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 392caauatcctg agtatcgauu 2039320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 393gugggatgct tcaatauccu 2039420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 394uagcctctgt agtgggaugc 2039520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 395cagagtttcc gtagccucug 2039620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 396gcguutgtaa tcagaguuuc 2039720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 397caugctacta tgcgtuugua 2039820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 398uguagctggc acatgcuacu 2039920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 399ccugctgggc ttgtagcugg 2040020DNAArtificial Sequencesource/note="Description of Artificial

Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 400gaugattctt ccctgcuggg 2040120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 401aggauccaga tgatgauucu 2040220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 402caccgcatga gaggauccag 2040320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 403gagaugtcca tcaccgcaug 2040420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 404caguaggcaa ggagaugucc 2040520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 405ugcactgatt ccagtaggca 2040620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 406ucuucttcat ttgcacugau 2040720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 407gggcutttaa gtcttcuuca 2040820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 408cccuuccaca agggcuuuua 2040920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 409aguugatcca cccctuccac 2041020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 410aaucagtgaa tagttgaucc 2041120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 411uuugatttgg taatcaguga 2041220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 412acaugtccat ctttgauuug 2041320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 413guugcagaat aacatgucca 2041420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 414aaucgaattc agttgcagaa 2041520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 415ucacuggagg gaatcgaauu 2041620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 416cacaaaggaa atcacuggag 2041720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 417ccggaatcgt acacaaagga 2041820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 418aguucaaata tccggaaucg 2041920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 419caacutcaaa gagttcaaau 2042020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 420acugagaaac ccaacuucaa 2042120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 421aaaguggcag gactgagaaa 2042220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 422cguacactgt gaaaguggca 2042320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 423ucuguggtat tcgtacacug 2042420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 424uguuuatctg gtctguggua 2042520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 425acauggtaca ctgttuaucu 2042620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 426agugctataa aacatgguac 2042720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 427uugauattgg aagtgcuaua 2042820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 428cuuuctgaat tttgauauug 2042920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 429uccuucacag actttcugaa 2043020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 430uugcacgcgg ctcctucaca 2043120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 431cuucuacaca cttgcacgcg 2043220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 432cccacaatca gcttcuacac 2043320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 433uccugcattt gcccacaauc 2043420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 434gauccaattc ttcctgcauu 2043520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 435agagattgtc agatccaauu 2043620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 436cuuguctctg cagagauugu 2043720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 437cuguutgttt tcttgucucu 2043820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 438ugguutacat gctgtuuguu 2043920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 439uaugcaatct ctggtuuaca 2044020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 440cuuuataagc atatgcaauc 2044120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 441ugugatgcta actttauaag 2044220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 442acagugatgg atgtgaugcu 2044320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 443aaacattttc tacagugaug 2044420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 444guacutgaca aaaacauuuu 2044520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 445agggutgcct tgtacuugac 2044620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 446agauatccag aaggguugcc 2044720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 447cccagttttg tagataucca 2044820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 448gcaacagctt ccccaguuuu 2044920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 449agucuttctc agcaacagcu 2045020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 450gguaatctca gagtcuuucu 2045120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 451uuuuuaatga aggtaaucuc 2045220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 452uacaggttac cttttuaaug 2045320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 453cucagcgtta gtacagguua 2045420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 454ccuuutacca gctcagcguu 2045520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 455aguactgtct tccttuuacc 2045620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 456acccataatt aagtacuguc 2045720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 457agggcttctt tacccauaau 2045820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 458auuuuatctg gagggcuucu 2045920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 459acugaaattg tatttuaucu 2046020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 460auguacctga aactgaaauu 2046120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 461cuaaagggta gatgtaccug 2046220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 462ggucaaggaa tctaaagggu

2046320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 463uauucaatcc aggtcaagga 2046420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 464cucuaggcca gtattcaauc 2046520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 465acaugttgtg tctctaggcc 2046620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 466ugacacgatg aacatguugu 2046720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 467cuaaaaatgc ttgacacgau 2046820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 468aucuaaatta gctaaaaaug 2046920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 469ucggcaaatt catctaaauu 2047020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 470aaaagatatc ttcggcaaau 2047120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 471gcauccattt aaaaagauau 2047220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 472caggaatttt agcatccauu 2047320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 473cagcugaact tcaggaauuu 2047420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 474caaactgtat gcagcugaac 2047520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 475guccataagt gcaaacugua 2047620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 476caacaacagg agtccauaag 2047720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 477aaaacgaact tcaacaacag 2047820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 478aagaaaacaa aaaaacgaac 2047920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 479uuuaaaaaaa gaagaaaaca 2048020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 480agcuatgaat gtttaaaaaa 2048120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 481caaauaagac cagctaugaa 2048220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 482agugagcttt acaaauaaga 2048320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 483auucuaagta aagtgagcuu 2048420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 484aagugccact aattcuaagu 2048520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 485cuaauaaaag caagtgccac 2048620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 486gaaaucattc tctaauaaaa 2048720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 487uuacagcatt tgaaaucauu 2048820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 488auuucagaaa gttacagcau 2048920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 489aaggccatgt tatttcagaa 2049020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 490ucaugccctc caaggccaug 2049120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 491aguauctgtc ttcatgcccu 2049220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 492aaccutggag gagtaucugu 2049320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 493cggugtccaa taaccuugga 2049420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 494uuuautgttt ccggtgucca 2049520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 495aggugttcca atttauuguu 2049620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 496uagguttgag gaggtguucc 2049720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 497uuccugagtg gtagguuuga 2049820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 498cccagcaaac attccugagu 2049920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 499guucuttcgg ccccagcaaa 2050020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 500uucaatggac tgttcuuucg 2050120DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 501uaauactccc tttcaaugga 2050220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 502ccaugttttt gtaatacucc 2050320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 503ucaagcaaag gccatguuuu 2050420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 504gguautttct ttcaagcaaa 2050520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 505uccugttcct tggtauuuuc 2050620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 506aaugatcagt ttcctguucc 2050720DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 507acucaggctt taatgaucag 2050820DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic oligonucleotide"source/note="Description of Combined DNA/RNA Molecule Synthetic oligonucleotide" 508uuugaaagca aactcaggcu 20

Claims

1. A single-stranded antisense polynucleotide agent for inhibiting expression of complement component C5, wherein the agent is 10-40 contiguous nucleotides in length, wherein at least one of the contiguous nucleotides is a modified nucleotide, and wherein the nucleotide sequence of the agent is about at least 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 1, wherein substantially all of the nucleotides of the single-stranded antisense polynucleotide agent are modified nucleotides.

2. The agent of claim 1, wherein the equivalent region is one of the target regions of SEQ ID NO:1 provided in Table 3.

3. (canceled)

4. (canceled)

5. The agent of claim 1, which is 10 to 30 nucleotides in length; 18 to 30 nucleotides in length; 10 to 24 nucleotides in length; 18 to 24 nucleotides in length; or 20 nucleotides in length.

6. The agent of claim 1, wherein the modified nucleotide comprises a modified sugar moiety selected from the group consisting of: a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety, a 5-methylcytosine, and a modified internucleoside linkage.

7. (canceled)

8. (canceled)

9. The agent of claim 1, comprising a plurality of 2'-deoxynucleotides forming a gap segment flanked on each side by at least one nucleotide having a modified sugar moiety forming a 5' wing segment and a 3' wing segment.

10. (canceled)

11. (canceled)

12. The agent of claim 9, wherein the gap segment is 5 to 14 nucleotides in length.

13. The agent of claim 1, wherein the agent further comprises a ligand.

14. The agent of claim 13, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

15. A pharmaceutical composition for inhibiting expression of a complement component C5 gene comprising the agent of claim 1.

16. A pharmaceutical composition comprising the agent of claim 1, and a lipid formulation.

17. A method of inhibiting complement component C5 expression in a cell, the method comprising: (a) contacting the cell with the agent of claim 1 or a pharmaceutical composition of claim 15 or 16; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain antisense inhibition of a complement component C5 gene, thereby inhibiting expression of the complement component C5 gene in the cell.

18. The method of claim 17, wherein the cell is within a subject.

19. The method of claim 18, wherein the subject is a human.

20.-25. (canceled)

26. The agent of claim 9, wherein the modified sugar moiety is selected from the group consisting of a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, and a bicyclic sugar moiety.

27. The agent of claim 9, wherein the 5'-wing segment is 1 to 6 nucleotides in length.

28. The agent of claim 9, wherein the 3'-wing segment is 1 to 6 nucleotides in length.

29. The agent of claim 13, wherein the antisense polynucleotide agent is conjugated to the ligand at the 3'-terminus.

30. The method of claim 17, wherein the complement component C5 expression is inhibited by at least 50%.