Patent application title: POLYNUCLEOTIDE AGENTS TARGETING SERPINC1 (AT3) AND METHODS OF USE THEREOF
Inventors:
IPC8 Class: AC12N15113FI
USPC Class:
1 1
Class name:
Publication date: 2021-04-29
Patent application number: 20210123049
Abstract:
The invention relates to polynucleotide agents targeting the Serpinc1
(AT3) gene, and methods of using such polynucleotide agents to inhibit
expression of Serpinc1 and to treat subjects having a bleeding disorder,
e.g., a hemophilia.Claims:
1. An antisense polynucleotide agent for inhibiting expression of
Serpinc1 (AT3), wherein the agent comprises 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.
2. (canceled)
3. An antisense polynucleotide agent for inhibiting expression of Serpinc1, 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 Tables 3 and 4.
4. The agent of claim 1, wherein substantially all of the nucleotides of the antisense polynucleotide agent are modified nucleotides; or all of the nucleotides of the antisense polynucleotide agent are modified nucleotides.
5. (canceled)
6. The agent of claim 1, which is 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.
7.-11. (canceled)
12. 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, and a bicyclic sugar moiety; a 5-methylcytosine; or a modified internucleoside linkage.
13.-31. (canceled)
32. An antisense polynucleotide agent for inhibiting Serpinc1 (AT3), 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.
33.-37. (canceled)
38. The agent of any one of claim 1 or 32, wherein the agent further comprises a ligand.
39. (canceled)
40. The agent of claim 38, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.
41. (canceled)
42. A pharmaceutical composition for inhibiting expression of a Serpinc1 (AT3) gene comprising the agent of claim 1 or 32.
43.-50. (canceled)
51. A method of inhibiting Serpinc1 (AT3) expression in a cell, the method comprising: (a) contacting the cell with the agent of claim 1 or 32; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain antisense inhibition of a Serpinc1 gene, thereby inhibiting expression of Serpinc1 gene in the cell.
52-54. (canceled)
55. A method of treating a subject having a disease or disorder that would benefit from reduction in Serpinc1 (AT3) expression, the method comprising administering to the subject a therapeutically effective amount of the agent of claim 1 or 32.
56. A method of preventing at least one symptom in a subject having a disease or disorder that would benefit from reduction in Serpinc1 (AT3) expression, the method comprising administering to the subject a prophylactically effective amount of the agent of claim 1 or 32, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in Serpinc1 expression.
57. (canceled)
58. The method of claim 55 or 56, wherein the disorder is a bleeding disorder.
59-74. (canceled)
Description:
RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No. 16/409,923, filed on May 13, 2019, which is a continuation application of U.S. patent application Ser. No. 15/499,981, filed on Apr. 28, 2017, now U.S. Pat. No. 10,344,278, issued on Jul. 9, 2019, which is a 35 .sctn. U.S.C. 111(a) continuation application which claims the benefit of priority to PCT/US2015/057717, filed on Oct. 28, 2015, which claims priority to U.S. Provisional Application No. 62/072,686, filed on Oct. 30, 2014. The entire contents of each of the foregoing applications are incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant 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 Jul. 10, 2020, is named 121301_02704_SL and is 299,773 bytes in size.
BACKGROUND OF THE INVENTION
[0003] Serpinc1 or antithrombin III (AT3) is a member of the serine proteinase inhibitor (serpin) superfamily. Serpinc1 is a plasma protease inhibitor that inhibits thrombin as well as other activated serine proteases of the coagulation system, such as factors X, IX, XI, XII and VII and, thus, regulates the blood coagulation cascade (see, e.g., FIG. 1). The anticoagulant activity of Serpinc1 is enhanced by the presence of heparin and other related glycosaminoglycans which catalyze the formation of thrombin:antithrombin (TAT) complexes.
[0004] Bleeding disorders, either inherited or acquired, are conditions in which there is inadequate blood clotting. For example, hemophilia is a group of hereditary genetic bleeding disorders that impair the body's ability to control blood clotting or coagulation. Hemophilia A is a recessive X-linked genetic disorder involving a lack of functional clotting Factor VIII and represents 80% of hemophilia cases. Hemophilia B is a recessive X-linked genetic disorder involving a lack of functional clotting Factor IX. It comprises approximately 20% of haemophilia cases. Hemophilia C is an autosomal genetic disorder involving a lack of functional clotting Factor XI. Hemophilia C is not completely recessive, as heterozygous individuals also show increased bleeding.
[0005] Although, at present there is no cure for hemophilia, it can be controlled with regular infusions of the deficient clotting factor, e.g., factor VIII in hemophilia A. However, some hemophiliacs develop antibodies (inhibitors) against the replacement factors given to them and, thus, become refractory to replacement coagulation factor. Accordingly, bleeds in such subjects cannot be properly controlled.
[0006] The development of high-titer inhibitors to, for example, factor VIII and other coagulation factors, is the most serious complication of hemophilia therapy and makes treatment of bleeds very challenging. Currently, the only strategies to stop bleeds in such subjects are the use of "bypassing agents" such as factor eight inhibitor bypass activity (FEIBA) and activated recombinant factor VII (rFVIIa), plasmapheresis, continuous factor replacement, and immune tolerance therapy, none of which are completely effective. Accordingly, there is a need in the art for alternative treatments for subjects having a bleeding disorder, such as hemophilia.
SUMMARY OF THE INVENTION
[0007] The present invention provides polynucleotide agents and compositions comprising such agents which target nucleic acids encoding Serpinc1 or antithrombin III (AT3) and interfere with the normal function of the targeted nucleic acid. The Serpinc1 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 Serpinc1 mRNA, e.g., a bleeding disorder, such a hemophila, using the polynucleotide agents and compositions of the invention.
[0008] Accordingly, in one aspect, the present invention provides antisense polynucleotide agents for inhibiting expression of Serpinc1 (AT3). 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.
[0009] In one embodiment, the equivalent region is one of the target regions of SEQ ID NO:1 provided in Tables 3 and 4.
[0010] In another aspect, the invention provides an antisense polynucleotide agent for inhibiting expression of Serpinc1, 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 Tables 3 and 4.
[0011] In one embodiment, substantially all of the nucleotides of the antisense polynucleotide agent are modified nucleotides.
[0012] In another embodiment, all of the nucleotides of the antisense polynucleotide agent are modified nucleotides.
[0013] The 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.
[0014] In some embodiments, 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.
[0015] In one embodiment, the bicyclic sugar moiety has a (--CRH--)n group forming a bridge between the 2' oxygen and the 4' carbon atoms of the sugar ring, wherein n is 1 or 2 and wherein R is H, CH.sub.3 or CH.sub.3OCH.sub.3.
[0016] In a further embodiment, n is 1 and R is CH.sub.3.
[0017] In one embodiment, the modified nucleotide is a 5-methylcytosine.
[0018] In another embodiment, the modified nucleotide includes a modified internucleoside linkage.
[0019] In one embodiment, the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
[0020] In one embodiment, an agent of the invention comprises one 2'-deoxynucleotide. In another embodiment, an agent of the invention comprises one 2'-deoxynucleotide flanked on each side by at least one nucleotide having a modified sugar moiety.
[0021] In one embodiment, an agent of the invention comprises a plurality, e.g., more than 1, e.g., 2, 3, 4, 5, 6, or 7, 2'-deoxynucleotides. In one embodiment, an agent of the invention comprises a plurality, e.g., more than 1, 2'-deoxynucleotides flanked on each side by at least one nucleotide having a modified sugar moiety.
[0022] 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.
[0023] In one embodiment, the agent including about 4 to about 50 contiguous nucleotides includes a plurality of 2'-deoxynucleotides flanked on each side by at least one nucleotide having a modified sugar moiety.
[0024] 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.
[0025] In one embodiment, the 5'-wing segment is 1 to 10 nucleotides in length.
[0026] In one embodiment, the 3'-wing segment is 1 to 10 nucleotides in length.
[0027] In one embodiment, the gap segment is 5 to 14 nucleotides in length.
[0028] In one embodiment, the 5'-wing segment is 4 nucleotides in length.
[0029] In one embodiment, the 3'-wing segment is 4 nucleotides in length.
[0030] In one embodiment, the 5'-wing segment is 5 nucleotides in length.
[0031] In one embodiment, the 3'-wing segment is 5 nucleotides in length.
[0032] In one embodiment, the 5'-wing segment is 6 nucleotides in length.
[0033] In one embodiment, the 3'-wing segment is 6 nucleotides in length.
[0034] In one embodiment, the 5'-wing segment is 7 nucleotides in length.
[0035] In one embodiment, the 3'-wing segment is 7 nucleotides in length.
[0036] In one embodiment, the gap segment is 10 nucleotides in length.
[0037] In another aspect the invention provides an antisense polynucleotide agent for inhibiting Serpinc1 (AT3), 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.
[0038] In one embodiment, the gap segment is ten 2'-deoxynucleotides in length and each of the wing segments is five nucleotides in length.
[0039] In one embodiment, the gap segment is eleven 2'-deoxynucleotides in length and each of the wing segments is five nucleotides in length.
[0040] In one embodiment, the gap segment is ten 2'-deoxynucleotides in length and each of the wing segments is six nucleotides in length.
[0041] In one embodiment, the gap segment is eleven 2'-deoxynucleotides in length and each of the wing segments is six nucleotides in length.
[0042] In one embodiment, the gap segment is ten 2'-deoxynucleotides in length and each of the wing segments is seven nucleotides in length.
[0043] In one embodiment, the gap segment is eleven 2'-deoxynucleotides in length and each of the wing segments is seven nucleotides in length.
[0044] In one embodiment, the gap segment is ten 2'-deoxynucleotides in length and each of the wing segments is four nucleotides in length.
[0045] In one embodiment, the gap segment is eleven 2'-deoxynucleotides in length and each of the wing segments is four nucleotides in length.
[0046] 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.
[0047] In one embodiment, the polynucleotide agent for inhibiting expression of Serpinc1 (AT3) further includes a ligand.
[0048] In one embodiment, the antisense polynucleotide agent is conjugated to the ligand at the 3'-terminus.
[0049] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.
[0050] For example, the ligand is
##STR00001##
[0051] Further, in another aspect, the invention provides a pharmaceutical composition for inhibiting expression of a Serpinc1 (AT3) gene including an antisense polynucleotide for inhibiting Serpinc1 expression as described herein.
[0052] In one embodiment, the agent is present in an unbuffered solution.
[0053] In one embodiment, the unbuffered solution is saline or water.
[0054] In another embodiment, the agent is present in a buffer solution.
[0055] In one embodiment, the buffer solution includes acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.
[0056] In one embodiment, the buffer solution is phosphate buffered saline (PBS).
[0057] In one embodiment, the pharmaceutical composition includes a lipid formulation.
[0058] In one embodiment, the lipid formulation includes a LNP.
[0059] In another embodiment, the lipid formulation includes a MC3.
[0060] In another aspect, the invention provides a method of inhibiting Serpinc1 (AT3) expression in a cell, the method including contacting the cell with any one of the agents or pharmaceutical compositions described above; and maintaining the cell produced for a time sufficient to obtain antisense inhibition of a Serpinc1 gene, thereby inhibiting expression of Serpinc1 gene in the cell.
[0061] In one embodiment, the cell is within a subject.
[0062] For example, the subject is a human.
[0063] In one embodiment, Serpinc1 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%.
[0064] In yet another aspect, the invention provides a method of treating a subject having a disease or disorder that would benefit from reduction in Serpinc1 (AT3) expression, the method including administering to the subject a therapeutically effective amount of any one of the agents or the pharmaceutical compositions described herein, thereby treating the subject.
[0065] In another aspect, the invention provides a method of preventing at least one symptom in a subject having a disease or disorder that would benefit from reduction in Serpinc1 (AT3) expression, the method including administering to the subject a prophylactically effective amount of any one of the agents or the pharmaceutical compositions described herein, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in Serpinc1 expression.
[0066] 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 Serpinc1 protein levels.
[0067] In one embodiment, the disorder is a bleeding disorder.
[0068] In one embodiment, the bleeding disorder is one of the inherited disorders hemophilia and von Willebrand's disease.
[0069] For example, the hemophelia is one of hemophilia A, hemophilia B, or hemophilia C.
[0070] In one embodiment, the subject having the inherited disorder has developed an inhibitor.
[0071] For example, the inhibitor is an alloantibody to a replacement coagulation therapy.
[0072] In one embodiment, the bleeding disorder is an acquired disorder associated with a disease or condition selected from the group consisting of disseminated intravascular coagulation, pregnancy-associated eclampsia, vitamin K deficiency, an autoimmune disorder, inflammatory bowel disease, ulcerative colitis, a dermatologic disorder, a respiratory disease, an allergic drug reaction, diabetes, acute hepatitis B infection, acute hepatitis C infection, and a malignancy or solid tumor.
[0073] For example, the dermatologic disorder is one of psoriasis or pemphigus.
[0074] For example, the respiratory disease is one of asthma and chronic obstructive pulmonary disease.
[0075] For example, the allergic reaction is a result of a medication.
[0076] For example, the medication is selected from the group consisting of aspirin, heparin, and warfarin.
[0077] For example, the malignancy or solid tumor is selected from the group consisting of a tumor of prostate, lung, colon, pancreas, stomach, bile duct, head and neck, cervix, breast, skin, kidney, and a hematologic malignancy.
[0078] 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.
[0079] In one embodiment, the agent is administered at a dose of about 10 mg/kg to about 30 mg/kg.
[0080] In one embodiment, the agent is administered to the subject once a week.
[0081] In one embodiment, the agent is administered to the subject twice a week.
[0082] In one embodiment, the agent is administered to the subject twice a month.
[0083] In one embodiment, the agent is administered to the subject subcutaneously.
BRIEF DESCRIPTION OF THE DRAWINGS
[0084] FIG. 1 is a schematic of the blood coagulation cascade.
[0085] FIG. 2A shows the nucleotide sequence of Homo sapiens serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) (SEQ ID NO:1); FIG. 2B shows the nucleotide sequence of Macaca mulatta serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) (SEQ ID NO:2); FIG. 2C shows the nucleotide sequence of Mus musculus serine (or cysteine) peptidase inhibitor, clade C (antithrombin), member 1 (Serpinc1) (SEQ ID NO:3); FIG. 2D shows the nucleotide sequence of Rattus norvegicus serpin peptidase inhibitor, clade C (antithrombin), member 1 (Serpinc1) (SEQ ID NO:4); FIG. 2E shows the reverse complement of SEQ ID NO:1 (SEQ ID NO:5); FIG. 2F shows the reverse complement of SEQ ID NO:2 (SEQ ID NO:6); FIG. 2G shows the reverse complement of SEQ ID NO:3 (SEQ ID NO:7); and FIG. 2H shows the reverse complement of SEQ ID NO:4 (SEQ ID NO:8).
DETAILED DESCRIPTION OF THE INVENTION
[0086] The present invention provides polynucleotide agents and compositions comprising such agents which target nucleic acids encoding Serpinc1 or antithrombin III (AT3) (e.g., mRNA encoding Serpinc1 as provided in, for example, any one of SEQ ID NOs:1-4). The Serpinc1 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the polynucleotide agents and compositions of the invention for inhibiting the expression of a Serpinc1 gene and/or for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a Serpinc1 gene, e.g., a bleeding disorder, such as hemophilia. The present invention further provides methods for preventing at least one symptom, e.g., bleeding, in a subject having a disorder that would benefit from inhibiting or reducing the expression of a Serpinc1 gene, e.g., a bleeding disorder, such as hemophilia.
[0087] The 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 Serpinc1 gene. The use of these polynucleotide agents enables the targeted inhibition of RNA expression and/or activity of a Serpinc1 gene in mammals.
[0088] The present inventors have demonstrated that polynucleotide agents targeting Serpinc1 can mediate antisense inhibition in vitro resulting in significant inhibition of expression of a Serpinc1 gene. Thus, methods and compositions including these polynucleotide agents are useful for treating a subject who would benefit by a reduction in the levels and/or activity of a Serpinc1 protein, such as a subject having a bleeding disorder, e.g. hemophilia.
[0089] 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 Serpinc1gene, e.g., an inherited or acquired bleeding disorder, by using the polynucleotide agents and compositions of the invention. Such combination therapies include one or more agents which function by a non-antisense polynucleotide mechanism and which are useful in treating a bleeding 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 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.
[0090] The present invention also provides methods for preventing at least one symptom, e.g., a bleed, in a subject having a disorder that would benefit from inhibiting or reducing the expression of a Serpinc1 gene, e.g., Hemophilia A, Hemophilia B, or Hemophilia C. The present invention further provides compositions comprising polynucleotide agents which effect antisense inhibition of a Serpinc1 gene. The Serpinc1 gene may be within a cell, e.g., a cell within a subject, such as a human.
[0091] The combination therapies of the present invention include administering to a subject having a bleeding disorder, a polynucleotide agent of the invention and an additional therapeutic agent useful in treating a bleeding disorder, such as fresh-frozen plasma (FFP), recombinant FVIIa, or recombinant FIX; FXI concentrates. The combination therapies of the invention reduce Serpinc1 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 Serpinc1 mRNA with a polynucleotide agent of the invention and, accordingly, allow the therapeutically (or prophylactically) effective amount of the additional therapeutic agent required to treat the subject to be reduced, thereby decreasing the costs of treatment and permitting easier and more convenient ways of administering the agent, such as subcutaneous administration.
[0092] The following detailed description discloses how to make and use polynucleotide agents to inhibit the mRNA and/or protein expression of a Serpinc1 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.
Definitions
[0093] 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.
[0094] 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.
[0095] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".
[0096] The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.
[0097] As used herein, "Serpinc1" refers to a particular polypeptide expressed in a cell. Serpinc1 is also known as serpin peptidase inhibitor, clade C (antithrombin), member 1; antithrombin III; AT3; antithrombin; and heparin cofactor 1. The sequence of a human Serpinc1 mRNA transcript can be found at, for example, GenBank Accession No. GI:254588059 (NM_000488; SEQ ID NO:1). The sequence of rhesus Serpinc1 mRNA can be found at, for example, GenBank Accession No. GI:157167169 (NM_001104583; SEQ ID NO:2). The sequence of mouse Serpinc1 mRNA can be found at, for example, GenBank Accession No. GI:237874216 (NM_080844; SEQ ID NO:3). The sequence of rat Serpinc1 mRNA can be found at, for example, GenBank Accession No. GI:58865629 (NM_001012027; SEQ ID NO:4).
[0098] Additional examples of Serpinc1 mRNA sequences are readily available using publicly available databases, e.g., GenBank, Prosite, OMIM.
[0099] The term"Serpinc1" as used herein also refers to a particular polypeptide expressed in a cell by naturally occurring DNA sequence variations of the Serpinc1 gene, such as a single nucleotide polymorphism in the Serpinc1 gene. Numerous SNPs within the Serpinc1 gene have been identified and may be found at, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within the Serpinc1 gene may be found at, NCBI dbSNP Accession Nos. rs677; rs5877; rs5878; rs5879; rs941988; rs941989; rs1799876; rs19637711; rs2008946; and rs2227586.
[0100] 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 Serpinc1 (e.g., mRNA encoding Serpinc1 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."
[0101] 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.
[0102] 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.
[0103] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a Serpinc1 gene, including mRNA that is a product of RNA processing of a primary transcription product
[0104] As used herein, "target nucleic acid" refers to a nucleic acid molecule to which an antisense polynucleotide agent specifically hybridizes.
[0105] 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.
[0106] 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 Serpinc1 gene. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
[0107] 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.
[0108] 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 Serpinc1). For example, a polynucleotide is complementary to at least a part of a Serpinc1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding Serpinc1.
[0109] 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 Serpinc1 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.
[0110] 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.
[0111] 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.
[0112] "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.
[0113] 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.
[0114] "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.
[0115] 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.
[0116] "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.
[0117] 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.
[0118] 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.
[0119] 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 Serpinc1 expression; a human at risk for a disease, disorder or condition that would benefit from reduction in Serpinc1 expression; a human having a disease, disorder or condition that would benefit from reduction in Serpinc1 expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in Serpinc1 expression as described herein.
[0120] 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, diminishing the extent of bleeding, stabilized (i.e., not worsening) state of bleeding, amelioration or palliation of the bleeding, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.
[0121] The term "lower" in the context of the level of Serpinc1 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.
[0122] 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 Sertpinc1 gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with a such a disease, disorder, or condition, e.g., a symptom such as a bleed. The likelihood of developing a bleed is reduced, for example, when an individual having one or more risk factors for a bleed either fails to develop a bleed or develops a bleed 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.
[0123] As used herein, the term "bleeding disorder" is a disease or disorder that results in poor blood clotting and/or excessive bleeding. A bleeding disorder may be an inherited disorder, such as a hemophilia or von Willebrand's disease, or an acquired disorder, associated with, for example, disseminated intravascular coagulation, pregnancy-associated eclampsia, vitamin K deficiency, an autoimmune disorder, inflammatory bowel disease, ulcerative colitis, a dermatologic disorder (e.g., psoriasis, pemphigus), a respiratory disease (e.g., asthma, chronic obstructive pulmonary disease), an allergic drug reaction, e.g., the result of medications, such as aspirin, heparin, and warfarin, diabetes, acute hepatitis B infection, acute hepatitis C infection, a malignancy or solid tumor (e.g., prostate, lung, colon, pancreas, stomach, bile duct, head and neck, cervix, breast, melanoma, kidney, and/or a hematologic malignancy). In one embodiment, an inherited bleeding disorder is a hemophilia, e.g., hemophilia A, B, or C. In one embodiment, a subject having an inherited bleeding disorder, e.g., a hemophilia, has developed inhibitors, e.g., alloantibody inhibitors, to replacement coagulation therapies and is referred to herein as an "inhibitor subject." In one embodiment, the inhibitor subject has hemophilia A. In another embodiment, the inhibitor subject has hemophilia B. In yet another embodiment, the inhibitor subject has hemophilia C.
II. Polynucleotide Agents of the Invention
[0124] The present invention provides polynucleotide agents, e.g., antisense polynucleotide agents, and compositions comprising such agents, which target a Serpinc1 gene and inhibit the expression of the Serpinc1 gene. In one embodiment, the antisense polynucleotide agents inhibit the expression of a Serpinc1 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having a bleeding disorder e.g., hemophilia.
[0125] 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 Serpinc1 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 Serpinc1 gene, the antisense polynucleotide agent inhibits the expression of the Serpinc1 gene (e.g., a human, a primate, a non-primate, or a bird Serpinc1 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.
[0126] 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 Serpinc1 gene.
[0127] Accordingly, in one aspect, an antisense polynucleotide agent of the invention specifically hybridizes to a target nucleic acid molecule, such as the mRNA encoding Serpinc1, 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.
[0128] 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 Serpinc1 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 Serpinc1 mRNA.
[0129] 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.
[0130] 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).
[0131] 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.
[0132] 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.
[0133] In one aspect, an antisense polynucleotide agent of the invention includes a sequence selected from the group of the sequences provided in Tables 3 and 4. It will be understood that, although some of the sequences in Tables 3 and 4 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 Tables 3 and 4 that is un-modified, un-conjugated, and/or modified and/or conjugated differently than described therein.
[0134] By virtue of the nature of the nucleotide sequences provided in Tables 3 and 4, antisense polynucleotide agents of the invention may include one of the sequences of Tables 3 and 4 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 Tables 3 and 4 and differing in their ability to inhibit the expression of a Serpinc1 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.
[0135] In addition, the antisense polynucleotide agents provided in Tables 3 and 4 identify a region(s) in a Serpinc1 transcript that is susceptible to antisense inhibition (e.g., the regions encompassed by the start and end positions relative to NM_000488.3 in Table 4). 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 Tables 3 or 4 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a Serpinc1 (AT3) gene.
[0136] 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 Tables 3 or 4 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.
[0137] Further, it is contemplated that for any sequence identified, e.g., in Tables 3 or 4, 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 Polynucleotide Agents of the Invention
[0138] In one embodiment, the nucleotides of a polynucleotide agent of the invention, e.g., 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 a polynucleotide agent of the invention, e.g., 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 a polynucleotide agent of the invention, e.g., an antisense polynucleotide agent of the invention, are modified. In other embodiments of the invention, all of the nucleotides of a polynucleotide agent of the invention, e.g., 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.
[0139] 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.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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).
[0151] 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.
[0152] 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.
[0153] 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).
[0154] 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."
[0155] 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).
[0156] 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."
[0157] 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'-CH2-0-2' 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).
[0158] 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).
[0159] 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.
[0160] 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.
[0161] One or more of the nucleotides of an agent 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.
[0162] Additional modifications 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.
[0163] Any of the antisense polynucleotide agents of the invention may be optionally conjugated with a GalNAc derivative ligand, as described in Section IV, below.
[0164] 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.
[0165] 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.
[0166] 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 and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.
[0167] 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 Tables 3 and 4. These agents may further comprise a ligand, as described in Section IV, below.
[0168] A. Polynucleotide Agents Comprising Motifs
[0169] In certain embodiments of the invention, at least one of the contiguous nucleotides of the the polynucleotide agent of the invention, e.g., 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.
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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).
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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 11, 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.
[0178] 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.
[0179] In some embodiments of the invention, antisense polynucleotide agents targeting Serpinc1 include a 5-10-5 gapmer motif. In other embodiments of the invention, antisense polynucleotide agents targeting Serpinc1 include a 4-10-4 gapmer motif. In another embodiment of the invention, antisense polynucleotide agents targeting Serpinc1 include a 3-10-3 gapmer motif. In yet other embodiments of the invention, antisense polynucleotide agents targeting Serpinc1 include a 2-10-2 gapmer motif.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] The 5'-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleotides.
[0185] 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.
[0186] 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.
[0187] 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.
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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.
[0192] The 3'-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleotides.
[0193] 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.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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 the gap of a gapmer are 5-methylcytosines.
[0202] 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 the gap of a gapmer are 2'-deoxynucleotides.
[0203] 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.
[0204] 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.
[0205] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0206] In another embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0207] In another embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0208] In another embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0209] 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 Serpinc1 gene is a 5-methylcytosine.
[0210] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0211] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0212] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0213] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0214] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0215] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0216] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0217] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0218] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0219] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0220] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0221] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0222] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0223] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0224] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0225] In one embodiment, an antisense polynucleotide agent targeting a Serpinc1 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.
[0226] 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. Polynucleotide Agents Conjugated to Ligands
[0227] 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 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).
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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.
[0237] 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.
[0238] 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.
[0239] A. Lipid Conjugates
[0240] 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.
[0241] 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.
[0242] 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.
[0243] 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.
[0244] 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).
[0245] B. Cell Permeation Agents
[0246] 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.
[0247] 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.
[0248] 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.
[0249] 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 peptidomimetics 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.
[0250] 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).
[0251] C. Carbohydrate Conjugates
[0252] 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).
[0253] 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##
[0254] 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##
[0255] 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.
[0256] 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.
[0257] D. Linkers
[0258] 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.
[0259] 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.
[0260] 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).
[0261] 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.
[0262] 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.
[0263] 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.
[0264] Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
[0265] 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).
[0266] i. Redox Cleavable Linking Groups
[0267] 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.
[0268] ii. Phosphate-Based Cleavable Linking Groups
[0269] 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.
[0270] iii. Acid Cleavable Linking Groups
[0271] 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.
[0272] iv. Ester-Based Linking Groups
[0273] 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.
[0274] v. Peptide-Based Cleaving Groups
[0275] 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--).
[0276] 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.
[0277] 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## ##STR00012##
[0278] when one of X or Y is an oligonucleotide, the other is a hydrogen.
[0279] 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.
[0280] 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):
##STR00013##
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.5C 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--
##STR00014##
or heterocyclyl;
[0281] 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):
##STR00015##
[0282] wherein L.sup.5A, L.sup.5B and L.sup.5C represent a monosaccharide, such as GalNAc derivative.
[0283] 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.
[0284] 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.
[0285] 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.
[0286] "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.
[0287] 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 a Polynucleotide Agent of the Invention
[0288] The delivery of a polynucleotide agent of the invention, e.g., 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 bleeding disorder) 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.
[0289] 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
[0290] The present invention also includes pharmaceutical compositions and formulations which include the polynucleotide agents of the invention, e.g., 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.
[0291] 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.
[0292] 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.
[0293] The pharmaceutical compositions containing the antisense polynucleotide agents are useful for treating a bleeding disorder that would benefit from inhibiting or reducing the expression of Serpinc1. 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 Serpinc1 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.
[0294] 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.
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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.
[0299] 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 regimine 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.
[0300] 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 regimine 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.
[0301] 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.
[0302] 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.
[0303] 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.
[0304] 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.
[0305] 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 Serpinc1. 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, Hemophilia A mouse models and Hemohphilia B mouse models, e.g., mice containing a knock-out of a clotting factor gene, such as those described in Bolliger, et al. (2010) Thromb Haemost 103:1233-1238, Bi L, et al. (1995) Nat Genet 10: 119-21, Lin et al. (1997) Blood 90: 3962-6, Kundu et al. (1998) Blood 92: 168-74, Wang et al. (1997) Proc Natl Acad Sci USA 94: 11563-6, and Jin, et al. (2004) Blood 104:1733.
[0306] 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.
[0307] 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).
[0308] 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.
[0309] A. Antisense Polynucleotide Agent Formulations Comprising Membranous Molecular Assemblies
[0310] 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.
[0311] 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.
[0312] 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.
[0313] 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.
[0314] 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).
[0315] 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).
[0316] 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.
[0317] 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.
[0318] 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).
[0319] 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).
[0320] 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. USA., 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).
[0321] 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.
[0322] 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.
[0323] 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).
[0324] 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.
[0325] 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).
[0326] 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.
[0327] 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).
[0328] 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.
[0329] 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. Transfersomes 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 transfersomes 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.
[0330] 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.
[0331] 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.
[0332] 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).
[0333] 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.
[0334] 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.
[0335] 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.
[0336] 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.
[0337] 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).
[0338] 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.
[0339] 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.
[0340] 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.
[0341] 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.
[0342] 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.
[0343] 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.
[0344] 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.
[0345] B. Lipid Particles
[0346] 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.
[0347] 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.
[0348] 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.
[0349] 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)tetrahydro- -3aH-cyclopenta[d][1,3]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 G1), 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.
[0350] 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.
[0351] 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.
[0352] 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.
[0353] 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.
[0354] 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.
[0355] 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.
##STR00016##
[0356] LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
[0357] 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. XTC comprising formulations are described, e.g., in U.S. Provisional Serial No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Serial No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun. 10, 2009; U.S. Provisional Serial No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Serial 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. 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. 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. C12-200 comprising formulations are described in U.S. Provisional Serial 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.
[0358] 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.
[0359] 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.
[0360] 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.
[0361] 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.
[0362] 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.
[0363] C. Additional Formulations
[0364] i. Emulsions
[0365] 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 antioxidants 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.
[0366] 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).
[0367] 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).
[0368] 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.
[0369] 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).
[0370] 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.
[0371] 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.
[0372] 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.
[0373] ii. Microemulsions
[0374] 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).
[0375] 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.
[0376] 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.
[0377] 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.
[0378] 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.
[0379] iii. Microparticles
[0380] 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.
[0381] iv. Penetration Enhancers
[0382] 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.
[0383] 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.
[0384] 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).
[0385] 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).
[0386] 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).
[0387] 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).
[0388] 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).
[0389] 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. MAX (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), TransPassa D1 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.
[0390] 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.
[0391] v. Carriers
[0392] 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.
[0393] vi. Excipients
[0394] 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).
[0395] 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.
[0396] 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.
[0397] 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.
[0398] vii. Other Components
[0399] 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.
[0400] 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.
[0401] 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.
[0402] 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.
[0403] 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.
[0404] 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 a bleeding disorder. 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 Serpinc1 Expression
[0405] The present invention provides methods of inhibiting expression of Serpinc1 in a cell. The methods include contacting a cell with a polynucleotide agent of the invention, e.g., an antisense polynucleotide agent of the invention, in an amount effective to inhibit expression of the Serpinc1 in the cell, thereby inhibiting expression of the Serpinc1 in the cell.
[0406] 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.
[0407] The term "inhibiting," as used herein, is used interchangeably with "reducing," "silencing," "downregulating" and other similar terms, and includes any level of inhibition.
[0408] The phrase "inhibiting expression of a Serpinc1" is intended to refer to inhibition of expression of any Serpinc1 gene (such as, e.g., a mouse Serpinc1 gene, a rat Serpinc1 gene, a monkey Serpinc1 gene, or a human Serpinc1 gene) as well as variants or mutants of a Serpinc1 gene. Thus, the Serpinc1 gene may be a wild-type Serpinc1 gene, a mutant Serpinc1 gene, or a transgenic Serpinc1 gene in the context of a genetically manipulated cell, group of cells, or organism.
[0409] "Inhibiting expression of a Serpinc1 gene" includes any level of inhibition of a Serpinc1 gene, e.g., at least partial suppression of the expression of a Serpinc1 gene. The expression of the Serpinc1 gene may be assessed based on the level, or the change in the level, of any variable associated with Serpinc1 gene expression, e.g., Serpinc1 mRNA level, Serpinc1 protein level. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.
[0410] Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with Serpinc1 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).
[0411] In some embodiments of the methods of the invention, expression of a Serpinc1 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%.
[0412] Inhibition of the expression of a Serpinc1 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 Serpinc1 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 Serpinc1 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 .times. .times. in .times. .times. control .times. .times. cells ) - ( mRNA .times. .times. in .times. .times. treated .times. .times. cells ) ( mRNA .times. .times. in .times. .times. control .times. .times. cells ) 100 .times. % ##EQU00001##
[0413] Alternatively, inhibition of the expression of a Serpinc1 gene may be assessed in terms of a reduction of a parameter that is functionally linked to Serpinc1 gene expression, e.g., Serpinc1 protein expression.
[0414] Inhibition of the expression of a Serpinc1 protein may be manifested by a reduction in the level of the Serpinc1 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.
[0415] A control cell or group of cells that may be used to assess the inhibition of the expression of a Serpinc1 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.
[0416] The level of Serpinc1 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 Serpinc1 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the Serpinc1 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.
[0417] In one embodiment, the level of expression of Serpinc1 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 Serpinc1. 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.
[0418] 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 Serpinc1 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 Serpinc1 mRNA.
[0419] An alternative method for determining the level of expression of Serpinc1 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 Serpinc1 is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan.TM. System).
[0420] The expression levels of Serpinc1 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 Serpinc1 expression level may also comprise using nucleic acid probes in solution.
[0421] 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.
[0422] The level of Serpinc1 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.
[0423] 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.
[0424] 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 Serpinc1 may be assessed using measurements of the level or change in the level of Serpinc1 mRNA or Serpinc1 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.
[0425] 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.
[0426] 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.
[0427] 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 Serpinc1-Associated Disorder
[0428] The present invention also provides therapeutic and prophylactic methods which include administering to a subject having a Serpinc1-associated disorder, such as a bleeding disorder, e.g., a hemophilia, 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, e.g., recombinant FVIIa, or recombinant FIX.
[0429] In one aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in Serpinc1 expression, e.g., a bleeding disorder e.g., hemophilia. 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 Serpinc1 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a Serpinc1 gene, thereby treating the subject having a disorder that would benefit from reduction in Serpinc1 expression.
[0430] In another aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in Serpinc1 expression, e.g., a bleeding disorder, e.g., a hemophilia, which include administering to the subject, e.g., a human, a therapeutically effective amount of an antisense polynucleotide agent targeting a Serpinc1 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a Serpinc1 gene, and an additional therapeutic agent, such as recombinant FVIIa, recombinant FIX, or a FXI concentrate, thereby treating the subject having a disorder that would benefit from reduction in Serpinc1 expression.
[0431] 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 Serpinc1 expression, e.g., a bleeding disorder, e.g., a hemophilia. The methods include administering to the subject a prohpylactically effective amount of an antisense polynucleotide agent targeting a Serpinc1 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a Serpinc1 gene, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in Serpinc1 expression. For example, the invention provides methods for preventing or decreasing the frequency of bleeding in a subject suffering from a bleeding disorder that would benefit from reduction in Serpinc1 expression, e.g., a hemophilia, ulcerative colitis, or an allergic drug reaction.
[0432] 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 Serpinc1 expression, e.g., a bleeding disorder, e.g., disseminated intravascular coagulation, pregnancy-associated eclampsia, or a dermatologic disorder. The methods include administering to the subject a prohpylactically effective amount of an antisense polynucleotide agent targeting a Serpinc1 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a Serpinc1 gene, and an additional therapeutic agent, such as an antifibrinolytic, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in Serpinc1 expression.
[0433] "Therapeutically effective amount," as used herein, is intended to include the amount of an antisense polynucleotide agent or an amount of another therapeutic agent, such as an antifibrinolytic, that may be used in combination with the polynucleotide agent, that, when administered to a subject having a bleeding disorder, 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 the other therapeutic agent, how the polynucleotide agent or the other therapeutic 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.
[0434] "Prophylactically effective amount," as used herein, is intended to include the amount of an antisense polynucleotide agent or an amount of another therapeutic agent, such as an antifibrinolytic, that may be used in combination with the polynucleotide agent, that, when administered to a subject having a bleeding disorder but not yet (or currently) experiencing or displaying symptoms of the disorder, and/or a subject at risk of developing a bleeding disorder, e.g., a subject having inflammatory bowel disease or a subject having ulcerative colitis, 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 the other therapeutic agent, how the polynucleotide agent or the other therapeutic agent 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.
[0435] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an antisense polynucleotide agent or an amount of another therapeutic agent, such as an antifibrinolytic, that may be used in combination with the polynucleotide agent, 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.
[0436] 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 Serpinc1 expression.
[0437] 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 recombinant FVIIa for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of Serpinc1 expression.
[0438] In yet another aspect, the present invention provides use of an antisense polynucleotide agent of the invention targeting a Serpinc1 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a Serpinc1 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 Serpinc1 expression, such as a subject having a disorder that would benefit from reduction in Serpinc1 expression, e.g., a bleeding disorder, e.g., a hemophilia.
[0439] In another aspect, the present invention provides uses of an antisense polynucleotide agent of the invention targeting a Serpinc1 gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a Serpinc1 gene in the manufacture of a medicament for use in combination with an additional therapeutic agent, such as an immunosuppressive agent, for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of Serpinc1 expression, e.g., a bleeding disorder associated with an allergic drug reaction.
[0440] 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 Serpinc1 expression, such as ulcerative colitis.
[0441] In yet another aspect, the invention provides uses of an antisense polynucleotide agent of the invention, and an additional therapeutic agent, such as an antifibrinolytic agent, for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of Serpinc1 expression, such as a bleeding disorder, e.g., hemophilia.
[0442] 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 Serpinc1 expression, such as a such as a bleeding disorder, e.g., hemophilia.
[0443] 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 recombinant FVIIa or recombinant FIX, for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of Serpinc1 expression, such as a a bleeding disorder, e.g., hemophilia.
[0444] In one embodiment, an antisense polynucleotide agent targeting Serpinc1 is administered to a subject having a bleeding disorder such that Serpinc1 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.
[0445] The additional therapeutic may be other pharmaceuticals such as, for example, those which are currently employed for treating these disorders. For example, additional therapeutics suitable for treating a subject that would benefit from reduction in Serpinc1 expression, e.g., a subject having a bleeding disorder, include fresh-frozen plasma (FFP); recombinant FVIIa; recombinant FIX; FXI concentrates; virus-inactivated vWF-containing FVIII concentrates; desmopressin acetate [DDAVP]; antifibrinolytics, such as aminocaproic acid and tranexamic acid; activated prothrombin complex concentrate (PCC); antihemophilic agents; corticosteroids; immunosuppressive agents; and estrogens. The polynucleotide agent 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. Examples of such another method are desensitization therapy which may include large doses of FVIII or FIX, along with steroids or intravenous immunoglobulin (IVIG) and cyclophosphamide; plasmapheresis in conjunction with immunosuppression and infusion of FVIII or FIX, with or without antifibrinolytic therapy; and immune tolerance induction (ITI), with or without immunosuppressive therapy (e.g., cyclophosphamide, prednisone, and/or anti-CD20).
[0446] Moreover, the additional therapeutic, e.g., recombinant FVIIa may be administered to the subject in the same formulation as the antisense polynucleotide agent targeting Serpinc1 or in a different formulation as the antisense polynucleotide agent targeting Serpinc1.
[0447] The methods and uses of the invention include administering a composition described herein such that expression of the target Serpinc1 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 Serpinc1 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.
[0448] 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 bleeding. 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%.
[0449] 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 Serpinc1 or pharmaceutical composition thereof, "effective against" a bleeding disorder 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 bleeding disorder.
[0450] 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.
[0451] 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. 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.
[0452] 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.
[0453] 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 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.
[0454] 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.
[0455] 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.
[0456] 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.
[0457] 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.
[0458] Administration of the antisense polynucleotide agent can reduce Serpinc1 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.
[0459] 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.
[0460] Owing to the inhibitory effects on Serpinc1 expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.
[0461] 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.
[0462] Alternatively, an antisense polynucleotide agent of the invention may be administered as a pharmaceutical composition, such as an antisense polynucleotide agent liposomal formulation.
[0463] Subjects that would benefit from a reduction and/or inhibition of Serpinc1 gene expression are those having a bleeding disorder as described herein. In one embodiment, a subject having a bleeding disorder has an acquired disorder associated with ulcerative colitis. In another embodiment, a subject having a bleeding disorder has an acquired disorder associated with pregnancy-associated eclampsia. In another embodiment, a subject having a bleeding disorder has an inherited disorder such as von Willebrand's disease. In another embodiment, a subject having a bleeding disorder has an acquired disorder associated with vitamin K deficiency. In yet another embodiment, a subject having a a bleeding disorder has an acquired disorder associated with disseminated intravascular coagulation. In one embodiment, a subject having a bleeding disorder has an acquired disorder associated with an autoimmune disorder. In one embodiment, a subject having a bleeding disorder has an acquired disorder associated with inflammatory bowel disease. In another embodiment, a subject having a bleeding disorder has has an acquired disorder associated with a dermatologic disorder, e.g., psoriasis or pemphigus. In yet another embodiment, a subject having a bleeding disorder has an acquired disorder associated with a respiratory disease (e.g., asthma or chronic obstructive pulmonary disease). In one embodiment, a subject having a bleeding disorder has an acquired disorder associated with an allergic drug reaction, e.g., the result of medications such as aspirin, heparin, or warfarin. In another embodiment, a subject having a bleeding disorder has an acquired disorder associated with diabetes. In another embodiment, a subject having a bleeding disorder has an acquired disorder associated with acute hepatitis B infection. In another embodiment, a subject having a bleeding disorder has an acquired disorder associated with acute hepatitis C infection. In another embodiment, a subject having a bleeding disorder has an acquired disorder associated with a malignancy or solid tumor, (e.g., prostate, lung, colon, colon, pancreas, stomach, bile duct, head and neck, cervix, breast, melanoma, kidney) or a hematologic malignancy. In another embodiment, a subject has an inherited bleeding disorder, e.g., hemophilia A, B, or C. In one embodiment, the subject having an inherited bleeding disorder, e.g., a hemophilia has developed inhibitors, e.g., alloantibody inhibitors to replacement coagulation therapies. Such a subject is referred to herein as an "inhibitor subject." In one embodiment, the inhibitor subject has hemophilia A. In another embodiment, the inhibitor subject has hemophilia B. In yet another embodiment, the inhibitor subject has hemophilia C.
[0464] Treatment of a subject that would benefit from a reduction and/or inhibition of Serpinc1 gene expression includes therapeutic and prophylactic (e.g., the subject is to undergo sensitized (or allogenic) transplant surgery) treatment.
[0465] The invention further provides methods and uses of an antisense polynucleotide agent or a pharmaceutical composition thereof for treating a subject that would benefit from reduction and/or inhibition of Serpinc1 expression, e.g., a subject having a bleeding disorder, 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 Serpinc1 is administered in combination with, e.g., an agent useful in treating a bleeding disorder as described elsewhere herein.
[0466] For example, additional therapeutics and therapeutic methods suitable for treating a subject that would benefit from reduction in Serpinc1 expression, e.g., a subject having a bleeding disorder, include 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 Serpinc1 is administered in combination with, e.g., an agent useful in treating a bleeding disorder as described elsewhere herein. For example, additional therapeutics and therapeutic methods suitable for treating a subject that would benefit from reduction in Serpinc1 expression, e.g., a subject having a bleeding disorder, include fresh-frozen plasma (FFP); recombinant FVIIa; recombinant FIX; FXI concentrates; virus-inactivated, vWF-containing FVIII concentrates; desensitization therapy which may include large doses of FVIII or FIX, along with steroids or intravenous immunoglobulin (IVIG) and cyclophosphamide; plasmapheresis in conjunction with immunosuppression and infusion of FVIII or FIX, with or without antifibrinolytic therapy; immune tolerance induction (ITI), with or without immunosuppressive therapy (e.g., cyclophosphamide, prednisone, and/or anti-CD20); desmopressin acetate [DDAVP]; antifibrinolytics, such as aminocaproic acid and tranexamic acid; activated prothrombin complex concentrate (PCC); antihemophilic agents; corticosteroids; immunosuppressive agents; and estrogens.
[0467] The antisense polynucleotide agent 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.
[0468] 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 Serpinc1 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 Serpinc1 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 Serpinc1 expression in a cell are provided.
[0469] 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 Serpinc1 gene, thereby inhibiting expression of the Serpinc1 gene in the cell.
[0470] Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of Serpinc1 may be determined by determining the mRNA expression level of Serpinc1 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, by determining the protein level of Serpinc1 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 Serpinc1 and/or by determining the biological activity of one or more molecules associated with Serpinc1 in the coagulation pathway.
[0471] 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.
[0472] A cell suitable for treatment using the methods of the invention may be any cell that expresses a Serpinc1 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.
[0473] Serpinc1 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%.
[0474] 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 Serpinc1 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.
[0475] 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 Serpinc1, 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.
[0476] 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.
[0477] 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.
[0478] In one aspect, the present invention also provides methods for inhibiting the expression of a Serpinc1 gene in a mammal, e.g., a human. The present invention also provides a composition comprising an antisense polynucleotide agent that targets a Serpinc1 gene in a cell of a mammal for use in inhibiting expression of the Serpinc1 gene in the mammal. In another aspect, the present invention provides use of an antisense polynucleotide agent that targets a Serpinc1 gene in a cell of a mammal in the manufacture of a medicament for inhibiting expression of the Serpinc1 gene in the mammal.
[0479] The methods and uses include administering to the mammal, e.g., a human, a composition comprising an antisense polynucleotide agent that targets a Serpinc1 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 Serpinc1 gene, thereby inhibiting expression of the Serpinc1 gene in the mammal.
[0480] 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 Serpinc1 gene and/or protein expression. In another embodiment, a blood sample serves as the tissue material for monitoring the reduction in Serpinc1 gene and/or protein expression. In other embodiments, inhibition of the expression of a Serpinc1 gene is monitored indirectly by, for example, determining the expression and/or activity of a gene in a Serpinc1 pathway (see, e.g., FIG. 1).
[0481] 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
[0482] The antisense polynucleotides targeting Serpinc1 were synthesized using standard synthesis methods well known in the art.
[0483] Design of antisense polynucleotides was carried out using the following transcripts from the NCBI RefSeq collection: Human--NM_000488.2, NM_000488.3; Rhesus--NM_001104583.1; Dog--XM_856414.1; Mouse--NM_080844.4; Rat--NM_001012027.1.
[0484] A detailed list of antisense molecules targeting Serpinc1 is shown in Tables 3 and 4 below.
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. Abbreviation 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) or (m5dC) 5'-methyl-deoxycytidine-3'-phosphate (5MdC)s or 5'-methyl-deoxycytidine-3'-phosphorothioate (m5dCs)
TABLE-US-00003 TABLE 3 Antisense polynucleotides targeting Serpinc1(AT3) SEQ SEQ Alternative ID Oligonucleotide ID Sequence ID Sequence ID Modified Sequence (5'-3') NO. Sequence (5'-3') NO. A-131024.1 X10631 csusasasasdTs(5MdC)sdTs(5MdC)sdGs(5MdC)sdAsd 13 CTAAATCTCGCAGAGG 197 GsdAsdGsdGsgsususcsc GTTCC A-131025.1 X10632 gsususcsusdTsdTs(5MdC)s(5MdC)sdTs(5MdC)sdTsd 14 GTTCTTTCCTCTAAAT 198 AsdAsdAsdTsuscsuscsg TCTCG A-131026.1 X10633 csusgsasasdAsdAs(5MdC)sdTsdGsdGsdTsdTs(5MdC) 15 CTGAAAACTGGTTCTT 199 sdTsdTsususcscsu TTCCT A-131027.1 X10634 gsgscsasasdTs(5MdC)s(5MdC)sdGs(5MdC)s(5MdC)s 16 GGCAATCCGCCTGAAA 200 dTsdGsdAsdAsdAsasascsusg AACTG A-131028.1 X10635 usgsusgsasdTs(5MdC)sdTsdGsdAsdGsdGs(5MdC)sdA 17 TGTGATCTGAGGCAAT 201 sdAsdTsuscscsgsc TCCGC A-131029.1 X10636 gsusgsgsasdGsdAsdTsdAsdGsdTsdGsdTsdGsdAsdTsu 18 GTGGAGATAGTGTGAT 202 scsusgsa TCTGA A-131030.1 X10637 gsgscsusgsdGsdGs(5MdC)sdAsdAsdGsdTsdGsdGsdAs 19 GGCTGGGCAAGTGGAG 203 dGsgsasusasg GATAG A-131031.1 X10638 uscsususcs(5MdC)sdAs(5MdC)sdAsdGsdGsdGs(5MdC) 20 TCTTCCACAGGGCTGG 204 sdTsdGsdGsgsgscsasa GGCAA A-131032.1 X10639 usgsgscscsdGs(5MdC)sdTsdAsdAsdTs(5MdC)sdTsdTs 21 TGGCCGCTAATCTTCC 205 (5MdC)s(5MdC)scsascsasg CACAG A-131033.1 X10640 ususgsgsasdAsdTsdAs(5MdC)sdAsdTsdGsdGs(5MdC)s 22 TTGGAATACATGGCCG 206 (5MdC)sdGsgscsusasa GCTAA A-131034.1 X10641 uscscsusasdTs(5MdC)sdAs(5MdC)sdAsdTsdTsdGsdGs 23 TCCTATCACATTGGAA 207 dAsdAsasusascsa ATACA A-131035.1 X10642 asgsgsususdAs(5MdC)sdAsdGsdTsdTs(5MdC)s(5MdC) 24 AGGTTACAGTTCCTAT 208 sdTsdAsdTsuscsascsa TCACA A-131036.1 X10643 csususususdTs(5MdC)s(5MdC)sdAsdGsdAsdGsdGsdTs 25 CTTTTTCCAGAGGTTA 209 dTsdAsascsasgsu ACAGT A-131037.1 X10644 asusasasas(5MdC)s(5MdC)sdTsdTs(5MdC)s(5MdC)sd 26 ATAAACCTTCCTTTTT 210 TsdTsdTsdTsdTsuscscsasg TCCAG A-131038.1 X10645 asgsgsascsdAsdAsdAsdAsdGsdAsdTsdAsdAsdAs(5MdC) 27 AGGACAAAAGATAAAC 211 scscsususc CCTTC A-131039.1 X10646 asusgsasgs(5MdC)sdAsdGs(5MdC)sdAsdAsdGsdGsdAs 28 ATGAGCAGCAAGGACA 212 (5MdC)sdAsasasasasg AAAAG A-131040.1 X10647 cscsasgsasdAsdGs(5MdC)s(5MdC)sdAsdAsdTsdGsdAs 29 CCAGAAGCCAATGAGC 213 dGs(5MdC)scsasgscsa CAGCA A-131041.1 X10648 uscsascsgs(5MdC)sdAsdGsdTs(5MdC)s(5MdC)s(5MdC) 30 TCACGCAGTCCCAGAA 214 sdAsdGsdAsdAsasgscscsa AGCCA A-131042.1 X10649 cscsgsusgsdAs(5MdC)sdAsdGsdGsdTs(5MdC)sdAs 31 CCGTGACAGGTCACGC 215 (5MdC)sdGs(5MdC)scsasgsusc CAGTC A-131043.1 X10650 csascsasgsdGsdGs(5MdC)sdTs(5MdC)s(5MdC)s(5MdC) 32 CACAGGGCTCCCGTGA 216 sdGsdTsdGsdAsascsasgsg ACAGG A-131044.1 X10651 usgscsasgsdAsdTsdGsdTs(5MdC)s(5MdC)sdAs(5MdC)s 33 TGCAGATGTCCACAGG 217 dAsdGsdGsgsgscsusc GGCTC A-131045.1 X10652 gsgscsususdGsdGs(5MdC)sdTsdGsdTsdGs(5MdC)sdAsd 34 GGCTTGGCTGTGCAGA 218 GsdAsasusgsusc ATGTC A-131046.1 X10653 asasusgsus(5MdC)s(5MdC)s(5MdC)sdGs(5MdC)sdGsdG 35 ATGTGCCCGCGGCTTG 219 s(5MdC)sdTsdTsdGsgsgscsusg GGCTG A-131047.1 X10654 gsasususcsdAsdTsdGsdGsdGsdAsdAsdTsdGsdTs(5MdC)s 36 GATTCATGGGAATGTC 220 cscscsgsc CCCGC A-131048.1 X10655 asusgscsas(5MdC)sdAsdTsdGsdGsdGsdAsdTsdTs(5MdC) 37 ATGCACATGGGATTCA 221 sdAsasusgsgsg ATGGG A-131049.1 X10656 gsgsasgscsdGsdGsdTsdAsdAsdAsdTsdGs(5MdC)sdAs 38 GGAGCGGTAAATGCAC 222 (5MdC)scsasusgsg CATGG A-131050.1 X10657 uscsususcsdTs(5MdC)s(5MdC)sdGsdGsdGsdGsdAsdGs 39 TCTTCTCCGGGGAGCG 223 (5MdC)sdGsgsgsusasa GGTAA A-131051.1 X10658 uscsasgsusdTsdGs(5MdC)s(5MdC)sdTsdTs(5MdC)sdT 40 TCAGTTGCCTTCTTCT 224 sdTs(5MdC)sdTsuscscsgsg TCCGG A-131052.1 X10659 gscscscsus(5MdC)sdAsdTs(5MdC)s(5MdC)sdTs(5MdC) 41 GCCCTCATCCTCAGTT 225 sdAsdGsdTsdTsusgscscsu TGCCT A-131053.1 X10660 uscsusgsusdTs(5MdC)sdTsdGsdAsdGs(5MdC)s(5MdC)s 42 TCTGTTCTGAGCCCTC 226 (5MdC)sdTs(5MdC)scsasuscsc CATCC A-131054.1 X10661 uscscsgsgsdGsdAsdTs(5MdC)sdTsdTs(5MdC)sdTsdGsd 43 TCCGGGATCTTCTGTT 227 TsdTsuscsusgsa TCTGA A-131055.1 X10662 gsususgsgsdTsdGsdGs(5MdC)s(5MdC)sdTs(5MdC)s 44 GTTGGTGGCCTCGGGG 228 (5MdC)sdGsdGsdGsgsasuscsu ATCT A-131056.1 X10663 asgsascsas(5MdC)sdGs(5MdC)s(5MdC)sdGsdGsdTsdTs 45 AGACACGCCGGTTGGT 229 dGsdGsdTsusgsgscsc TGGC A-131057.1 X10664 gsascsasgsdTsdTs(5MdC)s(5MdC)s(5MdC)sdAsdGsdAs 46 GACAGTTCCCAGACAC 230 (5MdC)sdAs(5MdC)scsgscscsg CGCCG A-131058.1 X10665 asususgsgs(5MdC)s(5MdC)sdTsdTsdGsdGsdAs(5MdC)s 47 ATTGGCCTTGGACAGT 231 dAsdGsdTsususcscsc TTCCC A-131059.1 X10666 csasasasgs(5MdC)sdGsdGsdGsdAsdAsdTsdTsdGsdGs 48 CAAAGCGGGAATTGGC 232 (5MdC)scscsususg CCTTG A-131060.1 X10667 asasasgsusdGsdGsdTsdAsdGs(5MdC)sdAsdAsdAsdGs 49 AAAGTGGTAGCAAAGC 233 (5MdC)scsgsgsgsa CGGGA A-131061.1 X10668 gsusgscsusdGsdAsdTsdAsdGsdAsdAsdAsdGsdTsdGsg 50 GTGCTGATAGAAAGTG 234 sgsusasg GGTAG A-131062.1 X10669 asasuscsusdGs(5MdC)s(5MdC)sdAsdGsdGsdTsdGs 51 AATCTGCCAGGTGCTG 235 (5MdC)sdTsdGsgsasusasg GATAG A-131063.1 X10670 uscsasusus(5MdC)sdTsdTsdGsdGsdAsdAsdTs(5MdC) 52 TCATTCTTGGAATCTG 236 sdTsdGsgscscsasg GCCAG A-131064.1 X10671 gsususasus(5MdC)sdAsdTsdTsdGsdTs(5MdC)sdAsdT 53 GTTATCATTGTCATTC 237 sdTs(5MdC)scsususgsg CTTGG A-131065.1 X10672 ascsasgsgsdAsdAsdAsdAsdTsdGsdTsdTsdAsdTs 54 ACAGGAAAATGTTATC 238 (5MdC)scsasususg CATTG A-131066.1 X10673 csuscsasgsdGsdGsdGsdTsdGsdAs(5MdC)sdAsdGsdGs 55 CTCAGGGGTGACAGGA 239 dAsasasasasu AAAAT A-131067.1 X10674 csgsusgsgsdAsdGsdAsdTsdAs(5MdC)sdTs(5MdC)sdA 56 CGTGGAGATACTCAGG 240 sdGsdGsgsgsgsusg GGGTG A-131068.1 X10675 usasgscsasdAsdAsdAsdGs(5MdC)s(5MdC)sdGsdTsdG 57 TAGCAAAAGCCGTGGA 241 sdGsdAsasgsasusa AGATA A-131069.1 X10676 asgscsususdGsdGsdTs(5MdC)sdAsdTsdAsdGs(5MdC) 58 AGCTTGGTCATAGCAA 242 sdAsdAsasasasgsc AAAGC A-131070.1 X10677 ascsasgsgs(5MdC)sdAs(5MdC)s(5MdC)s(5MdC)sdAs 59 ACAGGCACCCAGCTTG 243 dGs(5MdC)sdTsdTsdGsgsgsuscsa GGTCA A-131071.1 X10678 gsgsgsusgsdTs(5MdC)sdAsdTsdTsdAs(5MdC)sdAsdG 60 GGGTGTCATTACAGGC 244 sdGs(5MdC)scsascscsc CACCC A-131072.1 X10679 asgsususgs(5MdC)sdTsdGsdGsdAsdGsdGsdGsdTsdGs 61 AGTTGCTGGAGGGTGT 245 dTsuscsasusu TCATT A-131073.1 X10680 usascscsus(5MdC)s(5MdC)sdAsdTs(5MdC)sdAsdGsd 62 TACCTCCATCAGTTGC 246 TsdTsdGs(5MdC)scsusgsgsa CTGGA A-131074.1 X10681 csasasascsdTsdTsdAsdAsdAsdTsdAs(5MdC)s(5MdC)s 63 CAAACTTAAATACCTC 247 dTs(5MdC)scscsasusc CCATC A-131075.1 X10682 gsasusasusdGsdGsdTsdGsdTs(5MdC)sdAsdAsdAs 64 GATATGGTGTCAAACT 248 (5MdC)sdTsususasasa TTAAA A-131076.1 X10683 usgsusususdTs(5MdC)sdTs(5MdC)sdAsdGsdAsdTsdAs 65 TGTTTTCTCAGATATG 249 dTsdGsgsgsusgsu GGTGT A-131077.1 X10684 uscsusgsasdTs(5MdC)sdAsdGsdAsdTsdGsdTsdTsdTsd 66 TCTGATCAGATGTTTT 250 Tsuscsuscsa TCTCA A-131078.1 X10685 asasgsasasdGsdTsdGsdGsdAsdTs(5MdC)sdTsdGsdAsd 67 AAGAAGTGGATCTGAT 251 Tsuscsasgsa TCAGA A-131079.1 X10686 usususgsgs(5MdC)sdAsdAsdAsdGsdAsdAsdGsdAsdAsd 68 TTTGGCAAAGAAGAAG 252 Gsgsusgsgsa GTGGA A-131080.1 X10687 gsgscsasgsdTsdTs(5MdC)sdAsdGsdTsdTsdTsdGsdGs 69 GGCAGTTCAGTTTGGC 253 (5MdC)scsasasasg CAAAG A-131081.1 X10688 csgsasusasdGsdAsdGsdTs(5MdC)sdGsdGs(5MdC)sdAs 70 CGATAGAGTCGGCAGT 254 dGsdTsususcsasg TTCAG A-131082.1 X10689 gsususgsgs(5MdC)sdTsdTsdTsdTs(5MdC)sdGsdAsdTs 71 GTTGGCTTTTCGATAG 255 dAsdGsgsasgsusc GAGTC A-131083.1 X10690 usgsgsasgsdGsdAsdTsdTsdTsdGsdTsdTsdGsdGs(5MdC) 72 TGGAGGATTTGTTGGC 256 scsusususu CTTTT A-131084.1 X10691 gsasusascsdTsdAsdAs(5MdC)sdTsdTsdGsdGsdAsdGsdG 73 GATACTAACTTGGAGG 257
sgsasususu GATTT A-131085.1 X10692 gscsgsasusdTsdGsdGs(5MdC)sdTsdGsdAsdTsdAs(5MdC) 74 GCGATTGGCTGATACT 258 sdTsusasascsu TAACT A-131086.1 X10693 csuscscsasdAsdAsdAsdAsdGsdGs(5MdC)sdGsdAsdTsdTs 75 CTCCAAAAAGGCGATT 259 usgsgscsu TGGCT A-131087.1 X10694 asgsgsgsasdTsdTsdTsdGsdTs(5MdC)sdTs(5MdC)s 76 AGGGATTTGTCTCCAA 260 (5MdC)sdAsdAsasasasasg AAAAG A-131088.1 X10695 asususgsasdAsdGsdGsdTsdAsdAsdGsdGsdGsdAsdTsusus 77 ATTGAAGGTAAGGGAT 261 usgsu TTTGT A-131089.1 X10696 gsgsusasgsdGsdTs(5MdC)sdTs(5MdC)sdAsdTsdTsdGsdA 78 GGTAGGTCTCATTGAA 262 sdAsasgsgsusa AGGTA A-131090.1 X10697 csusgsasusdGsdTs(5MdC)s(5MdC)sdTsdGsdGsdTsdAsdG 79 CTGATGTCCTGGTAGG 263 sdGsgsuscsusc GTCTC A-131091.1 X10698 usascscsasdAs(5MdC)sdTs(5MdC)sdAs(5MdC)sdTsdGsd 80 TACCAACTCACTGATG 264 AsdTsdGsgsuscscsu GTCCT A-131092.1 X10699 usgsgscsus(5MdC)s(5MdC)sdAsdTsdAsdTsdAs(5MdC)s 81 TGGCTCCATATACCAA 265 (5MdC)sdAsdAsascsuscsa ACTCA A-131093.1 X10700 gsgscsusgsdGsdAsdGs(5MdC)sdTsdTsdGsdGs(5MdC)sdT 82 GGCTGGAGCTTGGCTC 266 s(5MdC)scscsasusa CCATA A-131094.1 X10701 gsasasgsus(5MdC)s(5MdC)sdAsdGsdGsdGsdGs(5MdC)sd 83 GAAGTCCAGGGGCTGG 267 TsdGsdGsgsasgscsu GAGCT A-131095.1 X10702 csasusususdTs(5MdC)s(5MdC)sdTsdTsdGsdAsdAsdGsdT 84 CATTTTCCTTGAAGTC 268 s(5MdC)scscsasgsg CCAGG A-131096.1 X10703 gsasususgs(5MdC)sdTs(5MdC)sdTsdGs(5MdC)sdAsdTsd 85 GATTGCTCTGCATTTT 269 TsdTsdTsuscscsusu TCCTT A-131097.1 X10704 gsgscscsgs(5MdC)sdTs(5MdC)sdTsdGsdGsdAsdTsdTsdG 86 GGCCGCTCTGGATTGC 270 s(5MdC)scsuscsusg CTCTG A-131098.1 X10705 asusususgsdTsdTsdGsdAsdTsdGsdGs(5MdC)s(5MdC)sdG 87 ATTTGTTGATGGCCGC 271 s(5MdC)scsuscsusg CTCTG A-131099.1 X10706 ususgsgsas(5MdC)sdAs(5MdC)s(5MdC)s(5MdC)sdAsdTs 88 TTGGACACCCATTTGT 272 dTsdTsdGsdTsususgsasu TTGAT A-131100.1 X10707 ususcsgsgsdTs(5MdC)sdTsdTsdAsdTsdTsdGsdGsdAs 89 TTCGGTCTTATTGGAC 273 (5MdC)scsascscsc CACCC A-131101.1 X10708 usgsasusus(5MdC)sdGsdGs(5MdC)s(5MdC)sdTsdTs 90 TGATTCGGCCTTCGGT 274 (5MdC)sdGsdGsdTsuscsususa TCTTA A-131102.1 X10709 asusgsascsdAsdTs(5MdC)sdGsdGsdTsdGsdAsdTsdTs 91 ATGACATCGGTGATTC 275 (5MdC)scsgsgscsc CGGCC A-131103.1 X10710 ususcscsgsdAsdGsdGsdGsdAsdAsdTsdGsdAs(5MdC)sdAs 92 TTCCGAGGGAATGACA 276 asuscsgsg ATCGG A-131104.1 X10711 csasususgsdAsdTsdGsdGs(5MdC)sdTsdTs(5MdC)s 93 CATTGATGGCTTCCGA 277 (5MdC)sdGsdAsasgsgsgsa AGGGA A-131105.1 X10712 ascsasgsusdGsdAsdGs(5MdC)sdTs(5MdC)sdAsdTsdTsdG 94 ACAGTGAGCTCATTGA 278 sdAsasusgsgsc ATGGC A-131106.1 X10713 csasgscsas(5MdC)s(5MdC)sdAsdGsdAsdAs(5MdC)sdAsd 95 CAGCACCAGAACAGTG 279 GsdTsdGsgsasgscsu GAGCT A-131107.1 X10714 asascscsasdGs(5MdC)sdAs(5MdC)s(5MdC)sdAsdGsdAsd 96 AACCAGCACCAGAACA 280 As(5MdC)sdAsasgsusgsa AGTGA A-131108.1 X10715 usasascscsdAsdGs(5MdC)sdAs(5MdC)s(5MdC)sdAsdGsd 97 TAACCAGCACCAGAAC 281 AsdAs(5MdC)scsasgsusg CAGTG A-131109.1 X10716 ususasascs(5MdC)sdAsdGs(5MdC)sdAs(5MdC)s(5MdC)s 98 TTAACCAGCACCAGAA 282 dAsdGsdAsdAsascsasgsu ACAGT A-131110.1 X10717 gsususasas(5MdC)s(5MdC)sdAsdGs(5MdC)sdAs(5MdC)s 99 GTTAACCAGCACCAGA 283 (5MdC)sdAsdGsdAsasascsasg AACAG A-131111.1 X10718 usgsususasdAs(5MdC)s(5MdC)sdAsdGs(5MdC)sdAs 100 TGTTAACCAGCACCAG 284 (5MdC)s(5MdC)sdAsdGsgsasascsa GAACA A-131112.1 X10719 gsusgsususdAsdAs(5MdC)s(5MdC)sdAsdGs(5MdC)sdAs 101 GTGTTAACCAGCACCA 285 (5MdC)s(5MdC)sdAsasgsasasc AGAAC A-131113.1 X10720 gsgsusgsusdTsdAsdAs(5MdC)s(5MdC)sdAsdGs(5MdC)sd 102 GGTGTTAACCAGCACC 286 As(5MdC)s(5MdC)scsasgsasa CAGAA A-131114.1 X10721 usgsgsusgsdTsdTsdAsdAs(5MdC)s(5MdC)sdAsdGs 103 TGGTGTTAACCAGCAC 287 (5MdC)sdAs(5MdC)scscsasgsa CCAGA A-131115.1 X10722 asusgsgsusdGsdTsdTsdAsdAs(5MdC)s(5MdC)sdAsdGs 104 ATGGTGTTAACCAGCA 288 (5MdC)sdAsascscsasg ACCAG A-131116.1 X10723 asasusgsgsdTsdGsdTsdTsdAsdAs(5MdC)s(5MdC)sdAsdG 105 AATGGTGTTAACCAGC 289 s(5MdC)scsascscsa CACCA A-131117.1 X10724 asasasusgsdGsdTsdGsdTsdTsdAsdAs(5MdC)s(5MdC)sdA 106 AAATGGTGTTAACCAG 290 sdGsgscsascsc GCACC A-131118.1 X10725 usasasasusdGsdGsdTsdGsdTsdTsdAsdAs(5MdC)s(5MdC) 107 TAAATGGTGTTAACCA 291 sdAsasgscsasc AGCAC A-131119.1 X10726 gsusasasasdTsdGsdGsdTsdGsdTsdTsdAsdAs(5MdC)s 108 GTAAATGGTGTTAACC 292 (5MdC)scsasgscsa CAGCA A-131120.1 X10727 asgsusasasdAsdTsdGsdGsdTsdGsdTsdTsdAsdAs(5MdC)s 109 AGTAAATGGTGTTAAC 293 cscsasgsc CCAGC A-131121.1 X10728 asasgsusasdAsdAsdTsdGsdGsdTsdGsdTsdTsdAsdAsascs 110 AAGTAAATGGTGTTAA 294 csasg ACCAG A-131122.1 X10729 gsasasgsusdAsdAsdAsdTsdGsdGsdTsdGsdTsdTsdAsasas 111 GAAGTAAATGGTGTTA 295 cscsa AACCA A-131123.1 X10730 usgsasasgsdTsdAsdAsdAsdTsdGsdGsdTsdGsdTsdTsusas 112 TGAAGTAAATGGTGTT 296 ascsc TAACC A-131124.1 X10731 ususgsasasdGsdTsdAsdAsdAsdTsdGsdGsdTsdGsdTsusus 113 TTGAAGTAAATGGTGT 297 asasc TTAAC A-131125.1 X10732 csususgsasdAsdGsdTsdAsdAsdAsdTsdGsdGsdTsdGsgsus 114 CTTGAAGTAAATGGTG 298 usasa GTTAA A-131126.1 X10733 cscsususgsdAsdAsdGsdTsdAsdAsdAsdTsdGsdGsdTsusgs 115 CCTTGAAGTAAATGGT 299 ususa TGTTA A-131127.1 X10734 cscscsususdGsdAsdAsdGsdTsdAsdAsdAsdTsdGsdGsgsus 116 CCCTTGAAGTAAATGG 300 gsusu GTGTT A-131128.1 X10735 gscscscsusdTsdGsdAsdAsdGsdTsdAsdAsdAsdTsdGsgsgs 117 GCCCTTGAAGTAAATG 301 usgsu GGTGT A-131129.1 X10736 gsgscscscsdTsdTsdGsdAsdAsdGsdTsdAsdAsdAsdTsusgs 118 GGCCCTTGAAGTAAAT 302 gsusg TGGTG A-131130.1 X10737 asgsgscscs(5MdC)sdTsdTsdGsdAsdAsdGsdTsdAsdAsdAs 119 AGGCCCTTGAAGTAAA 303 asusgsgsu ATGGT A-131131.1 X10738 csasgsgscs(5MdC)s(5MdC)sdTsdTsdGsdAsdAsdGsdTsdA 120 CAGGCCCTTGAAGTAA 304 sdAsasasusgsg AATGG A-131132.1 X10739 ascsasgsgs(5MdC)s(5MdC)s(5MdC)sdTsdTsdGsdAsdAsd 121 ACAGGCCCTTGAAGTA 305 GsdTsdAsasasasusg AAATG A-131133.1 X10740 csascsasgsdGs(5MdC)s(5MdC)s(5MdC)sdTsdTsdGsdAsd 122 CACAGGCCCTTGAAGT 306 AsdGsdTsusasasasu TAAAT A-131134.1 X10741 cscsascsasdGsdGs(5MdC)s(5MdC)s(5MdC)sdTsdTsdGsd 123 CCACAGGCCCTTGAAG 307 AsdAsdGsgsusasasa GTAAA A-131135.1 X10742 uscscsascsdAsdGsdGs(5MdC)s(5MdC)s(5MdC)sdTsdTsd 124 TCCACAGGCCCTTGAA 308 GsdAsdAsasgsusasa AGTAA A-131136.1 X10743 ususcscsas(5MdC)sdAsdGsdGs(5MdC)s(5MdC)s(5MdC)s 125 TTCCACAGGCCCTTGA 309 dTsdTsdGsdAsasasgsusa AAGTA A-131137.1 X10744 csususcscsdAs(5MdC)sdAsdGsdGs(5MdC)s(5MdC)s 126 CTTCCACAGGCCCTTG 310 (5MdC)sdTsdTsdGsgsasasgsu GAAGT A-131138.1 X10745 ascsusususdGsdAs(5MdC)sdTsdTs(5MdC)s(5MdC)sdAs 127 ACTTTGACTTCCACAG 311 (5MdC)sdAsdGsgsgscscsc GGCCC A-131139.1 X10746 uscsasgsgsdGs(5MdC)sdTsdGsdAsdAs(5MdC)sdTsdTsdT 128 TCAGGGCTGAACTTTG 312 sdGsgsascsusu GACTT A-131140.1 X10747 cscsususgsdTsdGsdTsdTs(5MdC)sdTs(5MdC)sdAsdGsdG 129 CCTTGTGTTCTCAGGG 313 sdGsgscsusgsa GCTGA A-131141.1 X10748 ascsasgsusdTs(5MdC)s(5MdC)sdTsdTs(5MdC)s(5MdC)s 130 ACAGTTCCTTCCTTGT 314 dTsdTsdGsdTsusgsususc TGTTC A-131142.1 X10749 gscscsususdGsdTsdAsdGsdAsdAs(5MdC)sdAsdGsdTsdTs 131 GCCTTGTAGAACAGTT 315 uscscsusu TCCTT A-131143.1 X10750 csuscsuscs(5MdC)sdAsdTs(5MdC)sdAsdGs(5MdC)s 132 CTCTCCATCAGCCTTG 316 (5MdC)sdTsdTsdGsgsusasgsa GTAGA A-131144.1 X10751 csusgsasas(5MdC)sdAs(5MdC)sdGsdAs(5MdC)sdTs 133 CTGAACACGACTCTCC 317 (5MdC)sdTs(5MdC)s(5MdC) CATCA A-131145.1 X10752 asuscsasusdAsdGsdAsdTsdGs(5MdC)sdTsdGsdAsdAs 134 ATCATAGATGCTGAAC 318 (5MdC)scsascsgsa CACGA A-131146.1 X10753 ususcscsusdGsdGsdTsdAs(5MdC)sdAsdTs(5MdC)sdAsdT 135 TTCCTGGTACATCATA 319 sdAsasgsasusg AGATG A-131147.1 X10754 gsgsasascsdTsdTsdGs(5MdC)s(5MdC)sdTsdTs(5MdC)s 136 GGAACTTGCCTTCCTG 320
(5MdC)sdTsdGsgsgsusasc GGTAC A-131148.1 X10755 csgscscsgsdAsdTsdAsdAs(5MdC)sdGsdGsdAsdAs(5MdC) 137 CGCCGATAACGGAACT 321 sdTsususgscsc TTGCC A-131149.1 X10756 ususcsasgs(5MdC)s(5MdC)sdAs(5MdC)sdGs(5MdC)sdGs 138 TTCAGCCACGCGCCGA 322 (5MdC)s(5MdC)sdGsdAsasusasasc ATAAC A-131150.1 X10757 cscsusgsgsdGsdTsdGs(5MdC)s(5MdC)sdTsdTs(5MdC)sd 139 CCTGGGTGCCTTCAGC 323 AsdGs(5MdC)scscsascsg CCACG A-131151.1 X10758 asascsuscsdAsdAsdGs (5MdC)sdAs(5MdC)s(5MdC)sdTs 140 AACTCAAGCACCTGGG 324 dGsdGsdGsgsusgscsc GTGCC A-131152.1 X10759 usususgsasdAsdGsdGsdGs(5MdC)sdAsdAs(5MdC)sdTs 141 TTTGAAGGGCAACTCA 325 (5MdC)sdAsasasgscsa AAGCA A-131153.1 X10760 usgsuscsasdTs(5MdC)sdAs(5MdC)s(5MdC)sdTsdTsdTsd 142 TGTCATCACCTTTGAA 326 GsdAsdAsasgsgsgsc AGGGC A-131154.1 X10761 ascscsasusdGsdGsdTsdGsdAsdTsdGsdTs(5MdC)sdAsdTs 143 ACCATGGTGATGTCAT 327 uscsascsc TCACC A-131155.1 X10762 csasasgsasdTsdGsdAsdGsdGsdAs(5MdC)s(5MdC)sdAsdT 144 CAAGATGAGGACCATG 328 sdGsgsgsusgsa GGTGA A-131156.1 X10763 csasgsgscsdTsdTsdGsdGsdGs(5MdC)sdAsdAsdGsdAsdTs 145 CAGGCTTGGGCAAGAT 329 usgsasgsg TGAGG A-131157.1 X10764 asgsgscsus(5MdC)sdTsdTs(5MdC)sdTs(5MdC)sdAsdGsd 146 AGGCTCTTCTCAGGCT 330 Gs(5MdC)sdTsususgsgsg TTGGG A-131158.1 X10765 usascscsusdTsdGsdGs(5MdC)s(5MdC)sdAsdGsdGs 147 TACCTTGGCCAGGCTC 331 (5MdC)sdTs(5MdC)scsususcsu CTTCT A-131159.1 X10766 gsususcscsdTsdTs(5MdC)sdTs(5MdC)sdTsdAs(5MdC)s 148 GTTCCTTCTCTACCTT 332 (5MdC)sdTsdTsusgsgscsc TGGCC A-131160.1 X10767 uscsusgsgsdGsdGsdTsdGsdAsdGsdTsdTs(5MdC)s(5MdC) 149 TCTGGGGTGAGTTCCT 333 sdTsususcsusc TTCTC A-131161.1 X10768 ususgscsasdGs(5MdC)sdAs(5MdC)s(5MdC)sdTs(5MdC)s 150 TTGCAGCACCTCTGGG 334 dTsdGsdGsdGsgsgsusgsa GGTGA A-131162.1 X10769 cscsasgscs(5MdC)sdAs(5MdC)sdTs(5MdC)sdTsdTsdGs 151 CCAGCCACTCTTGCAG 335 (5MdC)sdAsdGsgscsascsc GCACC A-131163.1 X10770 uscscsasasdTsdTs(5MdC)sdAsdTs(5MdC)s(5MdC)sdAsd 152 TCCAATTCATCCAGCC 336 Gs(5MdC)s(5MdC)scsascsusc CACTC A-131164.1 X10771 csasuscsasdTs(5MdC)sdTs(5MdC)s(5MdC)sdTs(5MdC)s 153 CATCATCTCCTCCAAT 337 (5MdC)sdAsdAsdTsususcsasu TTCAT A-131165.1 X10772 gsgsascscsdAs(5MdC)s(5MdC)sdAsdGs(5MdC)sdAsdTs 154 GGACCACCAGCATCAT 338 (5MdC)sdAsdTsuscsuscsc TCTCC A-131166.1 X10773 csgsgsgsgs(5MdC)sdAsdTsdGsdTsdGsdGsdAs(5MdC)s 155 CGGGGCATGTGGACCA 339 (5MdC)sdAsascscsasg ACCAG A-131167.1 X10774 asasusgscsdGsdGsdAsdAsdGs(5MdC)sdGsdGsdGsdGs 156 AATGCGGAAGCGGGGC 340 (5MdC)scsasusgsu CATGT A-131168.1 X10775 asgscscsgsdTs(5MdC)s(5MdC)sdTs(5MdC)sdAsdAsdTsd 157 AGCCGTCCTCAATGCG 341 Gs(5MdC)sdGsgsgsasasg GGAAG A-131169.1 X10776 ususcsasasdAs(5MdC)sdTsdGsdAsdAsdGs(5MdC)s 158 TTCAAACTGAAGCCGT 342 (5MdC)sdGsdTsuscscsusc TCCTC A-131170.1 X10777 csasgscsusdGs(5MdC)sdTs(5MdC)s(5MdC)sdTsdTs 159 CAGCTGCTCCTTCAAA 343 (5MdC)sdAsdAsdAsascsusgsa ACTGA A-131171.1 X10778 cscsasusgsdTs(5MdC)sdTsdTsdGs(5MdC)sdAsdGs 160 CCATGTCTTGCAGCTG 344 (5MdC)sdTsdGsgscsuscsc GCTCC A-131172.1 X10779 uscsgsascsdAsdAsdGsdGs(5MdC)s(5MdC)s(5MdC)sdAsd 161 TCGACAAGGCCCATGT 345 TsdGsdTsuscsususg TCTTG A-131173.1 X10780 gscsusgsasdAs(5MdC)sdAsdGsdAsdTs(5MdC)sdGsdAs 162 GCTGAACAGATCGACA 346 (5MdC)sdAsasasgsgsc AAGGC A-131174.1 X10781 ascsusususdTs(5MdC)sdAsdGsdGsdGs(5MdC)sdTsdGsdA 163 ACTTTTCAGGGCTGAA 347 sdAsascsasgsa ACAGA A-131175.1 X10782 gsgsgsasgsdTsdTsdTsdGsdGsdAs(5MdC)sdTsdTsdTsdTs 164 GGGAGTTTGGACTTTT 348 uscsasgsg TCAGG A-131176.1 X10783 asascsasasdTsdAs(5MdC)s(5MdC)sdTsdGsdGsdGsdAsdG 165 AACAATACCTGGGAGT 349 sdTsusususgsg TTTGG A-131177.1 X10784 gsgscscsusdTs(5MdC)sdTsdGs(5MdC)sdAsdAs(5MdC)sd 166 GGCCTTCTGCAACAAT 350 AsdAsdTsusascscsu TACCT A-131178.1 X10785 asgsgsuscsdAsdTs(5MdC)sdTs(5MdC)sdGsdGs(5MdC)s 167 AGGTCATCTCGGCCTT 351 (5MdC)sdTsdTsuscsusgsc TCTGC A-131179.1 X10786 usgsasgsas(5MdC)sdAsdTsdAsdGsdAsdGsdGsdTs(5MdC) 168 TGAGACATAGAGGTCA 352 sdAsasuscsusc ATCTC A-131180.1 X10787 gsgsasasusdGs(5MdC)sdAsdTs(5MdC)sdTsdGsdAsdGsdA 169 GGAATGCATCTGAGAC 353 s(5MdC)scsasusasg CATAG A-131181.1 X10788 asasusgscs(5MdC)sdTsdTsdAsdTsdGsdGsdAsdAsdTsdGs 170 AATGCCTTATGGAATG 354 gscsasusc GCATC A-131182.1 X10789 usascscsus(5MdC)sdAsdAsdGsdAsdAsdAsdTsdGs(5MdC) 171 TACCTCAAGAAATGCC 355 s(5MdC)scsususasu CTTAT A-131183.1 X10790 csususcsusdTs(5MdC)sdAsdTsdTsdTsdAs(5MdC)s 172 CTTCTTCATTTACCTC 356 (5MdC)sdTs(5MdC)scsasasgsa CAAGA A-131184.1 X10791 gscsususcsdAs(5MdC)sdTsdGs(5MdC)s(5MdC)sdTsdTs 173 GCTTCACTGCCTTCTT 357 (5MdC)sdTsdTsuscsasusu TCATT A-131185.1 X10792 ascsususgs(5MdC)sdAsdGs(5MdC)sdTsdGs(5MdC)sdTsd 174 ACTTGCAGCTGCTTCA 358 Ts(5MdC)sdAsascsusgsc ACTGC A-131186.1 X10793 csasascsasdGs(5MdC)sdGsdGsdTsdAs(5MdC)sdTsdTsdG 175 CAACAGCGGTACTTGC 359 s(5MdC)scsasgscsu CAGCT A-131187.1 X10794 cscsasgscsdAsdAsdTs(5MdC)sdAs(5MdC)sdAsdAs(5MdC) 176 CCAGCAATCACAACAG 360 sdAsdGsgscsgsgsu GCGGT A-131188.1 X10795 usasgscsgsdAsdAs(5MdC)sdGsdGs(5MdC)s(5MdC)sdAsd 177 TAGCGAACGGCCAGCA 361 Gs(5MdC)sdAsasasuscsa AATCA A-131189.1 X10796 usgsususgsdGsdGsdGsdTsdTsdTsdAsdGs(5MdC)sdGsdAs 178 TGTTGGGGTTTAGCGA 362 asascsgsg AACGG A-131190.1 X10797 asasasgsus(5MdC)sdAs(5MdC)s(5MdC)s(5MdC)sdTsdGs 179 AAAGTCACCCTGTTGG 363 dTsdTsdGsdGsgsgsgsusu GGGTT A-131191.1 X10798 gsususgsgs(5MdC)s(5MdC)sdTsdTsdGsdAsdAsdAsdGsdT 180 GTTGGCCTTGAAAGTC 364 s(5MdC)scsascscsc CACCC A-131192.1 X10799 gsgsasasasdGsdGs(5MdC)s(5MdC)sdTsdGsdTsdTsdGsdG 181 GGAAAGGCCTGTTGGC 365 s(5MdC)scscsususg CCTTG A-131193.1 X10800 asusasasasdAsdAs(5MdC)s(5MdC)sdAsdGsdGsdAsdAsdA 182 ATAAAAACCAGGAAAG 366 sdGsgsgscscsu GGCCT A-131194.1 X10801 asascsusus(5MdC)sdTs(5MdC)sdTsdTsdAsdTsdAsdAsdA 183 AACTTCTCTTATAAAA 367 sdAsasascscsa AACCA A-131195.1 X10802 usgsususcsdAsdGsdAsdGsdGsdAsdAs(5MdC)sdTsdTs 184 TGTTCAGAGGAACTTC 368 (5MdC)scsuscsusu CTCTT A-131196.1 X10803 asasgsasusdAsdAsdTsdAsdGsdTsdGsdTsdTs(5MdC)sdAs 185 AAGATAATAGTGTTCA 369 asgsasgsg AGAGG A-131197.1 X10804 uscsusgscs(5MdC)s(5MdC)sdAsdTsdGsdAsdAsdGsdAsdT 186 TCTGCCCATGAAGATA 370 sdAsasasusasg AATAG A-131198.1 X10805 gsgsususgsdGs(5MdC)sdTsdAs(5MdC)sdTs(5MdC)sdTsd 187 GGTTGGCTACTCTGCC 371 Gs(5MdC)s(5MdC)scscsasusg CCATG A-131199.1 X10806 ususasascsdAs(5MdC)sdAsdAsdGsdGsdGsdTsdTsdGsdGs 188 TTAACACAAGGGTTGG 372 gscsusasc GCTAC A-131200.1 X10807 asascsasusdTsdTsdTsdAs(5MdC)sdTsdTsdAsdAs(5MdC) 189 AACATTTTACTTAACA 373 sdAsascsasasg ACAAG A-131201.1 X10808 asasasgsasdAsdTsdAsdAsdGsdAsdAs(5MdC)sdAsdTsdTs 190 AAAGAATAAGAACATT 374 usususasc TTTAC A-131202.1 X10809 gsasasgsasdGsdGsdTsdGs(5MdC)sdAsdAsdAsdGsdAsdAs 191 GAAGAGGTGCAAAGAA 375 asusasasg ATAAG A-131203.1 X10810 cscsasasasdAsdAsdTsdAsdGsdGsdAsdAsdGsdAsdGsgsgs 192 CCAAAAATAGGAAGAG 376 usgsc GGTGC A-131204.1 X10811 usgsususcsdAs(5MdC)sdAsdAsdAs(5MdC)s(5MdC)sdAsd 193 TGTTCACAAACCAAAA 377 AsdAsdAsasasusasg AATAG A-131205.1 X10812 usususususdAs(5MdC)sdTsdTs(5MdC)sdTsdGsdTsdTs 194 TTTTTACTTCTGTTCA 378 (5MdC)sdAsascsasasa ACAAA A-131206.1 X10813 usususgsusdAsdTsdTsdTsdAsdTsdTsdTsdTsdTsdAsascs 195 TTTGTATTTATTTTTA 379 ususc ACTTC A-131207.1 X10814 asusgsgsasdAsdGsdTsdAsdGsdTsdTsdTsdGsdTsdAsasus 196 ATGGAAGTAGTTTGTA 380 ususa ATTTA
TABLE-US-00004 TABLE 4 Antisense polynucleotides targeting Serpinc1(AT3) Start Position SEQ Antisense SEQ Sense SEQ in NM_ ID Sequence ID Sequence ID Sequence ID 000488.3 Modified Sequence (5'-3') NO. (5'-3') NO. (5'-3') NO. NM_000488.3_ 20 csusasasasdTs(m5dCs)dTs(m5dCs)d 381 CUAAAUCUCGCAG 565 GGAACCUCUGCGAGA 749 20-39_aso Gs(m5dCs)dAsdGsdAsdGsgsususcsc AGGUUCC UUUAG NM_000488.3_ 30 gsususcsusdTsdTs(m5dCs)(m5dCs)d 382 GUUCUUUCCUCUA 566 CGAGAUUUAGAGGAA 750 30-49_aso Ts(m5dCs)dTsdAsdAsdAsuscsuscsg AAUCUCG AGAAC NM_000488.3_ 40 csusgsasasdAsdAs(m5dCs)dTsdGsdG 383 CUGAAAACUGGUU 567 AGGAAAGAACCAGUU 751 40-59_aso sdTsdTs(m5dCs)dTsususcscsu CUUUCCU UUCAG NM_000488.3_ 50 gsgscsasasdTs(m5dCs)(m5dCs)dGs 384 GGCAAUCCGCCUG 568 CAGUUUUCAGGCGGA 752 50-69_aso (m5dCs)(m5dCs)dTsdGsdAsdAsasasc AAAACUG UUGCC susg NM_000488.3_ 60 usgsusgsasdTs(m5dCs)dTsdGsdAsdG 385 UGUGAUCUGAGGC 569 GCGGAUUGCCUCAGA 753 60-79_aso sdGs(m5dCs)dAsdAsuscscsgsc AAUCCGC UCACA NM_000488.3_ 70 gsusgsgsasdGsdAsdTsdAsdGsdTsdGs 386 GUGGAGAUAGUGU 570 UCAGAUCACACUAUC 754 70-89_aso dTsdGsdAsuscsusgsa GAUCUGA UCCAC NM_000488.3_ 80 gsgscsusgsdGsdGs(m5dCs)dAsdAsdG 387 GGCUGGGCAAGUG 571 CUAUCUCCACUUGCC 755 80-99_aso sdTsdGsdGsdAsgsasusasg GAGAUAG CAGCC NM_000488.3_ 90 uscsususcs(m5dCs)dAs(m5dCs)dAsd 388 UCUUCCACAGGGC 572 UUGCCCAGCCCUGUG 756 90-109_aso GsdGsdGs(m5dCs)dTsdGsgsgscsasa UGGGCAA GAAGA NM_000488.3_ 100 usgsgscscsdGs(m5dCs)dTsdAsdAsdT 389 UGGCCGCUAAUCU 573 CUGUGGAAGAUUAGC 757 100-119_aso s(m5dCs)dTsdTs(m5dCs)csascsasg UCCACAG GGCCA NM_000488.3_ 110 ususgsgsasdAsdTsdAs(m5dCs)dAsdT 390 UUGGAAUACAUGG 574 UUAGCGGCCAUGUAU 758 110-129_aso sdGsdGs(m5dCs)(m5dCs)gscsusasa CCGCUAA UCCAA NM_000488.3_ 120 uscscsusasdTs(m5dCs)dAs(m5dCs)d 391 UCCUAUCACAUUG 575 UGUAUUCCAAUGUGA 759 120-139_aso AsdTsdTsdGsdGsdAsasusascsa GAAUACA UAGGA NM_000488.3 130 asgsgsususdAs(m5dCs)dAsdGsdTsdT 392 AGGUUACAGUUCC 576 UGUGAUAGGAACUGU 760 130-149_aso s(m5dCs)(m5dCs)dTsdAsuscsascsa UAUCACA AACCU NM_000488.3_ 140 csususususdTs(m5dCs)(m5dCs)dAsd 393 CUUUUUCCAGAGG 577 ACUGUAACCUCUGGA 761 140-159_aso GsdAsdGsdGsdTsdTsascsasgsu UUACAGU AAAAG NM_000488.3_ 150 asusasasas(m5dCs)(m5dCs)dTsdTs 394 AUAAACCUUCCUU 578 CUGGAAAAAGGAAGG 762 150-169_aso (m5dCs)(m5dCs)dTsdTsdTsdTsuscsc UUUCCAG UUUAU sasg NM_000488.3_ 160 asgsgsascsdAsdAsdAsdAsdGsdAsdTs 395 AGGACAAAAGAUA 579 GAAGGUUUAUCUUUU 763 160-179_aso dAsdAsdAscscsususc AACCUUC GUCCU NM_000488.3_ 170 asusgsasgs(m5dCs)dAsdGs(m5dCs)d 396 AUGAGCAGCAAGG 580 CUUUUGUCCUUGCUG 764 170-189_aso AsdAsdGsdGsdAs(m5dCs)asasasasg ACAAAAG CUCAU NM_000488.3_ 180 cscsasgsasdAsdGs(m5dCs)(m5dCs)d 397 CCAGAAGCCAAUG 581 UGCUGCUCAUUGGCU 765 180-199_aso AsdAsdTsdGsdAsdGscsasgscsa AGCAGCA UCUGG NM_000488.3_ 190 uscsascsgs(m5dCs)dAsdGsdTs 398 UCACGCAGUCCCA 582 UGGCUUCUGGGACUG 766 190-209_aso (m5dCs)(m5dCs)(m5dCs)dAsdGsdAsa GAAGCCA CGUGA sgscscsa NM_000488.3_ 200 cscsgsusgsdAs(m5dCs)dAsdGsdGsdT 399 CCGUGACAGGUCA 583 GACUGCGUGACCUGU 767 200-219_aso s(m5dCs)dAs(m5dCs)dGscsasgsusc CGCAGUC CACGG NM_000488.3_ 210 csascsasgsdGsdGs(m5dCs)dTs 400 CACAGGGCUCCCG 584 CCUGUCACGGGAGCC 768 210-229_aso (m5dCs)(m5dCs)(m5dCs)dGsdTsdGsa UGACAGG CUGUG scsasgsg NM_000488.3_ 220 usgscsasgsdAsdTsdGsdTs(m5dCs) 401 UGCAGAUGUCCAC 585 GAGCCCUGUGGACAU 769 220-239_aso (m5dCs)dAs(m5dCs)dAsdGsgsgscsus AGGGCUC CUGCA c NM_000488.3_ 230 gsgscsususdGsdGs(m5dCs)dTsdGsdT 402 GGCUUGGCUGUGC 586 GACAUCUGCACAGCC 770 230-249_aso sdGs(m5dCs)dAsdGsasusgsusc AGAUGUC AAGCC NM_000488.3_ 240 asasusgsus(m5dCs)(m5dCs)(m5dCs) 403 AAUGUCCCGCGGC 587 CAGCCAAGCCGCGGG 771 240-259_aso dGs(m5dCs)dGsdGs(m5dCs)dTsdTsgs UUGGCUG ACAUU gscsusg NM_000488.3_ 250 gsasususcsdAsdTsdGsdGsdGsdAsdAs 404 GAUUCAUGGGAAU 588 GCGGGACAUUCCCAU 772 250-269_aso dTsdGsdTscscscsgsc GUCCCGC GAAUC NM_000488.3_ 260 asusgscsas(m5dCs)dAsdTsdGsdGsdG 405 AUGCACAUGGGAU 589 CCCAUGAAUCCCAUG 773 260-279_aso sdAsdTsdTs(m5dCs)asusgsgsg UCAUGGG UGCAU NM_000488.3_ 270 gsgsasgscsdGsdGsdTsdAsdAsdAsdTs 406 GGAGCGGUAAAUG 590 CCAUGUGCAUUUACC 774 270-289_aso dGs(m5dCs)dAscsasusgsg CACAUGG GCUCC NM_000488.3_ 280 uscsususcsdTs(m5dCs)(m5dCs)dGsd 407 UCUUCUCCGGGGA 591 UUACCGCUCCCCGGA 775 280-299_aso GsdGsdGsdAsdGs(m5dCs)gsgsusasa GCGGUAA GAAGA NM_000488.3_ 290 uscsasgsusdTsdGs(m5dCs)(m5dCs)d 408 UCAGUUGCCUUCU 592 CCGGAGAAGAAGGCA 776 290-309_aso TsdTs(m5dCs)dTsdTs(m5dCs)uscscs UCUCCGG ACUGA gsg NM_000488.3_ 300 gscscscsus(m5dCs)dAsdTs(m5dCs) 409 GCCCUCAUCCUCA 593 AGGCAACUGAGGAUG 777 300-319_aso (m5dCs)dTs(m5dCs)dAsdGsdTsusgsc GUUGCCU AGGGC scsu NM_000488.3_ 310 uscsusgsusdTs(m5dCs)dTsdGsdAsdG 410 UCUGUUCUGAGCC 594 GGAUGAGGGCUCAGA 778 310-329_aso s(m5dCs)(m5dCs)(m5dCs)dTscsasus CUCAUCC ACAGA csc NM_000488.3_ 320 uscscsgsgsdGsdAsdTs(m5dCs)dTsdT 411 UCCGGGAUCUUCU 595 UCAGAACAGAAGAUC 779 320-339_aso s(m5dCs)dTsdGsdTsuscsusgsa GUUCUGA CCGGA NM_000488.3_ 330 gsususgsgsdTsdGsdGs(m5dCs) 412 GUUGGUGGCCUCC 596 AGAUCCCGGAGGCCA 780 330-349_aso (m5dCs)dTs(m5dCs)(m5dCs)dGsdGsg GGGAUCU CCAAC sasuscsu NM_000488.3_ 340 asgsascsas(m5dCs)dGs(m5dCs) 413 AGACACGCCGGUU 597 GGCCACCAACCGGCG 781 340-359_aso (m5dCs)dGsdGsdTsdTsdGsdGsusgsgs GGUGGCC UGUCU csc NM_000488.3_ 350 gsascsasgsdTsdTs(m5dCs)(m5dCs) 414 GACAGUUCCCAGA 598 CGGCGUGUCUGGGAA 782 350-369_aso (m5dCs)dAsdGsdAs(m5dCs)dAscsgsc CACGCCG CUGUC scsg NM_000488.3_ 360 asususgsgs(m5dCs)(m5dCs)dTsdTsd 415 AUUGGCCUUGGAC 599 GGGAACUGUCCAAGG 783 360-379_aso GsdGsdAs(m5dCs)dAsdGsususcscsc AGUUCCC CCAAU NM_000488.3_ 370 csasasasgs(m5dCs)dGsdGsdGsdAsdA 416 CAAAGCGGGAAUU 600 CAAGGCCAAUUCCCG 784 370-389_aso sdTsdTsdGsdGscscsususg GGCCUUG CUUUG NM_000488.3_ 380 asasasgsusdGsdGsdTsdAsdGs 417 AAAGUGGUAGCAA 601 UCCCGCUUUGCUACC 785 380-399_aso (m5dCs)dAsdAsdAsdGscsgsgsgsa AGCGGGA ACUUU NM_000488.3_ 390 gsusgscsusdGsdAsdTsdAsdGsdAsdAs 418 GUGCUGAUAGAAA 602 CUACCACUUUCUAUC 786 390-409_aso dAsdGsdTsgsgsusasg GUGGUAG AGCAC NM_000488.3_ 400 asasuscsusdGs(m5dCs)(m5dCs)dAsd 419 AAUCUGCCAGGUG 603 CUAUCAGCACCUGGC 787 400-419_aso GsdGsdTsdGs(m5dCs)dTsgsasusasg CUGAUAG AGAUU NM_000488.3_ 410 uscsasusus(m5dCs)dTsdTsdGsdGsdA 420 UCAUUCUUGGAAU 604 CUGGCAGAUUCCAAG 788 410-429_aso sdAsdTs(m5dCs)dTsgscscsasg CUGCCAG AAUGA NM_000488.3_ 420 gsususasus(m5dCs)dAsdTsdTsdGsdT 421 GUUAUCAUUGUCA 605 CCAAGAAUGACAAUG 789 420-439_aso s(m5dCs)dAsdTsdTscsususgsg UUCUUGG AUAAC NM_000488.3_ 430 ascsasgsgsdAsdAsdAsdAsdTsdGsdTs 422 ACAGGAAAAUGUU 606 CAAUGAUAACAUUUU 790 430-449_aso dTsdAsdTscsasususg AUCAUUG CCUGU NM_000488.3_ 440 csuscsasgsdGsdGsdGsdTsdGsdAs 423 CUCAGGGGUGACA 607 AUUUUCCUGUCACCC 791 440-459_aso (m5dCs)dAsdGsdGsasasasasu GGAAAAU CUGAG NM_000488.3_ 450 csgsusgsgsdAsdGsdAsdTsdAs 424 CGUGGAGAUACUC 608 CACCCCUGAGUAUCU 792 450-469_aso (m5dCs)dTs(m5dCs)dAsdGsgsgsgsus AGGGGUG CCACG g NM_000488.3_ 460 usasgscsasdAsdAsdAsdGs(m5dCs) 425 UAGCAAAAGCCGU 609 UAUCUCCACGGCUUU 793 460-479_aso (m5dCs)dGsdTsdGsdGsasgsasusa GGAGAUA UGCUA NM_000488.3_ 470 asgscsususdGsdGsdTs(m5dCs)dAsdT 426 AGCUUGGUCAUAG 610 GCUUUUGCUAUGACC 794 470-489_aso sdAsdGs(m5dCs)dAsasasasgsc CAAAAGC AAGCU NM_000488.3_ 480 ascsasgsgs(m5dCs)dAs(m5dCs) 427 ACAGGCACCCAGC 611 UGACCAAGCUGGGUG 795 480-499_aso (m5dCs)(m5dCs)dAsdGs(m5dCs)dTsd UUGGUCA CCUGU Tsgsgsuscsa NM_000488.3_ 490 gsgsgsusgsdTs(m5dCs)dAsdTsdTsdA 428 GGGUGUCAUUACA 612 GGGUGCCUGUAAUGA 796 490-509_aso s(m5dCs)dAsdGsdGscsascscsc GGCACCC CACCC NM_000488.3_ 500 asgsususgs(m5dCs)dTsdGsdGsdAsdG 429 AGUUGCUGGAGGG 613 AAUGACACCCUCCAG 797 500-519_aso sdGsdGsdTsdGsuscsasusu UGUCAUU CAACU NM_000488.3_ 510 usascscsus(m5dCs)(m5dCs)dAsdTs 430 UACCUCCAUCAGU 614 UCCAGCAACUGAUGG 798 510-529_aso (m5dCs)dAsdGsdTsdTsdGscsusgsgsa UGCUGGA AGGUA NM_000488.3_ 520 csasasascsdTsdTsdAsdAsdAsdTsdAs 431 CAAACUUAAAUAC 615 GAUGGAGGUAUUUAA 799 520-539_aso (m5dCs)(m5dCs)dTscscsasusc CUCCAUC GUUUG NM_000488.3_ 530 gsasusasusdGsdGsdTsdGsdTs 432 GAUAUGGUGUCAA 616 UUUAAGUUUGACACC 800 530-549_aso (m5dCs)dAsdAsdAs(m5dCs)ususasas ACUUAAA AUAUC a NM_000488.3_ 540 usgsusususdTs(m5dCs)dTs(m5dCs)d 433 UGUUUUCUCAGAU 617 ACACCAUAUCUGAGA 801 540-559_aso AsdGsdAsdTsdAsdTsgsgsusgsu AUGGUGU AAACA NM_000488.3_ 550 uscsusgsasdTs(m5dCs)dAsdGsdAsdT 434 UCUGAUCAGAUGU 618 UGAGAAAACAUCUGA 802 550-569_aso sdGsdTsdTsdTsuscsuscsa UUUCUCA UCAGA NM_000488.3_ 560 asasgsasasdGsdTsdGsdGsdAsdTs 435 AAGAAGUGGAUCU 619 UCUGAUCAGAUCCAC 803 560-579_aso (m5dCs)dTsdGsdAsuscsasgsa GAUCAGA UUCUU NM_000488.3_ 570 usususgsgs(m5dCs)dAsdAsdAsdGsdA 436 UUUGGCAAAGAAG 620 UCCACUUCUUCUUUG 804 570-589_aso sdAsdGsdAsdAsgsusgsgsa AAGUGGA CCAAA NM_000488.3_ 580 gsgscsasgsdTsdTs(m5dCs)dAsdGsdT 437 GGCAGUUCAGUUU 621 CUUUGCCAAACUGAA 805
580-599_aso sdTsdTsdGsdGscsasasasg GGCAAAG CUGCC NM_000488.3_ 590 csgsasusasdGsdAsdGsdTs(m5dCs)dG 438 CGAUAGAGUCGGC 622 CUGAACUGCCGACUC 806 590-609_aso sdGs(m5dCs)dAsdGsususcsasg AGUUCAG UAUCG NM_000488.3_ 600 gsususgsgs(m5dCs)dTsdTsdTsdTs 439 GUUGGCUUUUCGA 623 GACUCUAUCGAAAAG 807 600-619_aso (m5dCs)dGsdAsdTsdAsgsasgsusc UAGAGUC CCAAC NM_000488.3_ 610 usgsgsasgsdGsdAsdTsdTsdTsdGsdTs 440 UGGAGGAUUUGUU 624 AAAAGCCAACAAAUC 808 610-629_aso dTsdGsdGscsusususu GGCUUUU CUCCA NM_000488.3_ 620 gsasusascsdTsdAsdAs(m5dCs)dTsdT 441 GAUACUAACUUGG 625 AAAUCCUCCAAGUUA 809 620-639_aso sdGsdGsdAsdGsgsasususu AGGAUUU GUAUC NM_000488.3_ 630 gscsgsasusdTsdGsdGs(m5dCs)dTsdG 442 GCGAUUGGCUGAU 626 AGUUAGUAUCAGCCA 810 630-649_aso sdAsdTsdAs(m5dCs)usasascsu ACUAACU AUCGC NM_000488.3_ 640 csuscscsasdAsdAsdAsdAsdGsdGs 443 CUCCAAAAAGGCG 627 AGCCAAUCGCCUUUU 811 640-659_aso (m5dCs)dGsdAsdTsusgsgscsu AUUGGCU UGGAG NM_000488.3_ 650 asgsgsgsasdTsdTsdTsdGsdTs 444 AGGGAUUUGUCUC 628 CUUUUUGGAGACAAA 812 650-669_aso (m5dCs)dTs(m5dCs)(m5dCs)dAsasas CAAAAAG UCCCU asasg NM_000488.3_ 660 asususgsasdAsdGsdGsdTsdAsdAsdGs 445 AUUGAAGGUAAGG 629 ACAAAUCCCUUACCU 813 660-679_aso dGsdGsdAsusususgsu GAUUUGU UCAAU NM_000488.3_ 670 gsgsusasgsdGsdTs(m5dCs)dTs 446 GGUAGGUCUCAUU 630 UACCUUCAAUGAGAC 814 670-689_aso (m5dCs)dAsdTsdTsdGsdAsasgsgsusa GAAGGUA CUACC NM_000488.3_ 680 csusgsasusdGsdTs(m5dCs)(m5dCs)d 447 CUGAUGUCCUGGU 631 GAGACCUACCAGGAC 815 680-699_aso TsdGsdGsdTsdAsdGsgsuscsusc AGGUCUC AUCAG NM_000488.3_ 690 usascscsasdAs(m5dCs)dTs(m5dCs)d 448 UACCAACUCACUG 632 AGGACAUCAGUGAGU 816 690-709_aso As(m5dCs)dTsdGsdAsdTsgsuscscsu AUGUCCU UGGUA NM_000488.3_ 700 usgsgscsus(m5dCs)(m5dCs)dAsdTsd 449 UGGCUCCAUAUAC 633 UGAGUUGGUAUAUGG 817 700-719_aso AsdTsdAs(m5dCs)(m5dCs)dAsascsus CAACUCA AGCCA csa NM_000488.3_ 710 gsgscsusgsdGsdAsdGs(m5dCs)dTsdT 450 GGCUGGAGCUUGG 634 UAUGGAGCCAAGCUC 818 710-729_aso sdGsdGs(m5dCs)dTscscsasusa CUCCAUA CAGCC NM_000488.3_ 720 gsasasgsus(m5dCs)(m5dCs)dAsdGsd 451 GAAGUCCAGGGGC 635 AGCUCCAGCCCCUGG 819 720-739_aso GsdGsdGs(m5dCs)dTsdGsgsasgscsu UGGAGCU ACUUC NM_000488.3_ 730 csasusususdTs(m5dCs)(m5dCs)dTsd 452 CAUUUUCCUUGAA 636 CCUGGACUUCAAGGA 820 730-749_aso TsdGsdAsdAsdGsdTscscsasgsg GUCCAGG AAAUG NM_000488.3_ 740 gsasususgs(m5dCs)dTs(m5dCs)dTsd 453 GAUUGCUCUGCAU 637 AAGGAAAAUGCAGAG 821 740-759_aso Gs(m5dCs)dAsdTsdTsdTsuscscsusu UUUCCUU CAAUC NM_000488.3_ 750 gsgscscsgs(m5dCs)dTs(m5dCs)dTsd 454 GGCCGCUCUGGAU 638 CAGAGCAAUCCAGAG 822 750-769_aso GsdGsdAsdTsdTsdGscsuscsusg UGCUCUG CGGCC NM_000488.3_ 760 asusususgsdTsdTsdGsdAsdTsdGsdGs 455 AUUUGUUGAUGGC 639 CAGAGCGGCCAUCAA 823 760-779_aso (m5dCs)(m5dCs)dGscsuscsusg CGCUCUG CAAAU NM_000488.3_ 770 ususgsgsas(m5dCs)dAs(m5dCs) 456 UUGGACACCCAUU 640 AUCAACAAAUGGGUG 824 770-789_aso (m5dCs)(m5dCs)dAsdTsdTsdTsdGsus UGUUGAU UCCAA usgsasu NM_000488.3_ 780 ususcsgsgsdTs(m5dCs)dTsdTsdAsdT 457 UUCGGUCUUAUUG 641 GGGUGUCCAAUAAGA 825 780-799_aso sdTsdGsdGsdAscsascscsc GACACCC CCGAA NM_000488.3_ 790 usgsasusus(m5dCs)dGsdGs(m5dCs) 458 UGAUUCGGCCUUC 642 UAAGACCGAAGGCCG 826 790-809_aso (m5dCs)dTsdTs(m5dCs)dGsdGsuscsu GGUCUUA AAUCA susa NM_000488.3_ 800 asusgsascsdAsdTs(m5dCs)dGsdGsdT 459 AUGACAUCGGUGA 643 GGCCGAAUCACCGAU 827 800-819_aso sdGsdAsdTsdTscsgsgscsc UUCGGCC GUCAU NM_000488.3_ 810 ususcscsgsdAsdGsdGsdGsdAsdAsdTs 460 UUCCGAGGGAAUG 644 CCGAUGUCAUUCCCU 828 810-829_aso dGsdAs(m5dCs)asuscsgsg ACAUCGG CGGAA NM_000488.3_ 820 csasususgsdAsdTsdGsdGs(m5dCs)dT 461 CAUUGAUGGCUUC 645 UCCCUCGGAAGCCAU 829 820-839_aso sdTs(m5dCs)(m5dCs)dGsasgsgsgsa CGAGGGA CAAUG NM_000488.3_ 830 ascsasgsusdGsdAsdGs(m5dCs)dTs 462 ACAGUGAGCUCAU 646 GCCAUCAAUGAGCUC 830 830-849_aso (m5dCs)dAsdTsdTsdGsasusgsgsc UGAUGGC ACUGU NM_000488.3_ 840 csasgscsas(m5dCs)(m5dCs)dAsdGsd 463 CAGCACCAGAACA 647 AGCUCACUGUUCUGG 831 840-859_aso AsdAs(m5dCs)dAsdGsdTsgsasgscsu GUGAGCU UGCUG NM_000488.3_ 843 asascscsasdGs(m5dCs)dAs(m5dCs) 464 AACCAGCACCAGA 648 UCACUGUUCUGGUGC 832 843-862_aso (m5dCs)dAsdGsdAsdAs(m5dCs)asgsu ACAGUGA UGGUU sgsa NM_000488.3_ 844 usasascscsdAsdGs(m5dCs)dAs 465 UAACCAGCACCAG 649 CACUGUUCUGGUGCU 833 844-863_aso (m5dCs)(m5dCs)dAsdGsdAsdAscsasg AACAGUG GGUUA susg NM_000488.3_ 845 ususasascs(m5dCs)dAsdGs(m5dCs)d 466 UUAACCAGCACCA 650 ACUGUUCUGGUGCUG 834 845-864_aso As(m5dCs)(m5dCs)dAsdGsdAsascsas GAACAGU GUUAA gsu NM_000488.3_ 846 gsususasas(m5dCs)(m5dCs)dAsdGs 467 GUUAACCAGCACC 651 CUGUUCUGGUGCUGG 835 846-865_aso (m5dCs)dAs(m5dCs)(m5dCs)dAsdGsa AGAACAG UUAAC sascsasg NM_000488.3_ 847 usgsususasdAs(m5dCs)(m5dCs)dAsd 468 UGUUAACCAGCAC 652 UGUUCUGGUGCUGGU 836 847-866_aso Gs(m5dCs)dAs(m5dCs)(m5dCs)dAsgs CAGAACA UAACA asascsa NM_000488.3_ 848 gsusgsususdAsdAs(m5dCs)(m5dCs)d 469 GUGUUAACCAGCA 653 GUUCUGGUGCUGGUU 837 848-867_aso AsdGs(m5dCs)dAs(m5dCs)(m5dCs)as CCAGAAC AACAC gsasasc NM_000488.3_ 849 gsgsusgsusdTsdAsdAs(m5dCs) 470 GGUGUUAACCAGC 654 UUCUGGUGCUGGUUA 838 849-868_aso (m5dCs)dAsdGs(m5dCs)dAs(m5dCs)c ACCAGAA ACACC sasgsasa NM_000488.3_ 850 usgsgsusgsdTsdTsdAsdAs(m5dCs) 471 UGGUGUUAACCAG 655 UCUGGUGCUGGUUAA 839 850-869_aso (m5dCs)dAsdGs(m5dCs)dAscscsasgs CACCAGA CACCA a NM_000488.3_ 851 asusgsgsusdGsdTsdTsdAsdAs 472 AUGGUGUUAACCA 656 CUGGUGCUGGUUAAC 840 851-870_aso (m5dCs)(m5dCs)dAsdGs(m5dCs)ascs GCACCAG ACCAU csasg NM_000488.3_ 852 asasusgsgsdTsdGsdTsdTsdAsdAs 473 AAUGGUGUUAACC 657 UGGUGCUGGUUAACA 841 852-871_aso (m5dCs)(m5dCs)dAsdGscsascscsa AGCACCA CCAUU NM_000488.3_ 853 asasasusgsdGsdTsdGsdTsdTsdAsdAs 474 AAAUGGUGUUAAC 658 GGUGCUGGUUAACAC 842 853-872_aso (m5dCs)(m5dCs)dAsgscsascsc CAGCACC CAUUU NM_000488.3_ 854 usasasasusdGsdGsdTsdGsdTsdTsdAs 475 UAAAUGGUGUUAA 659 GUGCUGGUUAACACC 843 854-873_aso dAs(m5dCs)(m5dCs)asgscsasc CCAGCAC AUUUA NM_000488.3_ 855 gsusasasasdTsdGsdGsdTsdGsdTsdTs 476 GUAAAUGGUGUUA 660 UGCUGGUUAACACCA 844 855-874_aso dAsdAs(m5dCs)csasgscsa ACCAGCA UUUAC NM_000488.3_ 856 asgsusasasdAsdTsdGsdGsdTsdGsdTs 477 AGUAAAUGGUGUU 661 GCUGGUUAACACCAU 845 856-875_aso dTsdAsdAscscsasgsc AACCAGC UUACU NM_000488.3_ 857 asasgsusasdAsdAsdTsdGsdGsdTsdGs 478 AAGUAAAUGGUGU 662 CUGGUUAACACCAUU 846 857-876_aso dTsdTsdAsascscsasg UAACCAG UACUU NM_000488.3_ 858 gsasasgsusdAsdAsdAsdTsdGsdGsdTs 479 GAAGUAAAUGGUG 663 UGGUUAACACCAUUU 847 858-877_aso dGsdTsdTsasascscsa UUAACCA ACUUC NM_000488.3_ 859 usgsasasgsdTsdAsdAsdAsdTsdGsdGs 480 UGAAGUAAAUGGU 664 GGUUAACACCAUUUA 848 859-878_aso dTsdGsdTsusasascsc GUUAACC CUUCA NM_000488.3_ 860 ususgsasasdGsdTsdAsdAsdAsdTsdGs 481 UUGAAGUAAAUGG 665 GUUAACACCAUUUAC 849 860-879_aso dGsdTsdGsususasasc UGUUAAC UUCAA NM_000488.3_ 861 csususgsasdAsdGsdTsdAsdAsdAsdTs 482 CUUGAAGUAAAUG 666 UUAACACCAUUUACU 850 861-880_aso dGsdGsdTsgsususasa GUGUUAA UCAAG NM_000488.3_ 862 cscsususgsdAsdAsdGsdTsdAsdAsdAs 483 CCUUGAAGUAAAU 667 UAACACCAUUUACUU 851 862-881_aso dTsdGsdGsusgsususa GGUGUUA CAAGG NM_000488.3_ 863 cscscsususdGsdAsdAsdGsdTsdAsdAs 484 CCCUUGAAGUAAA 668 AACACCAUUUACUUC 852 863-882_aso dAsdTsdGsgsusgsusu UGGUGUU AAGGG NM_000488.3_ 864 gscscscsusdTsdGsdAsdAsdGsdTsdAs 485 GCCCUUGAAGUAA 669 ACACCAUUUACUUCA 853 864-883_aso dAsdAsdTsgsgsusgsu AUGGUGU AGGGC NM_000488.3_ 865 gsgscscscsdTsdTsdGsdAsdAsdGsdTs 486 GGCCCUUGAAGUA 670 CACCAUUUACUUCAA 854 865-884_aso dAsdAsdAsusgsgsusg AAUGGUG GGGCC NM_000488.3_ 866 asgsgscscs(m5dCs)dTsdTsdGsdAsdA 487 AGGCCCUUGAAGU 671 ACCAUUUACUUCAAG 855 866-885_aso sdGsdTsdAsdAsasusgsgsu AAAUGGU GGCCU NM_000488.3_ 867 csasgsgscs(m5dCs)(m5dCs)dTsdTsd 488 CAGGCCCUUGAAG 672 CCAUUUACUUCAAGG 856 867-886_aso GsdAsdAsdGsdTsdAsasasusgsg UAAAUGG GCCUG NM_000488.3_ 868 ascsasgsgs(m5dCs)(m5dCs)(m5dCs) 489 ACAGGCCCUUGAA 673 CAUUUACUUCAAGGG 857 868-887_aso dTsdTsdGsdAsdAsdGsdTsasasasusg GUAAAUG CCUGU NM_000488.3_ 869 csascsasgsdGs(m5dCs)(m5dCs) 490 CACAGGCCCUUGA 674 AUUUACUUCAAGGGC 858 869-888_aso (m5dCs)dTsdTsdGsdAsdAsdGsusasas AGUAAAU CUGUG asu NM_000488.3_ 870 cscsascsasdGsdGs(m5dCs)(m5dCs) 491 CCACAGGCCCUUG 675 UUUACUUCAAGGGCC 859 870-889_aso (m5dCs)dTsdTsdGsdAsdAsgsusasasa AAGUAAA UGUGG NM_000488.3_ 871 uscscsascsdAsdGsdGs(m5dCs) 492 UCCACAGGCCCUU 676 UUACUUCAAGGGCCU 860 87 1-890_aso (m5dCs)(m5dCs)dTsdTsdGsdAsasgsu GAAGUAA GUGGA sasa NM_000488.3_ 872 ususcscsas(m5dCs)dAsdGsdGs 493 UUCCACAGGCCCU 677 UACUUCAAGGGCCUG 861 872-891_aso (m5dCs)(m5dCs)(m5dCs)dTsdTsdGsa UGAAGUA UGGAA sasgsusa NM_000488.3_ 873 csususcscsdAs(m5dCs)dAsdGsdGs 494 CUUCCACAGGCCC 678 ACUUCAAGGGCCUGU 862 873-892_aso (m5dCs)(m5dCs)(m5dCs)dTsdTsgsas UUGAAGU GGAAG asgsu NM_000488.3_ 880 ascsusususdGsdAs(m5dCs)dTsdTs 495 ACUUUGACUUCCA 679 GGGCCUGUGGAAGUC 863 880-899_aso (m5dCs)(m5dCs)dAs(m5dCs)dAsgsgs CAGGCCC AAAGU cscsc
NM_000488.3_ 890 uscsasgsgsdGs(m5dCs)dTsdGsdAsdA 496 UCAGGGCUGAACU 680 AAGUCAAAGUUCAGC 864 890-909_aso s(m5dCs)dTsdTsdTsgsascsusu UUGACUU CCUGA NM_000488.3_ 900 cscsususgsdTsdGsdTsdTs(m5dCs)dT 497 CCUUGUGUUCUCA 681 UCAGCCCUGAGAACA 865 900-919_aso s(m5dCs)dAsdGsdGsgscsusgsa GGGCUGA CAAGG NM_000488.3_ 910 ascsasgsusdTs(m5dCs)(m5dCs)dTsd 498 ACAGUUCCUUCCU 682 GAACACAAGGAAGGA 866 910-929_aso Ts(m5dCs)(m5dCs)dTsdTsdGsusgsus UGUGUUC ACUGU usc NM_000488.3_ 920 gscscsususdGsdTsdAsdGsdAsdAs 499 GCCUUGUAGAACA 683 AAGGAACUGUUCUAC 867 920-939_aso (m5dCs)dAsdGsdTsuscscsusu GUUCCUU AAGGC NM_000488.3_ 930 csuscsuscs(m5dCs)dAsdTs(m5dCs)d 500 CUCUCCAUCAGCC 684 UCUACAAGGCUGAUG 868 930-949_aso AsdGs(m5dCs)(m5dCs)dTsdTsgsusas UUGUAGA GAGAG gsa NM_000488.3_ 940 csusgsasas(m5dCs)dAs(m5dCs)dGsd 501 CUGAACACGACUC 685 UGAUGGAGAGUCGUG 869 940-959_aso As(m5dCs)dTs(m5dCs)dTs(m5dCs)cs UCCAUCA UUCAG asuscsa NM_000488.3_ 950 asuscsasusdAsdGsdAsdTsdGs 502 AUCAUAGAUGCUG 686 UCGUGUUCAGCAUCU 870 950-969_aso (m5dCs)dTsdGsdAsdAscsascsgsa AACACGA AUGAU NM_000488.3_ 960 ususcscsusdGsdGsdTsdAs(m5dCs)dA 503 UUCCUGGUACAUC 687 CAUCUAUGAUGUACC 871 960-979_aso sdTs(m5dCs)dAsdTsasgsasusg AUAGAUG AGGAA NM_000488.3_ 970 gsgsasascsdTsdTsdGs(m5dCs) 504 GGAACUUGCCUUC 688 GUACCAGGAAGGCAA 872 970-989_aso (m5dCs)dTsdTs(m5dCs)(m5dCs)dTsg CUGGUAC GUUCC sgsusasc NM_000488.3_ 980 csgscscsgsdAsdTsdAsdAs(m5dCs)dG 505 CGCCGAUAACGGA 689 GGCAAGUUCCGUUAU 873 980-999_aso sdGsdAsdAs(m5dCs)ususgscsc ACUUGCC CGGCG NM_000488.3_ 990 ususcsasgs(m5dCs)(m5dCs)dAs 506 UUCAGCCACGCGC 690 GUUAUCGGCGCGUGG 874 990- (m5dCs)dGs(m5dCs)dGs(m5dCs) CGAUAAC CUGAA 1009_aso (m5dCs)dGsasusasasc NM_000488.3_ 1000 cscsusgsgsdGsdTsdGs(m5dCs) 507 CCUGGGUGCCUUC 691 CGUGGCUGAAGGCAC 875 1000- (m5dCs)dTsdTs(m5dCs)dAsdGscscsa AGCCACG CCAGG 1019_aso scsg NM_000488.3_ 1010 asascsuscsdAsdAsdGs(m5dCs)dAs 508 AACUCAAGCACCU 692 GGCACCCAGGUGCUU 876 1010- (m5dCs)(m5dCs)dTsdGsdGsgsusgscs GGGUGCC GAGUU 1029_aso c NM_000488.3_ 1020 usususgsasdAsdGsdGsdGs(m5dCs)dA 509 UUUGAAGGGCAAC 693 UGCUUGAGUUGCCCU 877 1020- sdAs(m5dCs)dTs(m5dCs)asasgscsa UCAAGCA UCAAA 1039_aso NM_000488.3_ 1030 usgsuscsasdTs(m5dCs)dAs(m5dCs) 510 UGUCAUCACCUUU 694 GCCCUUCAAAGGUGA 878 1030- (m5dCs)dTsdTsdTsdGsdAsasgsgsgsc GAAGGGC UGACA 1049_aso NM_000488.3_ 1040 ascscsasusdGsdGsdTsdGsdAsdTsdGs 511 ACCAUGGUGAUGU 695 GGUGAUGACAUCACC 879 1040- dTs(m5dCs)dAsuscsascsc CAUCACC AUGGU 1059_aso NM_000488.3_ 1050 csasasgsasdTsdGsdAsdGsdGsdAs 512 CAAGAUGAGGACC 696 UCACCAUGGUCCUCA 880 1050- (m5dCs)(m5dCs)dAsdTsgsgsusgsa AUGGUGA UCUUG 1069_aso NM_000488.3_ 1060 csasgsgscsdTsdTsdGsdGsdGs 513 CAGGCUUGGGCAA 697 CCUCAUCUUGCCCAA 881 1060- (m5dCs)dAsdAsdGsdAsusgsasgsg GAUGAGG GCCUG 1079_aso NM_000488.3_ 1070 asgsgscsus(m5dCs)dTsdTs(m5dCs)d 514 AGGCUCUUCUCAG 698 CCCAAGCCUGAGAAG 882 1070- Ts(m5dCs)dAsdGsdGs(m5dCs)ususgs GCUUGGG AGCCU 1089_aso gsg NM_000488.3_ 1080 usascscsusdTsdGsdGs(m5dCs) 515 UACCUUGGCCAGG 699 AGAAGAGCCUGGCCA 883 1080- (m5dCs)dAsdGsdGs(m5dCs)dTscsusu CUCUUCU AGGUA 1099_aso scsu NM_000488.3_ 1090 gsususcscsdTsdTs(m5dCs)dTs 516 GUUCCUUCUCUAC 700 GGCCAAGGUAGAGAA 884 1090- (m5dCs)dTsdAs(m5dCs)(m5dCs)dTsu CUUGGCC GGAAC 1109_aso sgsgscsc NM_000488.3_ 1100 uscsusgsgsdGsdGsdTsdGsdAsdGsdTs 517 UCUGGGGUGAGUU 701 GAGAAGGAACUCACC 885 1100- dTs(m5dCs)(m5dCs)ususcsusc CCUUCUC CCAGA 1119_aso NM_000488.3_ 1110 ususgscsasdGs(m5dCs)dAs(m5dCs) 518 UUGCAGCACCUCU 702 UCACCCCAGAGGUGC 886 1110- (m5dCs)dTs(m5dCs)dTsdGsdGsgsgsu GGGGUGA UGCAA 1129_aso sgsa NM_000488.3_ 1120 cscsasgscs(m5dCs)dAs(m5dCs)dTs 519 CCAGCCACUCUUG 703 GGUGCUGCAAGAGUG 887 1120- (m5dCs)dTsdTsdGs(m5dCs)dAsgscsa CAGCACC GCUGG 1139_aso scsc NM_000488.3_ 1130 uscscsasasdTsdTs(m5dCs)dAsdTs 520 UCCAAUUCAUCCA 704 GAGUGGCUGGAUGAA 888 1130- (m5dCs)(m5dCs)dAsdGs(m5dCs)csas GCCACUC UUGGA 1149_aso csusc NM_000488.3_ 1140 csasuscsasdTs(m5dCs)dTs(m5dCs) 521 CAUCAUCUCCUCC 705 AUGAAUUGGAGGAGA 889 1140- (m5dCs)dTs(m5dCs)(m5dCs)dAsdAsu AAUUCAU UGAUG 1159_aso suscsasu NM_000488.3_ 1150 gsgsascscsdAs(m5dCs)(m5dCs)dAsd 522 GGACCACCAGCAU 706 GGAGAUGAUGCUGGU 890 1150- Gs(m5dCs)dAsdTs(m5dCs)dAsuscsus CAUCUCC GGUCC 1169_aso csc NM_000488.3_ 1160 csgsgsgsgs(m5dCs)dAsdTsdGsdTsdG 523 CGGGGCAUGUGGA 707 CUGGUGGUCCACAUG 891 1160- sdGsdAs(m5dCs)(m5dCs)ascscsasg CCACCAG CCCCG 1179_aso NM_000488.3_ 1170 asasusgscsdGsdGsdAsdAsdGs 524 AAUGCGGAAGCGG 708 ACAUGCCCCGCUUCC 892 1170- (m5dCs)dGsdGsdGsdGscsasusgsu GGCAUGU GCAUU 1189_aso NM_000488.3_ 1180 asgscscsgsdTs(m5dCs)(m5dCs)dTs 525 AGCCGUCCUCAAU 709 CUUCCGCAUUGAGGA 893 1180- (m5dCs)dAsdAsdTsdGs(m5dCs)gsgsa GCGGAAG CGGCU 1199_aso sasg NM_000488.3_ 1190 ususcsasasdAs(m5dCs)dTsdGsdAsdA 526 UUCAAACUGAAGC 710 GAGGACGGCUUCAGU 894 1190- sdGs(m5dCs)(m5dCs)dGsuscscsusc CGUCCUC UUGAA 1209_aso NM_000488.3_ 1200 csasgscsusdGs(m5dCs)dTs(m5dCs) 527 CAGCUGCUCCUUC 711 UCAGUUUGAAGGAGC 895 1200- (m5dCs)dTsdTs(m5dCs)dAsdAsascsu AAACUGA AGCUG 1219_aso sgsa NM_000488.3_ 1210 cscsasusgsdTs(m5dCs)dTsdTsdGs 528 CCAUGUCUUGCAG 712 GGAGCAGCUGCAAGA 896 1210- (m5dCs)dAsdGs(m5dCs)dTsgscsuscs CUGCUCC CAUGG 1229_aso c NM_000488.3_ 1220 uscsgsascsdAsdAsdGsdGs(m5dCs) 529 UCGACAAGGCCCA 713 CAAGACAUGGGCCUU 897 1220- (m5dCs)(m5dCs)dAsdTsdGsuscsusus UGUCUUG GUCGA 1239_aso g NM_000488.3_ 1230 gscsusgsasdAs(m5dCs)dAsdGsdAsdT 530 GCUGAACAGAUCG 714 GCCUUGUCGAUCUGU 898 1230- s(m5dCs)dGsdAs(m5dCs)asasgsgsc ACAAGGC UCAGC 1249_aso NM_000488.3_ 1240 ascsusususdTs(m5dCs)dAsdGsdGsdG 531 ACUUUUCAGGGCU 715 UCUGUUCAGCCCUGA 899 1240- s(m5dCs)dTsdGsdAsascsasgsa GAACAGA AAAGU 1259_aso NM_000488.3_ 1250 gsgsgsasgsdTsdTsdTsdGsdGsdAs 532 GGGAGUUUGGACU 716 CCUGAAAAGUCCAAA 900 1250- (m5dCs)dTsdTsdTsuscsasgsg UUUCAGG CUCCC 1269_aso NM_000488.3_ 1260 asascsasasdTsdAs(m5dCs)(m5dCs) 533 AACAAUACCUGGG 717 CCAAACUCCCAGGUA 901 1260- TsdGsdGsdGsdAsdGsusususgsg AGUUUGG UUGUU 1279_aso NM_000488.3_ 1270 gsgscscsusdTs(m5dCs)dTsdGs 534 GGCCUUCUGCAAC 718 AGGUAUUGUUGCAGA 902 1270- (m5dCs)dAsdAs(m5dCs)dAsdAsusasc AAUACCU AGGCC 1289_aso scsu NM_000488.3_ 1280 asgsgsuscsdAsdTs(m5dCs)dTs 535 AGGUCAUCUCGGC 719 GCAGAAGGCCGAGAU 903 1280- (m5dCs)dGsdGs(m5dCs)(m5dCs)dTsu CUUCUGC GACCU 1299_aso scsusgsc NM_000488.3_ 1290 usgsasgsas(m5dCs)dAsdTsdAsdGsdA 536 UGAGACAUAGAGG 720 GAGAUGACCUCUAUG 904 1290- sdGsdGsdTs(m5dCs)asuscsusc UCAUCUC UCUCA 1309_aso NM_000488.3_ 1300 gsgsasasusdGs(m5dCs)dAsdTs 537 GGAAUGCAUCUGA 721 CUAUGUCUCAGAUGC 905 1300- (m5dCs)dTsdGsdAsdGsdAscsasusasg GACAUAG AUUCC 1319_aso NM_000488.3_ 1310 asasusgscs(m5dCs)dTsdTsdAsdTsdG 538 AAUGCCUUAUGGA 722 GAUGCAUUCCAUAAG 906 1310- sdGsdAsdAsdTsgscsasusc AUGCAUC GCAUU 1329_aso NM_000488.3_ 1320 usascscsus(m5dCs)dAsdAsdGsdAsdA 539 UACCUCAAGAAAU 723 AUAAGGCAUUUCUUG 907 1320- sdAsdTsdGs(m5dCs)csususasu GCCUUAU AGGUA 1339_aso NM_000488.3_ 1330 csususcsusdTs(m5dCs)dAsdTsdTsdT 540 CUUCUUCAUUUAC 724 UCUUGAGGUAAAUGA 908 1330- sdAs(m5dCs)(m5dCs)dTscsasasgsa CUCAAGA AGAAG 1349_aso NM_000488.3_ 1340 gscsususcsdAs(m5dCs)dTsdGs 541 GCUUCACUGCCUU 725 AAUGAAGAAGGCAGU 909 1340- (m5dCs)(m5dCs)dTsdTs(m5dCs)dTsu CUUCAUU GAAGC 1359_aso scsasusu NM_000488.3_ 1350 ascsususgs(m5dCs)dAsdGs(m5dCs)d 542 ACUUGCAGCUGCU 726 GCAGUGAAGCAGCUG 910 1350- TsdGs(m5dCs)dTsdTs(m5dCs)ascsus UCACUGC CAAGU 1369_aso gsc NM_000488.3_ 1360 csasascsasdGs(m5dCs)dGsdGsdTsdA 543 CAACAGCGGUACU 727 AGCUGCAAGUACCGC 911 1360- s(m5dCs)dTsdTsdGscsasgscsu UGCAGCU UGUUG 1379_aso NM_000488.3_ 1370 cscsasgscsdAsdAsdTs(m5dCs)dAs 544 CCAGCAAUCACAA 728 ACCGCUGUUGUGAUU 912 1370- (m5dCs)dAsdAs(m5dCs)dAsgscsgsgs CAGCGGU GCUGG 1389_aso u NM_000488.3_ 1380 usasgscsgsdAsdAs(m5dCs)dGsdGs 545 UAGCGAACGGCCA 729 UGAUUGCUGGCCGUU 913 1380- (m5dCs)(m5dCs)dAsdGs(m5dCs)asas GCAAUCA CGCUA 1399_aso uscsa NM_000488.3_ 1390 usgsususgsdGsdGsdGsdTsdTsdTsdAs 546 UGUUGGGGUUUAG 730 CCGUUCGCUAAACCC 914 1390- dGs(m5dCs)dGsasascsgsg CGAACGG CAACA 1409_aso NM_000488.3_ 1400 asasasgsus(m5dCs)dAs(m5dCs) 547 AAAGUCACCCUGU 731
AACCCCAACAGGGUG 915 1400- (m5dCs)(m5dCs)dTsdGsdTsdTsdGsgs UGGGGUU ACUUU 1419_aso gsgsusu NM_000488.3_ 1410 gsususgsgs(m5dCs)(m5dCs)dTsdTsd 548 GUUGGCCUUGAAA 732 GGGUGACUUUCAAGG 916 1410- GsdAsdAsdAsdGsdTscsascscsc GUCACCC CCAAC 1429_aso NM_000488.3_ 1420 gsgsasasasdGsdGs(m5dCs)(m5dCs)d 549 GGAAAGGCCUGUU 733 CAAGGCCAACAGGCC 917 1420- TsdGsdTsdTsdGsdGscscsususg GGCCUUG UUUCC 1439_aso NM_000488.3_ 1430 asusasasasdAsdAs(m5dCs)(m5dCs)d 550 AUAAAAACCAGGA 734 AGGCCUUUCCUGGUU 918 1430- AsdGsdGsdAsdAsdAsgsgscscsu AAGGCCU UUUAU 1449_aso NM_000488.3_ 1440 asascsusus(m5dCs)dTs(m5dCs)dTsd 551 AACUUCUCUUAUA 735 UGGUUUUUAUAAGAG 919 1440- TsdAsdTsdAsdAsdAsasascscsa AAAACCA AAGUU 1459_aso NM_000488.3_ 1450 usgsususcsdAsdGsdAsdGsdGsdAsdAs 552 UGUUCAGAGGAAC 736 AAGAGAAGUUCCUCU 920 1450- (m5dCs)dTsdTscsuscsusu UUCUCUU GAACA 1469_aso NM_000488.3_ 1460 asasgsasusdAsdAsdTsdAsdGsdTsdGs 553 AAGAUAAUAGUGU 737 CCUCUGAACACUAUU 921 1460- dTsdTs(m5dCs)asgsasgsg UCAGAGG AUCUU 1479_aso NM_000488.3_ 1470 uscsusgscs(m5dCs)(m5dCs)dAsdTsd 554 UCUGCCCAUGAAG 738 CUAUUAUCUUCAUGG 922 1470- GsdAsdAsdGsdAsdTsasasusasg AUAAUAG GCAGA 1489_aso NM_000488.3_ 1480 gsgsususgsdGs(m5dCs)dTsdAs 555 GGUUGGCUACUCU 739 CAUGGGCAGAGUAGC 923 1480- (m5dCs)dTs(m5dCs)dTsdGs(m5dCs)c GCCCAUG CAACC 1499_aso scsasusg NM_000488.3_ 1490 ususasascsdAs(m5dCs)dAsdAsdGsdG 556 UUAACACAAGGGU 740 GUAGCCAACCCUUGU 924 1490- sdGsdTsdTsdGsgscsusasc UGGCUAC GUUAA 1509_aso NM_000488.3_ 1500 asascsasusdTsdTsdTsdAs(m5dCs)dT 557 AACAUUUUACUUA 741 CUUGUGUUAAGUAAA 925 1500- sdTsdAsdAs(m5dCs)ascsasasg ACACAAG AUGUU 1519_aso NM_000488.3_ 1510 asasasgsasdAsdTsdAsdAsdGsdAsdAs 558 AAAGAAUAAGAAC 742 GUAAAAUGUUCUUAU 926 1510- (m5dCs)dAsdTsusususasc AUUUUAC UCUUU 1529_aso NM_000488.3_ 1520 gsasasgsasdGsdGsdTsdGs(m5dCs)dA 559 GAAGAGGUGCAAA 743 CUUAUUCUUUGCACC 927 1520- sdAsdAsdGsdAsasusasasg GAAUAAG UCUUC 1539_aso NM_000488.3_ 1530 cscsasasasdAsdAsdTsdAsdGsdGsdAs 560 CCAAAAAUAGGAA 744 GCACCUCUUCCUAUU 928 1530- dAsdGsdAsgsgsusgsc GAGGUGC UUUGG 1549_aso NM_000488.3_ 1540 usgsususcsdAs(m5dCs)dAsdAsdAs 561 UGUUCACAAACCA 745 CUAUUUUUGGUUUGU 929 1540- (m5dCs)(m5dCs)dAsdAsdAsasasusas AAAAUAG GAACA 1559_aso g NM_000488.3_ 1550 usususususdAs(m5dCs)dTsdTs 562 UUUUUACUUCUGU 746 UUUGUGAACAGAAGU 930 1550- (m5dCs)dTsdGsdTsdTs(m5dCs)ascsa UCACAAA AAAAA 1569_aso sasa NM_000488.3_ 1560 usususgsusdAsdTsdTsdTsdAsdTsdTs 563 UUUGUAUUUAUUU 747 GAAGUAAAAAUAAAU 931 1560- dTsdTsdTsascsususc UUACUUC ACAAA 1579_aso NM_000488.3_ 1570 asusgsgsasdAsdGsdTsdAsdGsdTsdTs 564 AUGGAAGUAGUUU 748 UAAAUACAAACUACU 932 1570- dTsdGsdTsasusususa GUAUUUA UCCAU 1589_aso
Example 2. In Vitro Screening
[0485] Serpinc1 (AT3) gapmer transfections were done at 5 nM. Single dose screen of 184 Serpinc1 oligos was performed in Huh7 cells, directly after seeding 25,000 cells per well on 96 well plates. Each oligo was transfected in quadruplicate with 0.5 .mu.l Lipofectamine 2000/well. Transfections were harvested 24 h after seeding/transfection. Transfection of an Aha1-LNA gapmer, and mock transfections were performed quadruplicate on each plate as control. Mean values of Serpinc1/GAPDH from Aha1-LNA transfection was set as 100% Serpinc1 expression, which is the reference for all other mean values shown in Table 5. At the same time, the Aha1-LNA also served as a transfection control on each plate.
[0486] The complete screen was performed in two transfection "sessions". Overall, transfection efficiency with Aha1-oligo was between .about.60-70% at 5 nM. All Serpinc1 oligos were less efficient than the Aha1-LNA at the same concentration.
[0487] Transfection efficiency for each plate was determined, and compared with one another in order to correct the results for the difference in transfection efficiency between the plates.
TABLE-US-00005 TABLE 5 Transfection efficiency of antisense polynucleotides targeting Serpinc1 (AT3) Mean val Corrected % (w/o transfection correction) sd % efficiency X10631K1 85 13 76 X10632K1 92 17 83 X10633K1 85 9 76 X10634K1 81 10 72 X10635K1 71 3 62 X10636K1 91 18 83 X10637K1 90 2 81 X10638K1 82 14 73 X10639K1 72 24 63 X10640K1 71 7 62 X10641K1 86 18 77 X10642K1 105 14 96 X10643K1 99 4 90 X10644K1 90 11 81 X10645K1 113 15 104 X10646K1 87 18 78 X10647K1 100 27 91 X10648K1 96 17 87 X10649K1 105 39 96 X10650K1 105 30 96 X10651K1 103 15 103 X10652K1 76 7 76 X10653K1 90 12 90 X10654K1 95 10 95 X10655K1 84 5 84 X10656K1 89 8 89 X10657K1 104 13 104 X10658K1 89 14 89 X10659K1 69 6 69 X10660K1 80 3 80 X10661K1 95 19 95 X10662K1 88 14 88 X10663K1 106 15 106 X10664K1 91 7 91 X10665K1 79 12 79 X10666K1 96 7 96 X10667K1 101 14 101 X10668K1 96 13 96 X10669K1 74 7 74 X10670K1 69 8 69 X10671K1 79 6 78 X10672K1 117 10 117 X10673K1 86 8 86 X10674K1 96 10 95 X10675K1 100 5 99 X10676K1 96 16 95 X10677K1 103 10 102 X10678K1 118 11 117 X10679K1 95 12 94 X10680K1 90 5 89 X10681K1 99 15 99 X10682K1 93 8 92 X10683K1 105 7 105 X10684K1 99 8 98 X10685K1 97 12 96 X10686K1 102 3 102 X10687K1 77 12 76 X10688K1 81 7 81 X10689K1 69 16 68 X10690K1 78 8 78 X10691K1 126 16 124 X10692K1 105 13 103 X10693K1 107 24 105 X10694K1 91 17 89 X10695K1 138 19 136 X10696K1 110 8 108 X10697K1 103 17 101 X10698K1 101 13 99 X10699K1 106 10 104 X10700K1 86 5 84 X10701K1 82 3 80 X10702K1 95 15 93 X10703K1 96 16 94 X10704K1 85 7 83 X10705K1 102 9 100 X10706K1 111 11 109 X10707K1 100 27 98 X10708K1 122 6 120 X10709K1 85 9 83 X10710K1 82 26 80 X10711K1 102 19 94 X10712K1 105 5 97 X10713K1 97 8 89 X10714K1 107 5 99 X10715K1 104 19 96 X10716K1 103 3 95 X10717K1 107 15 99 X10718K1 115 20 107 X10719K1 96 16 88 X10720K1 96 11 88 X10721K1 75 8 66 X10722K1 76 5 67 X10723K1 85 3 77 X10724K1 84 12 76 X10725K1 93 6 85 X10726K1 92 16 84 X10727K2 99 6 91 X10728K2 101 11 93 X10729K2 96 2 87 X10730K2 98 8 90 X10731K2 168 5 160 X10732K2 160 15 152 X10733K2 155 9 147 X10734K2 139 6 131 X10735K2 123 12 115 X10736K2 133 31 125 X10737K2 115 15 107 X10738K2 116 25 108 X10739K2 96 9 88 X10740K2 96 6 88 X10741K2 109 10 102 X10742K2 109 15 101 X10743K2 147 41 139 X10744K2 192 33 185 X10745K2 163 67 155 X10746K2 145 21 138 X10747K2 148 55 140 X10748K2 139 23 131 X10749K1 117 15 109 X10750K1 93 7 85 X10751K1 114 19 113 X10752K1 139 24 138 X10753K1 133 30 132 X10754K1 87 14 86 X10755K1 91 11 90 X10756K1 98 19 97 X10757K1 105 9 104 X10758K1 97 7 96 X10759K1 99 11 98 X10760K1 82 5 81 X10761K1 118 36 117 X10762K1 135 22 133 X10763K2 129 31 128 X10764K2 111 19 110 X10765 103 9 102 X10766K2 106 15 105 X10767K1 100 6 99 X10768K1 106 16 104 X10769K1 85 11 84 X10770K1 88 12 87 X10771K1 110 10 104 X10772K1 82 27 76 X10773K2 97 18 92 X10774K1 119 14 113 X10775K1 108 12 103 X10776K1 96 5 91 X10777K1 100 10 95 X10778K1 86 5 81 X10779K1 103 14 98 X10780K1 105 12 100 X10781K1 139 5 134 X10782K1 115 9 109 X10783K1 132 35 127 X10784K1 96 10 90 X10785K1 86 5 81 X10786K1 113 8 108 X10787K1 110 3 105 X10788K1 105 10 100 X10789K1 114 7 109 X10790K1 114 8 109 X10791K1 92 4 84 X10792K1 90 8 81 X10793K1 91 11 83 X10794K1 94 5 86 X10795K1 99 14 91 X10796K1 127 9 119 X10797K1 105 6 97 X10798K1 126 13 117 X10799K1 115 13 107 X10800K1 119 7 110 X10801K1 106 21 98 X10802K1 94 11 86 X10803K1 111 23 103 X10804K1 102 9 93 X10805K1 79 16 70 X10806K1 108 12 99 X10807K1 116 11 108 X10808K1 123 14 115 X10809K1 97 16 89 X10810K1 90 12 82 X10811K1 133 20 122 X10812K1 133 20 122 X10813K1 116 9 104 X10814K1 97 13 86
EQUIVALENTS
[0488] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.
Sequence CWU
1
1
93211599DNAHomo sapiens 1tctgccccac cctgtcctct ggaacctctg cgagatttag
aggaaagaac cagttttcag 60gcggattgcc tcagatcaca ctatctccac ttgcccagcc
ctgtggaaga ttagcggcca 120tgtattccaa tgtgatagga actgtaacct ctggaaaaag
gaaggtttat cttttgtcct 180tgctgctcat tggcttctgg gactgcgtga cctgtcacgg
gagccctgtg gacatctgca 240cagccaagcc gcgggacatt cccatgaatc ccatgtgcat
ttaccgctcc ccggagaaga 300aggcaactga ggatgagggc tcagaacaga agatcccgga
ggccaccaac cggcgtgtct 360gggaactgtc caaggccaat tcccgctttg ctaccacttt
ctatcagcac ctggcagatt 420ccaagaatga caatgataac attttcctgt cacccctgag
tatctccacg gcttttgcta 480tgaccaagct gggtgcctgt aatgacaccc tccagcaact
gatggaggta tttaagtttg 540acaccatatc tgagaaaaca tctgatcaga tccacttctt
ctttgccaaa ctgaactgcc 600gactctatcg aaaagccaac aaatcctcca agttagtatc
agccaatcgc ctttttggag 660acaaatccct taccttcaat gagacctacc aggacatcag
tgagttggta tatggagcca 720agctccagcc cctggacttc aaggaaaatg cagagcaatc
cagagcggcc atcaacaaat 780gggtgtccaa taagaccgaa ggccgaatca ccgatgtcat
tccctcggaa gccatcaatg 840agctcactgt tctggtgctg gttaacacca tttacttcaa
gggcctgtgg aagtcaaagt 900tcagccctga gaacacaagg aaggaactgt tctacaaggc
tgatggagag tcgtgttcag 960catctatgat gtaccaggaa ggcaagttcc gttatcggcg
cgtggctgaa ggcacccagg 1020tgcttgagtt gcccttcaaa ggtgatgaca tcaccatggt
cctcatcttg cccaagcctg 1080agaagagcct ggccaaggta gagaaggaac tcaccccaga
ggtgctgcaa gagtggctgg 1140atgaattgga ggagatgatg ctggtggtcc acatgccccg
cttccgcatt gaggacggct 1200tcagtttgaa ggagcagctg caagacatgg gccttgtcga
tctgttcagc cctgaaaagt 1260ccaaactccc aggtattgtt gcagaaggcc gagatgacct
ctatgtctca gatgcattcc 1320ataaggcatt tcttgaggta aatgaagaag gcagtgaagc
agctgcaagt accgctgttg 1380tgattgctgg ccgttcgcta aaccccaaca gggtgacttt
caaggccaac aggcctttcc 1440tggtttttat aagagaagtt cctctgaaca ctattatctt
catgggcaga gtagccaacc 1500cttgtgttaa gtaaaatgtt cttattcttt gcacctcttc
ctatttttgg tttgtgaaca 1560gaagtaaaaa taaatacaaa ctacttccat ctcacatta
159921545DNAMacaca mulatta 2ggcacgagga ccatctccac
ttgcccagcc ctgtggaaga ttagcgacca tgtattccaa 60tgtgatagga accgtagcct
ctggaaaaag gaaggtttat cttctgtcct tgctgctcat 120tggcctctgg gactgtatga
cctgtcacgg gagccctgtg gacatctgca cagccaagcc 180gcgggacatt cccatgaatc
ccatgtgcat ttaccgctcc ccggagaaga aggcaactga 240ggatgagggc tcagaacaga
agatccccga ggccaccaac cggcgcgtct gggaactgtc 300caaggccaat tcccgctttg
ctaccacttt ctatcagcac ctggcagatt ccaagaacga 360caaggataac attttcctgt
cacccctgag tgtctccacg gcttttgcta tgaccaagct 420gggtgcctgt aatgacaccc
tcaagcaact gatggaggta tttaagtttg acaccatatc 480tgagaaaaca tctgatcaga
tccacttctt ctttgccaaa ctgaactgcc gactctatcg 540aaaagccaac aaatcctcca
agttagtatc agccaatcgc ctttttggag acaaatccct 600taccttcaat gagacctacc
aggacatcag tgagttggta tacggagcca agctccagcc 660cctggacttc aaggaaaatg
cagagcaatc cagagcggcc atcaacaaat gggtgtccaa 720taagaccgaa ggccgaatca
ccgatgtcat tcccccggaa gccatcaacg agctcactgt 780tctggtgctg gttaacacca
tttacttcaa gggcctgtgg aagtcaaagt ttagccctga 840gaacacaagg atggaaccgt
tctacaaggc tgatggagag tcgtgttcag cgtctatgat 900gtaccaggaa ggcaagttct
gttatcggcg cgtggctgaa ggcacccagg tgcttgagtt 960gcccttcaag ggtgatgaca
tcaccatggt gctcatcctg cccaagcctg agaagagcct 1020gaccaaggtg gagcaggaac
tcaccccaga ggtgctgcag gagtggctgg atgagttgga 1080ggagatgatg ctggtggttc
acatgccccg cttccgcatt gaggacggct tcagtttgaa 1140ggagcagctg caagacatgg
gccttgtcga tctgttcagc cctgaaaagt ccaaactccc 1200aggtattgtt gcagaaggcc
gggatgacct ctatgtctcc gatgcattcc ataaggcatt 1260tcttgaggta aatgaagaag
gcagtgaagc agctgcaagt accgccattg ggattgctgg 1320ccgttcgcta aaccccaaca
gggtgacctt caaggccaac aggcctttcc tggtttttat 1380aagagaagtt cctctgaaca
ctattatctt catgggcaga gtagccaacc cttgtgtgag 1440ctaaactgtt cttattcttt
gtacctcttc ctattttggt ttgtgaatag aagtaaaaat 1500aaatacaact actcccatct
tacattaaaa aaaaaaaaaa aaaaa 154532171DNAMus musculus
3ataggtaatt ttagaaatag atctgatttg tatctgagac attttagtga agtggtgaga
60tataagacat aatcagaaga catatctacc tgaagacttt aaggggagag ctccctcccc
120cacctggcct ctggacctct cagatttagg ggaaagaacc agttttcgga gtgatcgtct
180cagtcagcac catctctgta ggagcatcgg ccatgtattc ccctggggca ggaagtgggg
240ctgctggtga gaggaagctt tgtctcctct ctctgctcct catcggtgcc ttgggctgtg
300ctatctgtca cggaaaccct gtggacgaca tctgcatagc gaagccccga gacatccccg
360tgaatccctt gtgcatttac cgctcccctg ggaagaaggc caccgaggag gatggctcag
420agcagaaggt tccagaagcc accaaccggc gggtctggga actgtccaag gccaattcgc
480gatttgccac taacttctac cagcacctgg cagactccaa gaatgacaac gacaacattt
540tcctgtcacc cttgagcatc tccactgctt ttgctatgac caagctgggt gcctgtaacg
600acactctcaa gcagctgatg gaggttttta aatttgatac catctccgag aagacatccg
660accagatcca cttcttcttt gccaaactga actgccgact ctatcgaaaa gccaacaagt
720cctctgactt ggtatcagcc aaccgccttt ttggagacaa atccctcacc ttcaacgaga
780gctatcaaga tgttagtgag gttgtctatg gagccaagct ccagcccctg gacttcaagg
840agaatccgga gcaatccaga gtgaccatca acaactgggt agctaataag actgaaggcc
900gcatcaaaga tgtcatccca cagggcgcca ttaacgagct cactgccctg gttctggtta
960acaccattta cttcaagggc ctgtggaagt caaagttcag ccctgagaac acaaggaagg
1020aaccgttcta taaggtcgat gggcagtcat gcccagtgcc tatgatgtac caggaaggca
1080aattcaaata ccggcgcgtg gcagagggca cccaggtgct agagctgccc ttcaaggggg
1140atgacatcac catggtgctc atcctgccca agcctgagaa gagcctggcc aaggtggagc
1200aggagctcac cccagagctg ctgcaggagt ggctggatga gctgtcagag actatgcttg
1260tggtccacat gccccgcttc cgcaccgagg atggcttcag tctgaaggag cagctgcaag
1320acatgggcct cattgatctc ttcagccctg aaaagtccca actcccaggg atcgttgctg
1380gaggcaggga cgacctctat gtctccgacg cattccacaa agcatttctt gaggtaaatg
1440aggaaggcag tgaagcagca gcgagtactt ctgtcgtgat tactggccgg tcactgaacc
1500ccaatagggt gaccttcaag gccaacaggc ccttcctggt tcttataagg gaagttgcac
1560tgaacactat tatattcatg gggagagtgg ctaatccttg tgtgaactaa aatattctta
1620atctttgcac cttttcctac tttggtgttt gtgaatagaa gtaaaaataa atacgactgc
1680cacctcacga gaatggactt ttccacttga agacgagaga ctggagtaca gatgctacac
1740cacttttggg caagtgaagg gggagcagcc agccacggtg gcacaaacct atatcctggt
1800gcttttgaag gtagaagcag ggcggtcagg agttaaggcc agttgaggct gggctgcaga
1860gtgaaagacc atgtctcaag atggtctttc tcctccccaa agtagaaaag aaaaccataa
1920aaacaagagg taaatatatt actatttcat cttagaggat agcaggcatc ttgaaagggt
1980agagggacct taaattctca ttattgcccc catactacaa actaaaaaac aaacccgaat
2040caatctccca taaagacaga gattcaaata agagtattaa acgttttatt tctcaaacca
2100ctcacatgca taatgttctt atacacagtg tcaaaataaa gagaaatgca tttttataca
2160aaaaaaaaaa a
217141561DNARattus norvegicus 4cggagggatt gctcagcact gtctccacgg
cttctctgca gaagcgtcca ccatgtattc 60cccgggaata ggaagtgcgg ttgctggaga
gaggaagctt tgtctcctct ctctgctact 120cattggtgcc ttgggctgtg ctgtctgtca
tggaaaccct gtggacgaca tctgcatagc 180gaagccccga gacatccccg tgaaccccat
gtgcatttac cgctcccctg cgaagaaggc 240cacggaggag gatgtcctag agcagaaggt
tccggaagcc accaaccggc gggtctggga 300actgtccaag gccaattctc gatttgccac
taacttctat cagcacctgg cagactccaa 360gaacgacaac gacaacattt tcctgtcacc
cttgagcatc tccacggcgt ttgctatgac 420caagctgggt gcttgtaata acaccctcaa
gcagctgatg gaggttttta aatttgatac 480catctccgag aagacatccg accagatcca
cttcttcttt gccaaactga actgccgact 540ctatcgaaaa gccaacaagt cctctaactt
ggtgtcagcc aaccgccttt ttggagacaa 600atcccttacc ttcaatgaga gctatcaaga
cgttagtgag attgtctatg gagccaagct 660tcagcccctg gacttcaagg agaatccgga
gcaatccaga gtgaccatca acaactgggt 720agctaataag actgaaggcc gcatcaaaga
cgtcatcccc caaggagcca ttgatgagct 780cactgccctg gtgctggtta acaccattta
cttcaagggc ctgtggaagt caaagttcag 840ccctgagaac acaaggaagg aaccattcca
caaagttgat gggcagtcat gcctggtgcc 900catgatgtac caggaaggca aattcaaata
caggcgtgtg ggagagggta cccaggtgct 960agagatgccc ttcaaggggg acgacatcac
catggtgctc atcctgccca agcctgagaa 1020gagcctggct aaggtggagc aggaactcac
cccggagctg ctgcaggagt ggctggatga 1080gctgtcggag gtcatgcttg tggtccacgt
gccccgcttc cgcatcgagg acagcttcag 1140tctgaaggag cagctgcaag acatgggcct
tgttgatctc ttcagccctg agaagtccca 1200actcccaggg atcattgctg aaggcaggga
cgacctcttt gtctccgatg cattccacaa 1260agcgtttctt gaggtaaatg aggaaggcag
tgaagcagca gcgagtactt ctgtcgtgat 1320tactggccgg tcactgaacc ccagtagggt
gaccttcaag gccaacaggc ccttcctggt 1380tcttataagg gaagtcgcac tgaacactat
tatattcatg gggagagtgt ctaatccttg 1440tgtgaactaa aatattctta atctttgcac
cttttcctat ctcggtgttt gttaatggaa 1500gtaaaaataa atatgactgc cacctcaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560a
156151599DNAHomo sapiens 5taatgtgaga
tggaagtagt ttgtatttat ttttacttct gttcacaaac caaaaatagg 60aagaggtgca
aagaataaga acattttact taacacaagg gttggctact ctgcccatga 120agataatagt
gttcagagga acttctctta taaaaaccag gaaaggcctg ttggccttga 180aagtcaccct
gttggggttt agcgaacggc cagcaatcac aacagcggta cttgcagctg 240cttcactgcc
ttcttcattt acctcaagaa atgccttatg gaatgcatct gagacataga 300ggtcatctcg
gccttctgca acaatacctg ggagtttgga cttttcaggg ctgaacagat 360cgacaaggcc
catgtcttgc agctgctcct tcaaactgaa gccgtcctca atgcggaagc 420ggggcatgtg
gaccaccagc atcatctcct ccaattcatc cagccactct tgcagcacct 480ctggggtgag
ttccttctct accttggcca ggctcttctc aggcttgggc aagatgagga 540ccatggtgat
gtcatcacct ttgaagggca actcaagcac ctgggtgcct tcagccacgc 600gccgataacg
gaacttgcct tcctggtaca tcatagatgc tgaacacgac tctccatcag 660ccttgtagaa
cagttccttc cttgtgttct cagggctgaa ctttgacttc cacaggccct 720tgaagtaaat
ggtgttaacc agcaccagaa cagtgagctc attgatggct tccgagggaa 780tgacatcggt
gattcggcct tcggtcttat tggacaccca tttgttgatg gccgctctgg 840attgctctgc
attttccttg aagtccaggg gctggagctt ggctccatat accaactcac 900tgatgtcctg
gtaggtctca ttgaaggtaa gggatttgtc tccaaaaagg cgattggctg 960atactaactt
ggaggatttg ttggcttttc gatagagtcg gcagttcagt ttggcaaaga 1020agaagtggat
ctgatcagat gttttctcag atatggtgtc aaacttaaat acctccatca 1080gttgctggag
ggtgtcatta caggcaccca gcttggtcat agcaaaagcc gtggagatac 1140tcaggggtga
caggaaaatg ttatcattgt cattcttgga atctgccagg tgctgataga 1200aagtggtagc
aaagcgggaa ttggccttgg acagttccca gacacgccgg ttggtggcct 1260ccgggatctt
ctgttctgag ccctcatcct cagttgcctt cttctccggg gagcggtaaa 1320tgcacatggg
attcatggga atgtcccgcg gcttggctgt gcagatgtcc acagggctcc 1380cgtgacaggt
cacgcagtcc cagaagccaa tgagcagcaa ggacaaaaga taaaccttcc 1440tttttccaga
ggttacagtt cctatcacat tggaatacat ggccgctaat cttccacagg 1500gctgggcaag
tggagatagt gtgatctgag gcaatccgcc tgaaaactgg ttctttcctc 1560taaatctcgc
agaggttcca gaggacaggg tggggcaga
159961545DNAMacaca mulatta 6tttttttttt ttttttttta atgtaagatg ggagtagttg
tatttatttt tacttctatt 60cacaaaccaa aataggaaga ggtacaaaga ataagaacag
tttagctcac acaagggttg 120gctactctgc ccatgaagat aatagtgttc agaggaactt
ctcttataaa aaccaggaaa 180ggcctgttgg ccttgaaggt caccctgttg gggtttagcg
aacggccagc aatcccaatg 240gcggtacttg cagctgcttc actgccttct tcatttacct
caagaaatgc cttatggaat 300gcatcggaga catagaggtc atcccggcct tctgcaacaa
tacctgggag tttggacttt 360tcagggctga acagatcgac aaggcccatg tcttgcagct
gctccttcaa actgaagccg 420tcctcaatgc ggaagcgggg catgtgaacc accagcatca
tctcctccaa ctcatccagc 480cactcctgca gcacctctgg ggtgagttcc tgctccacct
tggtcaggct cttctcaggc 540ttgggcagga tgagcaccat ggtgatgtca tcacccttga
agggcaactc aagcacctgg 600gtgccttcag ccacgcgccg ataacagaac ttgccttcct
ggtacatcat agacgctgaa 660cacgactctc catcagcctt gtagaacggt tccatccttg
tgttctcagg gctaaacttt 720gacttccaca ggcccttgaa gtaaatggtg ttaaccagca
ccagaacagt gagctcgttg 780atggcttccg ggggaatgac atcggtgatt cggccttcgg
tcttattgga cacccatttg 840ttgatggccg ctctggattg ctctgcattt tccttgaagt
ccaggggctg gagcttggct 900ccgtatacca actcactgat gtcctggtag gtctcattga
aggtaaggga tttgtctcca 960aaaaggcgat tggctgatac taacttggag gatttgttgg
cttttcgata gagtcggcag 1020ttcagtttgg caaagaagaa gtggatctga tcagatgttt
tctcagatat ggtgtcaaac 1080ttaaatacct ccatcagttg cttgagggtg tcattacagg
cacccagctt ggtcatagca 1140aaagccgtgg agacactcag gggtgacagg aaaatgttat
ccttgtcgtt cttggaatct 1200gccaggtgct gatagaaagt ggtagcaaag cgggaattgg
ccttggacag ttcccagacg 1260cgccggttgg tggcctcggg gatcttctgt tctgagccct
catcctcagt tgccttcttc 1320tccggggagc ggtaaatgca catgggattc atgggaatgt
cccgcggctt ggctgtgcag 1380atgtccacag ggctcccgtg acaggtcata cagtcccaga
ggccaatgag cagcaaggac 1440agaagataaa ccttcctttt tccagaggct acggttccta
tcacattgga atacatggtc 1500gctaatcttc cacagggctg ggcaagtgga gatggtcctc
gtgcc 154572171DNAMus musculus 7tttttttttt ttgtataaaa
atgcatttct ctttattttg acactgtgta taagaacatt 60atgcatgtga gtggtttgag
aaataaaacg tttaatactc ttatttgaat ctctgtcttt 120atgggagatt gattcgggtt
tgttttttag tttgtagtat gggggcaata atgagaattt 180aaggtccctc taccctttca
agatgcctgc tatcctctaa gatgaaatag taatatattt 240acctcttgtt tttatggttt
tcttttctac tttggggagg agaaagacca tcttgagaca 300tggtctttca ctctgcagcc
cagcctcaac tggccttaac tcctgaccgc cctgcttcta 360ccttcaaaag caccaggata
taggtttgtg ccaccgtggc tggctgctcc cccttcactt 420gcccaaaagt ggtgtagcat
ctgtactcca gtctctcgtc ttcaagtgga aaagtccatt 480ctcgtgaggt ggcagtcgta
tttattttta cttctattca caaacaccaa agtaggaaaa 540ggtgcaaaga ttaagaatat
tttagttcac acaaggatta gccactctcc ccatgaatat 600aatagtgttc agtgcaactt
cccttataag aaccaggaag ggcctgttgg ccttgaaggt 660caccctattg gggttcagtg
accggccagt aatcacgaca gaagtactcg ctgctgcttc 720actgccttcc tcatttacct
caagaaatgc tttgtggaat gcgtcggaga catagaggtc 780gtccctgcct ccagcaacga
tccctgggag ttgggacttt tcagggctga agagatcaat 840gaggcccatg tcttgcagct
gctccttcag actgaagcca tcctcggtgc ggaagcgggg 900catgtggacc acaagcatag
tctctgacag ctcatccagc cactcctgca gcagctctgg 960ggtgagctcc tgctccacct
tggccaggct cttctcaggc ttgggcagga tgagcaccat 1020ggtgatgtca tcccccttga
agggcagctc tagcacctgg gtgccctctg ccacgcgccg 1080gtatttgaat ttgccttcct
ggtacatcat aggcactggg catgactgcc catcgacctt 1140atagaacggt tccttccttg
tgttctcagg gctgaacttt gacttccaca ggcccttgaa 1200gtaaatggtg ttaaccagaa
ccagggcagt gagctcgtta atggcgccct gtgggatgac 1260atctttgatg cggccttcag
tcttattagc tacccagttg ttgatggtca ctctggattg 1320ctccggattc tccttgaagt
ccaggggctg gagcttggct ccatagacaa cctcactaac 1380atcttgatag ctctcgttga
aggtgaggga tttgtctcca aaaaggcggt tggctgatac 1440caagtcagag gacttgttgg
cttttcgata gagtcggcag ttcagtttgg caaagaagaa 1500gtggatctgg tcggatgtct
tctcggagat ggtatcaaat ttaaaaacct ccatcagctg 1560cttgagagtg tcgttacagg
cacccagctt ggtcatagca aaagcagtgg agatgctcaa 1620gggtgacagg aaaatgttgt
cgttgtcatt cttggagtct gccaggtgct ggtagaagtt 1680agtggcaaat cgcgaattgg
ccttggacag ttcccagacc cgccggttgg tggcttctgg 1740aaccttctgc tctgagccat
cctcctcggt ggccttcttc ccaggggagc ggtaaatgca 1800caagggattc acggggatgt
ctcggggctt cgctatgcag atgtcgtcca cagggtttcc 1860gtgacagata gcacagccca
aggcaccgat gaggagcaga gagaggagac aaagcttcct 1920ctcaccagca gccccacttc
ctgccccagg ggaatacatg gccgatgctc ctacagagat 1980ggtgctgact gagacgatca
ctccgaaaac tggttctttc ccctaaatct gagaggtcca 2040gaggccaggt gggggaggga
gctctcccct taaagtcttc aggtagatat gtcttctgat 2100tatgtcttat atctcaccac
ttcactaaaa tgtctcagat acaaatcaga tctatttcta 2160aaattaccta t
217181561DNARattus norvegicus
8tttttttttt tttttttttt tttttttttt tttttgaggt ggcagtcata tttattttta
60cttccattaa caaacaccga gataggaaaa ggtgcaaaga ttaagaatat tttagttcac
120acaaggatta gacactctcc ccatgaatat aatagtgttc agtgcgactt cccttataag
180aaccaggaag ggcctgttgg ccttgaaggt caccctactg gggttcagtg accggccagt
240aatcacgaca gaagtactcg ctgctgcttc actgccttcc tcatttacct caagaaacgc
300tttgtggaat gcatcggaga caaagaggtc gtccctgcct tcagcaatga tccctgggag
360ttgggacttc tcagggctga agagatcaac aaggcccatg tcttgcagct gctccttcag
420actgaagctg tcctcgatgc ggaagcgggg cacgtggacc acaagcatga cctccgacag
480ctcatccagc cactcctgca gcagctccgg ggtgagttcc tgctccacct tagccaggct
540cttctcaggc ttgggcagga tgagcaccat ggtgatgtcg tcccccttga agggcatctc
600tagcacctgg gtaccctctc ccacacgcct gtatttgaat ttgccttcct ggtacatcat
660gggcaccagg catgactgcc catcaacttt gtggaatggt tccttccttg tgttctcagg
720gctgaacttt gacttccaca ggcccttgaa gtaaatggtg ttaaccagca ccagggcagt
780gagctcatca atggctcctt gggggatgac gtctttgatg cggccttcag tcttattagc
840tacccagttg ttgatggtca ctctggattg ctccggattc tccttgaagt ccaggggctg
900aagcttggct ccatagacaa tctcactaac gtcttgatag ctctcattga aggtaaggga
960tttgtctcca aaaaggcggt tggctgacac caagttagag gacttgttgg cttttcgata
1020gagtcggcag ttcagtttgg caaagaagaa gtggatctgg tcggatgtct tctcggagat
1080ggtatcaaat ttaaaaacct ccatcagctg cttgagggtg ttattacaag cacccagctt
1140ggtcatagca aacgccgtgg agatgctcaa gggtgacagg aaaatgttgt cgttgtcgtt
1200cttggagtct gccaggtgct gatagaagtt agtggcaaat cgagaattgg ccttggacag
1260ttcccagacc cgccggttgg tggcttccgg aaccttctgc tctaggacat cctcctccgt
1320ggccttcttc gcaggggagc ggtaaatgca catggggttc acggggatgt ctcggggctt
1380cgctatgcag atgtcgtcca cagggtttcc atgacagaca gcacagccca aggcaccaat
1440gagtagcaga gagaggagac aaagcttcct ctctccagca accgcacttc ctattcccgg
1500ggaatacatg gtggacgctt ctgcagagaa gccgtggaga cagtgctgag caatccctcc
1560g
1561916PRTUnknownsource/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
151321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 13cuaaatctcg
cagaggguuc c
211421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 14guucuttcct
ctaaatucuc g
211521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 15cugaaaactg
gttcttuucc u
211621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 16ggcaatccgc
ctgaaaaacu g
211721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 17ugugatctga
ggcaatuccg c
211821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 18guggagatag
tgtgatucug a
211921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 19ggcugggcaa
gtggaggaua g
212021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 20ucuuccacag
ggctggggca a
212121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 21uggccgctaa
tcttcccaca g
212221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 22uuggaataca
tggccggcua a
212321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 23uccuatcaca
ttggaaauac a
212421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 24agguuacagt
tcctatucac a
212521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 25cuuuutccag
aggttaacag u
212621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 26auaaaccttc
ctttttucca g
212721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 27aggacaaaag
ataaacccuu c
212821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 28augagcagca
aggacaaaaa g
212921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 29ccagaagcca
atgagccagc a
213021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 30ucacgcagtc
ccagaaagcc a
213121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 31ccgugacagg
tcacgccagu c
213221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 32cacagggctc
ccgtgaacag g
213321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 33ugcagatgtc
cacaggggcu c
213421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 34ggcuuggctg
tgcagaaugu c
213521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 35aaugucccgc
ggcttgggcu g
213621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 36gauucatggg
aatgtccccg c
213721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 37augcacatgg
gattcaaugg g
213821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 38ggagcggtaa
atgcaccaug g
213921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 39ucuuctccgg
ggagcgggua a
214021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 40ucagutgcct
tcttctuccg g
214121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 41gcccucatcc
tcagttugcc u
214221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 42ucugutctga
gccctccauc c
214321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 43uccgggatct
tctgttucug a
214421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 44guuggtggcc
tccggggauc u
214521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 45agacacgccg
gttggtuggc c
214621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 46gacagttccc
agacaccgcc g
214721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 47auuggccttg
gacagtuucc c
214821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 48caaagcggga
attggcccuu g
214921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 49aaaguggtag
caaagccggg a
215021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 50gugcugatag
aaagtgggua g
215121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 51aaucugccag
gtgctggaua g
215221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 52ucauucttgg
aatctggcca g
215321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 53guuaucattg
tcattccuug g
215421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 54acaggaaaat
gttatccauu g
215521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 55cucaggggtg
acaggaaaaa u
215621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 56cguggagata
ctcagggggu g
215721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 57uagcaaaagc
cgtggaagau a
215821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 58agcuuggtca
tagcaaaaag c
215921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 59acaggcaccc
agcttggguc a
216021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 60gggugtcatt
acaggccacc c
216121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 61aguugctgga
gggtgtucau u
216221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 62uaccuccatc
agttgccugg a
216321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 63caaacttaaa
tacctcccau c
216421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 64gauauggtgt
caaactuuaa a
216521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 65uguuutctca
gatatgggug u
216621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 66ucugatcaga
tgttttucuc a
216721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 67aagaagtgga
tctgatucag a
216821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 68uuuggcaaag
aagaaggugg a
216921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 69ggcagttcag
tttggccaaa g
217021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 70cgauagagtc
ggcagtuuca g
217121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 71guuggctttt
cgataggagu c
217221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 72uggaggattt
gttggccuuu u
217321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 73gauactaact
tggagggauu u
217421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 74gcgautggct
gatactuaac u
217521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 75cuccaaaaag
gcgattuggc u
217621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 76agggatttgt
ctccaaaaaa g
217721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 77auugaaggta
agggatuuug u
217821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 78gguaggtctc
attgaaaggu a
217921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 79cugaugtcct
ggtagggucu c
218021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 80uaccaactca
ctgatggucc u
218121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 81uggcuccata
taccaaacuc a
218221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 82ggcuggagct
tggctcccau a
218321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 83gaaguccagg
ggctgggagc u
218421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 84cauuutcctt
gaagtcccag g
218521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 85gauugctctg
cattttuccu u
218621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 86ggccgctctg
gattgccucu g
218721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 87auuugttgat
ggccgccucu g
218821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 88uuggacaccc
atttgtuuga u
218921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 89uucggtctta
ttggaccacc c
219021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 90ugauucggcc
ttcggtucuu a
219121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 91augacatcgg
tgattccggc c
219221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 92uuccgaggga
atgacaaucg g
219321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 93cauugatggc
ttccgaaggg a
219421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 94acagugagct
cattgaaugg c
219521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 95cagcaccaga
acagtggagc u
219621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 96aaccagcacc
agaacaagug a
219721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 97uaaccagcac
cagaaccagu g
219821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 98uuaaccagca
ccagaaacag u
219921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 99guuaaccagc
accagaaaca g
2110021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 100uguuaaccag
caccaggaac a
2110121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 101guguuaacca
gcaccaagaa c
2110221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 102ggugutaacc
agcacccaga a
2110321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 103uggugttaac
cagcacccag a
2110421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 104auggugttaa
ccagcaacca g
2110521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 105aauggtgtta
accagccacc a
2110621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 106aaauggtgtt
aaccaggcac c
2110721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 107uaaauggtgt
taaccaagca c
2110821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 108guaaatggtg
ttaacccagc a
2110921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 109aguaaatggt
gttaacccag c
2111021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 110aaguaaatgg
tgttaaacca g
2111121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 111gaaguaaatg
gtgttaaacc a
2111221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 112ugaagtaaat
ggtgttuaac c
2111321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 113uugaagtaaa
tggtgtuuaa c
2111421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 114cuugaagtaa
atggtgguua a
2111521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 115ccuugaagta
aatggtuguu a
2111621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 116cccuugaagt
aaatgggugu u
2111721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 117gcccutgaag
taaatgggug u
2111821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 118ggcccttgaa
gtaaatuggu g
2111921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 119aggcccttga
agtaaaaugg u
2112021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 120caggcccttg
aagtaaaaug g
2112121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 121acaggccctt
gaagtaaaau g
2112221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 122cacaggccct
tgaagtuaaa u
2112321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 123ccacaggccc
ttgaagguaa a
2112421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 124uccacaggcc
cttgaaagua a
2112521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 125uuccacaggc
ccttgaaagu a
2112621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 126cuuccacagg
cccttggaag u
2112721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 127acuuugactt
ccacagggcc c
2112821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 128ucagggctga
actttggacu u
2112921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 129ccuugtgttc
tcaggggcug a
2113021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 130acagutcctt
ccttgtuguu c
2113121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 131gccuugtaga
acagttuccu u
2113221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 132cucuccatca
gccttgguag a
2113321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 133cugaacacga
ctctcccauc a
2113421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 134aucauagatg
ctgaaccacg a
2113521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 135uuccuggtac
atcataagau g
2113621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 136ggaacttgcc
ttcctgggua c
2113721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 137cgccgataac
ggaactuugc c
2113821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 138uucagccacg
cgccgaauaa c
2113921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 139ccugggtgcc
ttcagcccac g
2114021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 140aacucaagca
cctggggugc c
2114121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 141uuugaagggc
aactcaaagc a
2114221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 142ugucatcacc
tttgaaaggg c
2114321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 143accauggtga
tgtcatucac c
2114421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 144caagatgagg
accatgggug a
2114521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 145caggcttggg
caagatugag g
2114621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 146aggcucttct
caggctuugg g
2114721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 147uaccutggcc
aggctccuuc u
2114821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 148guuccttctc
taccttuggc c
2114921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 149ucuggggtga
gttcctuucu c
2115021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 150uugcagcacc
tctgggggug a
2115121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 151ccagccactc
ttgcaggcac c
2115221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 152uccaattcat
ccagcccacu c
2115321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 153caucatctcc
tccaatuuca u
2115421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 154ggaccaccag
catcatucuc c
2115521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 155cggggcatgt
ggaccaacca g
2115621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 156aaugcggaag
cggggccaug u
2115721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 157agccgtcctc
aatgcgggaa g
2115821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 158uucaaactga
agccgtuccu c
2115921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 159cagcugctcc
ttcaaaacug a
2116021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 160ccaugtcttg
cagctggcuc c
2116121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 161ucgacaaggc
ccatgtucuu g
2116221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 162gcugaacaga
tcgacaaagg c
2116321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 163acuuutcagg
gctgaaacag a
2116421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 164gggagtttgg
acttttucag g
2116521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 165aacaatacct
gggagtuuug g
2116621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 166ggccutctgc
aacaatuacc u
2116721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 167aggucatctc
ggccttucug c
2116821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 168ugagacatag
aggtcaaucu c
2116921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 169ggaaugcatc
tgagaccaua g
2117021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 170aaugccttat
ggaatggcau c
2117121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 171uaccucaaga
aatgcccuua u
2117221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 172cuucutcatt
tacctccaag a
2117321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 173gcuucactgc
cttcttucau u
2117421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 174acuugcagct
gcttcaacug c
2117521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 175caacagcggt
acttgccagc u
2117621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 176ccagcaatca
caacaggcgg u
2117721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 177uagcgaacgg
ccagcaaauc a
2117821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 178uguuggggtt
tagcgaaacg g
2117921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 179aaagucaccc
tgttgggggu u
2118021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 180guuggccttg
aaagtccacc c
2118121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 181ggaaaggcct
gttggcccuu g
2118221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 182auaaaaacca
ggaaagggcc u
2118321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 183aacuuctctt
ataaaaaacc a
2118421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 184uguucagagg
aacttccucu u
2118521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 185aagauaatag
tgttcaagag g
2118621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 186ucugcccatg
aagataaaua g
2118721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 187gguuggctac
tctgccccau g
2118821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 188uuaacacaag
ggttgggcua c
2118921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 189aacautttac
ttaacaacaa g
2119021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 190aaagaataag
aacattuuua c
2119121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 191gaagaggtgc
aaagaaauaa g
2119221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 192ccaaaaatag
gaagagggug c
2119321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 193uguucacaaa
ccaaaaaaua g
2119421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 194uuuuuacttc
tgttcaacaa a
2119521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 195uuuguattta
tttttaacuu c
2119621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 196auggaagtag
tttgtaauuu a
2119721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 197ctaaatctcg cagagggttc c
2119821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 198gttctttcct ctaaattctc g
2119921DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 199ctgaaaactg
gttcttttcc t
2120021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 200ggcaatccgc ctgaaaaact g
2120121DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 201tgtgatctga ggcaattccg c
2120221DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 202gtggagatag
tgtgattctg a
2120321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 203ggctgggcaa gtggaggata g
2120421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 204tcttccacag ggctggggca a
2120521DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 205tggccgctaa
tcttcccaca g
2120621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 206ttggaataca tggccggcta a
2120721DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 207tcctatcaca ttggaaatac a
2120821DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 208aggttacagt
tcctattcac a
2120921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 209ctttttccag aggttaacag t
2121021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 210ataaaccttc cttttttcca g
2121121DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 211aggacaaaag
ataaaccctt c
2121221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 212atgagcagca aggacaaaaa g
2121321DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 213ccagaagcca atgagccagc a
2121421DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 214tcacgcagtc
ccagaaagcc a
2121521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 215ccgtgacagg tcacgccagt c
2121621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 216cacagggctc ccgtgaacag g
2121721DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 217tgcagatgtc
cacaggggct c
2121821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 218ggcttggctg tgcagaatgt c
2121921DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 219aatgtcccgc ggcttgggct g
2122021DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 220gattcatggg
aatgtccccg c
2122121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 221atgcacatgg gattcaatgg g
2122221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 222ggagcggtaa atgcaccatg g
2122321DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 223tcttctccgg
ggagcgggta a
2122421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 224tcagttgcct tcttcttccg g
2122521DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 225gccctcatcc tcagtttgcc t
2122621DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 226tctgttctga
gccctccatc c
2122721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 227tccgggatct tctgtttctg a
2122821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 228gttggtggcc tccggggatc t
2122921DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 229agacacgccg
gttggttggc c
2123021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 230gacagttccc agacaccgcc g
2123121DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 231attggccttg gacagtttcc c
2123221DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 232caaagcggga
attggccctt g
2123321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 233aaagtggtag caaagccggg a
2123421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 234gtgctgatag aaagtgggta g
2123521DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 235aatctgccag
gtgctggata g
2123621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 236tcattcttgg aatctggcca g
2123721DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 237gttatcattg tcattccttg g
2123821DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 238acaggaaaat
gttatccatt g
2123921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 239ctcaggggtg acaggaaaaa t
2124021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 240cgtggagata ctcagggggt g
2124121DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 241tagcaaaagc
cgtggaagat a
2124221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 242agcttggtca tagcaaaaag c
2124321DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 243acaggcaccc agcttgggtc a
2124421DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 244gggtgtcatt
acaggccacc c
2124521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 245agttgctgga gggtgttcat t
2124621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 246tacctccatc agttgcctgg a
2124721DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 247caaacttaaa
tacctcccat c
2124821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 248gatatggtgt caaactttaa a
2124921DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 249tgttttctca gatatgggtg t
2125021DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 250tctgatcaga
tgtttttctc a
2125121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 251aagaagtgga tctgattcag a
2125221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 252tttggcaaag aagaaggtgg a
2125321DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 253ggcagttcag
tttggccaaa g
2125421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 254cgatagagtc ggcagtttca g
2125521DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 255gttggctttt cgataggagt c
2125621DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 256tggaggattt
gttggccttt t
2125721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 257gatactaact tggagggatt t
2125821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 258gcgattggct gatacttaac t
2125921DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 259ctccaaaaag
gcgatttggc t
2126021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 260agggatttgt ctccaaaaaa g
2126121DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 261attgaaggta agggattttg t
2126221DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 262ggtaggtctc
attgaaaggt a
2126321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 263ctgatgtcct ggtagggtct c
2126421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 264taccaactca ctgatggtcc t
2126521DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 265tggctccata
taccaaactc a
2126621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 266ggctggagct tggctcccat a
2126721DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 267gaagtccagg ggctgggagc t
2126821DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 268cattttcctt
gaagtcccag g
2126921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 269gattgctctg catttttcct t
2127021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 270ggccgctctg gattgcctct g
2127121DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 271atttgttgat
ggccgcctct g
2127221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 272ttggacaccc atttgtttga t
2127321DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 273ttcggtctta ttggaccacc c
2127421DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 274tgattcggcc
ttcggttctt a
2127521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 275atgacatcgg tgattccggc c
2127621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 276ttccgaggga atgacaatcg g
2127721DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 277cattgatggc
ttccgaaggg a
2127821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 278acagtgagct cattgaatgg c
2127921DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 279cagcaccaga acagtggagc t
2128021DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 280aaccagcacc
agaacaagtg a
2128121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 281taaccagcac cagaaccagt g
2128221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 282ttaaccagca ccagaaacag t
2128321DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 283gttaaccagc
accagaaaca g
2128421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 284tgttaaccag caccaggaac a
2128521DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 285gtgttaacca gcaccaagaa c
2128621DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 286ggtgttaacc
agcacccaga a
2128721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 287tggtgttaac cagcacccag a
2128821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 288atggtgttaa ccagcaacca g
2128921DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 289aatggtgtta
accagccacc a
2129021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 290aaatggtgtt aaccaggcac c
2129121DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 291taaatggtgt taaccaagca c
2129221DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 292gtaaatggtg
ttaacccagc a
2129321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 293agtaaatggt gttaacccag c
2129421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 294aagtaaatgg tgttaaacca g
2129521DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 295gaagtaaatg
gtgttaaacc a
2129621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 296tgaagtaaat ggtgtttaac c
2129721DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 297ttgaagtaaa tggtgtttaa c
2129821DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 298cttgaagtaa
atggtggtta a
2129921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 299ccttgaagta aatggttgtt a
2130021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 300cccttgaagt aaatgggtgt t
2130121DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 301gcccttgaag
taaatgggtg t
2130221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 302ggcccttgaa gtaaattggt g
2130321DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 303aggcccttga agtaaaatgg t
2130421DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 304caggcccttg
aagtaaaatg g
2130521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 305acaggccctt gaagtaaaat g
2130621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 306cacaggccct tgaagttaaa t
2130721DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 307ccacaggccc
ttgaaggtaa a
2130821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 308tccacaggcc cttgaaagta a
2130921DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 309ttccacaggc ccttgaaagt a
2131021DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 310cttccacagg
cccttggaag t
2131121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 311actttgactt ccacagggcc c
2131221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 312tcagggctga actttggact t
2131321DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 313ccttgtgttc
tcaggggctg a
2131421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 314acagttcctt ccttgttgtt c
2131521DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 315gccttgtaga acagtttcct t
2131621DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 316ctctccatca
gccttggtag a
2131721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 317ctgaacacga ctctcccatc a
2131821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 318atcatagatg ctgaaccacg a
2131921DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 319ttcctggtac
atcataagat g
2132021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 320ggaacttgcc ttcctgggta c
2132121DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 321cgccgataac ggaactttgc c
2132221DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 322ttcagccacg
cgccgaataa c
2132321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 323cctgggtgcc ttcagcccac g
2132421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 324aactcaagca cctggggtgc c
2132521DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 325tttgaagggc
aactcaaagc a
2132621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 326tgtcatcacc tttgaaaggg c
2132721DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 327accatggtga tgtcattcac c
2132821DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 328caagatgagg
accatgggtg a
2132921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 329caggcttggg caagattgag g
2133021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 330aggctcttct caggctttgg g
2133121DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 331taccttggcc
aggctccttc t
2133221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 332gttccttctc tacctttggc c
2133321DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 333tctggggtga gttcctttct c
2133421DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 334ttgcagcacc
tctgggggtg a
2133521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 335ccagccactc ttgcaggcac c
2133621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 336tccaattcat ccagcccact c
2133721DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 337catcatctcc
tccaatttca t
2133821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 338ggaccaccag catcattctc c
2133921DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 339cggggcatgt ggaccaacca g
2134021DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 340aatgcggaag
cggggccatg t
2134121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 341agccgtcctc aatgcgggaa g
2134221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 342ttcaaactga agccgttcct c
2134321DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 343cagctgctcc
ttcaaaactg a
2134421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 344ccatgtcttg cagctggctc c
2134521DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 345tcgacaaggc ccatgttctt g
2134621DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 346gctgaacaga
tcgacaaagg c
2134721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 347acttttcagg gctgaaacag a
2134821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 348gggagtttgg actttttcag g
2134921DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 349aacaatacct
gggagttttg g
2135021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 350ggccttctgc aacaattacc t
2135121DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 351aggtcatctc ggcctttctg c
2135221DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 352tgagacatag
aggtcaatct c
2135321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 353ggaatgcatc tgagaccata g
2135421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 354aatgccttat ggaatggcat c
2135521DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 355tacctcaaga
aatgccctta t
2135621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 356cttcttcatt tacctccaag a
2135721DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 357gcttcactgc cttctttcat t
2135821DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 358acttgcagct
gcttcaactg c
2135921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 359caacagcggt acttgccagc t
2136021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 360ccagcaatca caacaggcgg t
2136121DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 361tagcgaacgg
ccagcaaatc a
2136221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 362tgttggggtt tagcgaaacg g
2136321DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 363aaagtcaccc tgttgggggt t
2136421DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 364gttggccttg
aaagtccacc c
2136521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 365ggaaaggcct gttggccctt g
2136621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 366ataaaaacca ggaaagggcc t
2136721DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 367aacttctctt
ataaaaaacc a
2136821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 368tgttcagagg aacttcctct t
2136921DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 369aagataatag tgttcaagag g
2137021DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 370tctgcccatg
aagataaata g
2137121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 371ggttggctac tctgccccat g
2137221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 372ttaacacaag ggttgggcta c
2137321DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 373aacattttac
ttaacaacaa g
2137421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 374aaagaataag aacattttta c
2137521DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 375gaagaggtgc aaagaaataa g
2137621DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 376ccaaaaatag
gaagagggtg c
2137721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 377tgttcacaaa ccaaaaaata g
2137821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 378tttttacttc tgttcaacaa a
2137921DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 379tttgtattta
tttttaactt c
2138021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 380atggaagtag tttgtaattt a
2138120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide" 381cuaaatctcg cagagguucc
2038220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 382guucuttcct ctaaaucucg
2038320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 383cugaaaactg gttctuuccu
2038420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 384ggcaatccgc ctgaaaacug
2038520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 385ugugatctga ggcaauccgc
2038620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 386guggagatag tgtgaucuga
2038720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 387ggcugggcaa gtggagauag
2038820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 388ucuuccacag ggctgggcaa
2038920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 389uggccgctaa tcttccacag
2039020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 390uuggaataca tggccgcuaa
2039120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 391uccuatcaca ttggaauaca
2039220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 392agguuacagt tcctaucaca
2039320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 393cuuuutccag aggttacagu
2039420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 394auaaaccttc cttttuccag
2039520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 395aggacaaaag ataaaccuuc
2039620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 396augagcagca aggacaaaag
2039720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 397ccagaagcca atgagcagca
2039820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 398ucacgcagtc ccagaagcca
2039920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 399ccgugacagg tcacgcaguc
2040020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 400cacagggctc ccgtgacagg
2040120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 401ugcagatgtc cacagggcuc
2040220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 402ggcuuggctg tgcagauguc
2040320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 403aaugucccgc ggcttggcug
2040420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 404gauucatggg aatgtcccgc
2040520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 405augcacatgg gattcauggg
2040620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 406ggagcggtaa atgcacaugg
2040720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 407ucuuctccgg ggagcgguaa
2040820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 408ucagutgcct tcttcuccgg
2040920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 409gcccucatcc tcagtugccu
2041020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 410ucugutctga gccctcaucc
2041120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 411uccgggatct tctgtucuga
2041220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 412guuggtggcc tccgggaucu
2041320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 413agacacgccg gttgguggcc
2041420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 414gacagttccc agacacgccg
2041520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 415auuggccttg gacaguuccc
2041620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 416caaagcggga attggccuug
2041720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 417aaaguggtag caaagcggga
2041820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 418gugcugatag aaagtgguag
2041920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 419aaucugccag gtgctgauag
2042020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 420ucauucttgg aatctgccag
2042120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 421guuaucattg tcattcuugg
2042220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 422acaggaaaat gttatcauug
2042320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 423cucaggggtg acaggaaaau
2042420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 424cguggagata ctcaggggug
2042520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 425uagcaaaagc cgtggagaua
2042620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 426agcuuggtca tagcaaaagc
2042720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 427acaggcaccc agcttgguca
2042820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 428gggugtcatt acaggcaccc
2042920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 429aguugctgga gggtgucauu
2043020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 430uaccuccatc agttgcugga
2043120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 431caaacttaaa tacctccauc
2043220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 432gauauggtgt caaacuuaaa
2043320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 433uguuutctca gatatggugu
2043420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 434ucugatcaga tgtttucuca
2043520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 435aagaagtgga tctgaucaga
2043620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 436uuuggcaaag aagaagugga
2043720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 437ggcagttcag tttggcaaag
2043820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 438cgauagagtc ggcaguucag
2043920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 439guuggctttt cgatagaguc
2044020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 440uggaggattt gttggcuuuu
2044120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 441gauactaact tggaggauuu
2044220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 442gcgautggct gatacuaacu
2044320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 443cuccaaaaag gcgatuggcu
2044420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 444agggatttgt ctccaaaaag
2044520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 445auugaaggta agggauuugu
2044620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 446gguaggtctc attgaaggua
2044720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 447cugaugtcct ggtaggucuc
2044820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 448uaccaactca ctgatguccu
2044920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 449uggcuccata taccaacuca
2045020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 450ggcuggagct tggctccaua
2045120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 451gaaguccagg ggctggagcu
2045220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 452cauuutcctt gaagtccagg
2045320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 453gauugctctg catttuccuu
2045420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 454ggccgctctg gattgcucug
2045520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 455auuugttgat ggccgcucug
2045620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 456uuggacaccc atttguugau
2045720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 457uucggtctta ttggacaccc
2045820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 458ugauucggcc ttcggucuua
2045920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 459augacatcgg tgattcggcc
2046020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 460uuccgaggga atgacaucgg
2046120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 461cauugatggc ttccgaggga
2046220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 462acagugagct cattgauggc
2046320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 463cagcaccaga acagtgagcu
2046420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 464aaccagcacc agaacaguga
2046520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 465uaaccagcac cagaacagug
2046620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 466uuaaccagca ccagaacagu
2046720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 467guuaaccagc accagaacag
2046820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 468uguuaaccag caccagaaca
2046920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 469guguuaacca gcaccagaac
2047020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 470ggugutaacc agcaccagaa
2047120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 471uggugttaac cagcaccaga
2047220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 472auggugttaa ccagcaccag
2047320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 473aauggtgtta accagcacca
2047420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 474aaauggtgtt aaccagcacc
2047520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 475uaaauggtgt taaccagcac
2047620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 476guaaatggtg ttaaccagca
2047720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 477aguaaatggt gttaaccagc
2047820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 478aaguaaatgg tgttaaccag
2047920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 479gaaguaaatg gtgttaacca
2048020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 480ugaagtaaat ggtgtuaacc
2048120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 481uugaagtaaa tggtguuaac
2048220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 482cuugaagtaa atggtguuaa
2048320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 483ccuugaagta aatgguguua
2048420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 484cccuugaagt aaatgguguu
2048520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 485gcccutgaag taaatggugu
2048620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 486ggcccttgaa gtaaauggug
2048720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 487aggcccttga agtaaauggu
2048820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 488caggcccttg aagtaaaugg
2048920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 489acaggccctt gaagtaaaug
2049020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 490cacaggccct tgaaguaaau
2049120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 491ccacaggccc ttgaaguaaa
2049220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 492uccacaggcc cttgaaguaa
2049320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 493uuccacaggc ccttgaagua
2049420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 494cuuccacagg cccttgaagu
2049520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 495acuuugactt ccacaggccc
2049620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 496ucagggctga actttgacuu
2049720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 497ccuugtgttc tcagggcuga
2049820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 498acagutcctt ccttguguuc
2049920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 499gccuugtaga acagtuccuu
2050020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 500cucuccatca gccttguaga
2050120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 501cugaacacga ctctccauca
2050220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 502aucauagatg ctgaacacga
2050320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 503uuccuggtac atcatagaug
2050420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 504ggaacttgcc ttcctgguac
2050520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 505cgccgataac ggaacuugcc
2050620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 506uucagccacg cgccgauaac
2050720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 507ccugggtgcc ttcagccacg
2050820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 508aacucaagca cctgggugcc
2050920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 509uuugaagggc aactcaagca
2051020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 510ugucatcacc tttgaagggc
2051120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 511accauggtga tgtcaucacc
2051220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 512caagatgagg accatgguga
2051320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 513caggcttggg caagaugagg
2051420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 514aggcucttct caggcuuggg
2051520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 515uaccutggcc aggctcuucu
2051620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 516guuccttctc tacctuggcc
2051720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 517ucuggggtga gttccuucuc
2051820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 518uugcagcacc tctgggguga
2051920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 519ccagccactc ttgcagcacc
2052020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 520uccaattcat ccagccacuc
2052120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 521caucatctcc tccaauucau
2052220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 522ggaccaccag catcaucucc
2052320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 523cggggcatgt ggaccaccag
2052420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 524aaugcggaag cggggcaugu
2052520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 525agccgtcctc aatgcggaag
2052620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 526uucaaactga agccguccuc
2052720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 527cagcugctcc ttcaaacuga
2052820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 528ccaugtcttg cagctgcucc
2052920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 529ucgacaaggc ccatgucuug
2053020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 530gcugaacaga tcgacaaggc
2053120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 531acuuutcagg gctgaacaga
2053220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 532gggagtttgg actttucagg
2053320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 533aacaatacct gggaguuugg
2053420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 534ggccutctgc aacaauaccu
2053520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 535aggucatctc ggcctucugc
2053620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 536ugagacatag aggtcaucuc
2053720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 537ggaaugcatc tgagacauag
2053820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 538aaugccttat ggaatgcauc
2053920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 539uaccucaaga aatgccuuau
2054020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 540cuucutcatt tacctcaaga
2054120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 541gcuucactgc cttctucauu
2054220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 542acuugcagct gcttcacugc
2054320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 543caacagcggt acttgcagcu
2054420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 544ccagcaatca caacagcggu
2054520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 545uagcgaacgg ccagcaauca
2054620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 546uguuggggtt tagcgaacgg
2054720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 547aaagucaccc tgttgggguu
2054820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 548guuggccttg aaagtcaccc
2054920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 549ggaaaggcct gttggccuug
2055020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 550auaaaaacca ggaaaggccu
2055120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 551aacuuctctt ataaaaacca
2055220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 552uguucagagg aacttcucuu
2055320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 553aagauaatag tgttcagagg
2055420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 554ucugcccatg aagataauag
2055520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 555gguuggctac tctgcccaug
2055620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 556uuaacacaag ggttggcuac
2055720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 557aacautttac ttaacacaag
2055820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 558aaagaataag aacatuuuac
2055920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 559gaagaggtgc aaagaauaag
2056020DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 560ccaaaaatag gaagaggugc
2056120DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 561uguucacaaa ccaaaaauag
2056220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 562uuuuuacttc tgttcacaaa
2056320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 563uuuguattta tttttacuuc
2056420DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 564auggaagtag tttgtauuua
2056520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 565cuaaaucucg cagagguucc
2056620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 566guucuuuccu
cuaaaucucg
2056720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 567cugaaaacug guucuuuccu
2056820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 568ggcaauccgc cugaaaacug
2056920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 569ugugaucuga
ggcaauccgc
2057020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 570guggagauag ugugaucuga
2057120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 571ggcugggcaa guggagauag
2057220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 572ucuuccacag
ggcugggcaa
2057320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 573uggccgcuaa ucuuccacag
2057420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 574uuggaauaca uggccgcuaa
2057520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 575uccuaucaca
uuggaauaca
2057620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 576agguuacagu uccuaucaca
2057720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 577cuuuuuccag agguuacagu
2057820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 578auaaaccuuc
cuuuuuccag
2057920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 579aggacaaaag auaaaccuuc
2058020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 580augagcagca aggacaaaag
2058120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 581ccagaagcca
augagcagca
2058220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 582ucacgcaguc ccagaagcca
2058320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 583ccgugacagg ucacgcaguc
2058420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 584cacagggcuc
ccgugacagg
2058520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 585ugcagauguc cacagggcuc
2058620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 586ggcuuggcug ugcagauguc
2058720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 587aaugucccgc
ggcuuggcug
2058820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 588gauucauggg aaugucccgc
2058920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 589augcacaugg gauucauggg
2059020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 590ggagcgguaa
augcacaugg
2059120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 591ucuucuccgg ggagcgguaa
2059220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 592ucaguugccu ucuucuccgg
2059320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 593gcccucaucc
ucaguugccu
2059420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 594ucuguucuga gcccucaucc
2059520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 595uccgggaucu ucuguucuga
2059620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 596guugguggcc
uccgggaucu
2059720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 597agacacgccg guugguggcc
2059820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 598gacaguuccc agacacgccg
2059920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 599auuggccuug
gacaguuccc
2060020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 600caaagcggga auuggccuug
2060120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 601aaagugguag caaagcggga
2060220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 602gugcugauag
aaagugguag
2060320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 603aaucugccag gugcugauag
2060420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 604ucauucuugg aaucugccag
2060520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 605guuaucauug
ucauucuugg
2060620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 606acaggaaaau guuaucauug
2060720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 607cucaggggug acaggaaaau
2060820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 608cguggagaua
cucaggggug
2060920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 609uagcaaaagc cguggagaua
2061020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 610agcuugguca uagcaaaagc
2061120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 611acaggcaccc
agcuugguca
2061220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 612gggugucauu acaggcaccc
2061320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 613aguugcugga gggugucauu
2061420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 614uaccuccauc
aguugcugga
2061520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 615caaacuuaaa uaccuccauc
2061620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 616gauauggugu caaacuuaaa
2061720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 617uguuuucuca
gauauggugu
2061820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 618ucugaucaga uguuuucuca
2061920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 619aagaagugga ucugaucaga
2062020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 620uuuggcaaag
aagaagugga
2062120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 621ggcaguucag uuuggcaaag
2062220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 622cgauagaguc ggcaguucag
2062320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 623guuggcuuuu
cgauagaguc
2062420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 624uggaggauuu guuggcuuuu
2062520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 625gauacuaacu uggaggauuu
2062620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 626gcgauuggcu
gauacuaacu
2062720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 627cuccaaaaag gcgauuggcu
2062820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 628agggauuugu cuccaaaaag
2062920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 629auugaaggua
agggauuugu
2063020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 630gguaggucuc auugaaggua
2063120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 631cugauguccu gguaggucuc
2063220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 632uaccaacuca
cugauguccu
2063320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 633uggcuccaua uaccaacuca
2063420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 634ggcuggagcu uggcuccaua
2063520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 635gaaguccagg
ggcuggagcu
2063620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 636cauuuuccuu gaaguccagg
2063720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 637gauugcucug cauuuuccuu
2063820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 638ggccgcucug
gauugcucug
2063920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 639auuuguugau ggccgcucug
2064020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 640uuggacaccc auuuguugau
2064120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 641uucggucuua
uuggacaccc
2064220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 642ugauucggcc uucggucuua
2064320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 643augacaucgg ugauucggcc
2064420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 644uuccgaggga
augacaucgg
2064520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 645cauugauggc uuccgaggga
2064620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 646acagugagcu cauugauggc
2064720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 647cagcaccaga
acagugagcu
2064820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 648aaccagcacc agaacaguga
2064920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 649uaaccagcac cagaacagug
2065020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 650uuaaccagca
ccagaacagu
2065120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 651guuaaccagc accagaacag
2065220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 652uguuaaccag caccagaaca
2065320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 653guguuaacca
gcaccagaac
2065420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 654gguguuaacc agcaccagaa
2065520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 655ugguguuaac cagcaccaga
2065620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 656augguguuaa
ccagcaccag
2065720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 657aaugguguua accagcacca
2065820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 658aaaugguguu aaccagcacc
2065920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 659uaaauggugu
uaaccagcac
2066020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 660guaaauggug uuaaccagca
2066120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 661aguaaauggu guuaaccagc
2066220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 662aaguaaaugg
uguuaaccag
2066320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 663gaaguaaaug guguuaacca
2066420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 664ugaaguaaau gguguuaacc
2066520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 665uugaaguaaa
ugguguuaac
2066620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 666cuugaaguaa augguguuaa
2066720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 667ccuugaagua aaugguguua
2066820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 668cccuugaagu
aaaugguguu
2066920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 669gcccuugaag uaaauggugu
2067020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 670ggcccuugaa guaaauggug
2067120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 671aggcccuuga
aguaaauggu
2067220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 672caggcccuug aaguaaaugg
2067320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 673acaggcccuu gaaguaaaug
2067420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 674cacaggcccu
ugaaguaaau
2067520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 675ccacaggccc uugaaguaaa
2067620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 676uccacaggcc cuugaaguaa
2067720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 677uuccacaggc
ccuugaagua
2067820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 678cuuccacagg cccuugaagu
2067920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 679acuuugacuu ccacaggccc
2068020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 680ucagggcuga
acuuugacuu
2068120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 681ccuuguguuc ucagggcuga
2068220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 682acaguuccuu ccuuguguuc
2068320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 683gccuuguaga
acaguuccuu
2068420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 684cucuccauca gccuuguaga
2068520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 685cugaacacga cucuccauca
2068620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 686aucauagaug
cugaacacga
2068720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 687uuccugguac aucauagaug
2068820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 688ggaacuugcc uuccugguac
2068920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 689cgccgauaac
ggaacuugcc
2069020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 690uucagccacg cgccgauaac
2069120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 691ccugggugcc uucagccacg
2069220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 692aacucaagca
ccugggugcc
2069320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 693uuugaagggc aacucaagca
2069420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 694ugucaucacc uuugaagggc
2069520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 695accaugguga
ugucaucacc
2069620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 696caagaugagg accaugguga
2069720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 697caggcuuggg caagaugagg
2069820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 698aggcucuucu
caggcuuggg
2069920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 699uaccuuggcc aggcucuucu
2070020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 700guuccuucuc uaccuuggcc
2070120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 701ucugggguga
guuccuucuc
2070220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 702uugcagcacc ucugggguga
2070320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 703ccagccacuc uugcagcacc
2070420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 704uccaauucau
ccagccacuc
2070520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 705caucaucucc uccaauucau
2070620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 706ggaccaccag caucaucucc
2070720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 707cggggcaugu
ggaccaccag
2070820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 708aaugcggaag cggggcaugu
2070920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 709agccguccuc aaugcggaag
2071020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 710uucaaacuga
agccguccuc
2071120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 711cagcugcucc uucaaacuga
2071220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 712ccaugucuug cagcugcucc
2071320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 713ucgacaaggc
ccaugucuug
2071420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 714gcugaacaga ucgacaaggc
2071520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 715acuuuucagg gcugaacaga
2071620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 716gggaguuugg
acuuuucagg
2071720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 717aacaauaccu gggaguuugg
2071820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 718ggccuucugc aacaauaccu
2071920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 719aggucaucuc
ggccuucugc
2072020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 720ugagacauag aggucaucuc
2072120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 721ggaaugcauc ugagacauag
2072220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 722aaugccuuau
ggaaugcauc
2072320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 723uaccucaaga aaugccuuau
2072420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 724cuucuucauu uaccucaaga
2072520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 725gcuucacugc
cuucuucauu
2072620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 726acuugcagcu gcuucacugc
2072720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 727caacagcggu acuugcagcu
2072820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 728ccagcaauca
caacagcggu
2072920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 729uagcgaacgg ccagcaauca
2073020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 730uguugggguu uagcgaacgg
2073120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 731aaagucaccc
uguugggguu
2073220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 732guuggccuug aaagucaccc
2073320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 733ggaaaggccu guuggccuug
2073420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 734auaaaaacca
ggaaaggccu
2073520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 735aacuucucuu auaaaaacca
2073620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 736uguucagagg aacuucucuu
2073720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 737aagauaauag
uguucagagg
2073820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 738ucugcccaug aagauaauag
2073920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 739gguuggcuac ucugcccaug
2074020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 740uuaacacaag
gguuggcuac
2074120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 741aacauuuuac uuaacacaag
2074220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 742aaagaauaag aacauuuuac
2074320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 743gaagaggugc
aaagaauaag
2074420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 744ccaaaaauag gaagaggugc
2074520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 745uguucacaaa ccaaaaauag
2074620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 746uuuuuacuuc
uguucacaaa
2074720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 747uuuguauuua uuuuuacuuc
2074820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 748auggaaguag uuuguauuua
2074920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 749ggaaccucug
cgagauuuag
2075020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 750cgagauuuag aggaaagaac
2075120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 751aggaaagaac caguuuucag
2075220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 752caguuuucag
gcggauugcc
2075320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 753gcggauugcc ucagaucaca
2075420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 754ucagaucaca cuaucuccac
2075520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 755cuaucuccac
uugcccagcc
2075620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 756uugcccagcc cuguggaaga
2075720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 757cuguggaaga uuagcggcca
2075820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 758uuagcggcca
uguauuccaa
2075920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 759uguauuccaa ugugauagga
2076020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 760ugugauagga acuguaaccu
2076120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 761acuguaaccu
cuggaaaaag
2076220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 762cuggaaaaag gaagguuuau
2076320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 763gaagguuuau cuuuuguccu
2076420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 764cuuuuguccu
ugcugcucau
2076520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 765ugcugcucau uggcuucugg
2076620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 766uggcuucugg gacugcguga
2076720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 767gacugcguga
ccugucacgg
2076820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 768ccugucacgg gagcccugug
2076920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 769gagcccugug gacaucugca
2077020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 770gacaucugca
cagccaagcc
2077120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 771cagccaagcc gcgggacauu
2077220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 772gcgggacauu cccaugaauc
2077320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 773cccaugaauc
ccaugugcau
2077420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 774ccaugugcau uuaccgcucc
2077520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 775uuaccgcucc ccggagaaga
2077620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 776ccggagaaga
aggcaacuga
2077720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 777aggcaacuga ggaugagggc
2077820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 778ggaugagggc ucagaacaga
2077920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 779ucagaacaga
agaucccgga
2078020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 780agaucccgga ggccaccaac
2078120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 781ggccaccaac cggcgugucu
2078220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 782cggcgugucu
gggaacuguc
2078320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 783gggaacuguc caaggccaau
2078420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 784caaggccaau ucccgcuuug
2078520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 785ucccgcuuug
cuaccacuuu
2078620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 786cuaccacuuu cuaucagcac
2078720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 787cuaucagcac cuggcagauu
2078820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 788cuggcagauu
ccaagaauga
2078920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 789ccaagaauga caaugauaac
2079020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 790caaugauaac auuuuccugu
2079120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 791auuuuccugu
caccccugag
2079220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 792caccccugag uaucuccacg
2079320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 793uaucuccacg gcuuuugcua
2079420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 794gcuuuugcua
ugaccaagcu
2079520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 795ugaccaagcu gggugccugu
2079620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 796gggugccugu aaugacaccc
2079720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 797aaugacaccc
uccagcaacu
2079820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 798uccagcaacu gauggaggua
2079920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 799gauggaggua uuuaaguuug
2080020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 800uuuaaguuug
acaccauauc
2080120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 801acaccauauc ugagaaaaca
2080220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 802ugagaaaaca ucugaucaga
2080320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 803ucugaucaga
uccacuucuu
2080420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 804uccacuucuu cuuugccaaa
2080520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 805cuuugccaaa cugaacugcc
2080620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 806cugaacugcc
gacucuaucg
2080720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 807gacucuaucg aaaagccaac
2080820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 808aaaagccaac aaauccucca
2080920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 809aaauccucca
aguuaguauc
2081020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 810aguuaguauc agccaaucgc
2081120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 811agccaaucgc cuuuuuggag
2081220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 812cuuuuuggag
acaaaucccu
2081320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 813acaaaucccu uaccuucaau
2081420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 814uaccuucaau gagaccuacc
2081520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 815gagaccuacc
aggacaucag
2081620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 816aggacaucag ugaguuggua
2081720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 817ugaguuggua uauggagcca
2081820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 818uauggagcca
agcuccagcc
2081920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 819agcuccagcc ccuggacuuc
2082020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 820ccuggacuuc aaggaaaaug
2082120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 821aaggaaaaug
cagagcaauc
2082220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 822cagagcaauc cagagcggcc
2082320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 823cagagcggcc aucaacaaau
2082420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 824aucaacaaau
ggguguccaa
2082520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 825ggguguccaa uaagaccgaa
2082620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 826uaagaccgaa ggccgaauca
2082720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 827ggccgaauca
ccgaugucau
2082820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 828ccgaugucau ucccucggaa
2082920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 829ucccucggaa gccaucaaug
2083020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 830gccaucaaug
agcucacugu
2083120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 831agcucacugu ucuggugcug
2083220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 832ucacuguucu ggugcugguu
2083320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 833cacuguucug
gugcugguua
2083420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 834acuguucugg ugcugguuaa
2083520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 835cuguucuggu gcugguuaac
2083620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 836uguucuggug
cugguuaaca
2083720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 837guucuggugc ugguuaacac
2083820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 838uucuggugcu gguuaacacc
2083920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 839ucuggugcug
guuaacacca
2084020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 840cuggugcugg uuaacaccau
2084120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 841uggugcuggu uaacaccauu
2084220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 842ggugcugguu
aacaccauuu
2084320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 843gugcugguua acaccauuua
2084420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 844ugcugguuaa caccauuuac
2084520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 845gcugguuaac
accauuuacu
2084620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 846cugguuaaca ccauuuacuu
2084720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 847ugguuaacac cauuuacuuc
2084820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 848gguuaacacc
auuuacuuca
2084920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 849guuaacacca uuuacuucaa
2085020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 850uuaacaccau uuacuucaag
2085120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 851uaacaccauu
uacuucaagg
2085220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 852aacaccauuu acuucaaggg
2085320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 853acaccauuua cuucaagggc
2085420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 854caccauuuac
uucaagggcc
2085520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 855accauuuacu ucaagggccu
2085620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 856ccauuuacuu caagggccug
2085720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 857cauuuacuuc
aagggccugu
2085820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 858auuuacuuca agggccugug
2085920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 859uuuacuucaa gggccugugg
2086020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 860uuacuucaag
ggccugugga
2086120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 861uacuucaagg gccuguggaa
2086220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 862acuucaaggg ccuguggaag
2086320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 863gggccugugg
aagucaaagu
2086420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 864aagucaaagu ucagcccuga
2086520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 865ucagcccuga gaacacaagg
2086620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 866gaacacaagg
aaggaacugu
2086720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 867aaggaacugu ucuacaaggc
2086820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 868ucuacaaggc ugauggagag
2086920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 869ugauggagag
ucguguucag
2087020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 870ucguguucag caucuaugau
2087120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 871caucuaugau guaccaggaa
2087220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 872guaccaggaa
ggcaaguucc
2087320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 873ggcaaguucc guuaucggcg
2087420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 874guuaucggcg cguggcugaa
2087520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 875cguggcugaa
ggcacccagg
2087620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 876ggcacccagg ugcuugaguu
2087720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 877ugcuugaguu gcccuucaaa
2087820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 878gcccuucaaa
ggugaugaca
2087920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 879ggugaugaca ucaccauggu
2088020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 880ucaccauggu ccucaucuug
2088120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 881ccucaucuug
cccaagccug
2088220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 882cccaagccug agaagagccu
2088320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 883agaagagccu ggccaaggua
2088420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 884ggccaaggua
gagaaggaac
2088520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 885gagaaggaac ucaccccaga
2088620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 886ucaccccaga ggugcugcaa
2088720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 887ggugcugcaa
gaguggcugg
2088820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 888gaguggcugg augaauugga
2088920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 889augaauugga ggagaugaug
2089020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 890ggagaugaug
cugguggucc
2089120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 891cugguggucc acaugccccg
2089220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 892acaugccccg cuuccgcauu
2089320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 893cuuccgcauu
gaggacggcu
2089420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 894gaggacggcu ucaguuugaa
2089520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 895ucaguuugaa ggagcagcug
2089620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 896ggagcagcug
caagacaugg
2089720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 897caagacaugg gccuugucga
2089820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 898gccuugucga ucuguucagc
2089920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 899ucuguucagc
ccugaaaagu
2090020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 900ccugaaaagu ccaaacuccc
2090120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 901ccaaacuccc agguauuguu
2090220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 902agguauuguu
gcagaaggcc
2090320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 903gcagaaggcc gagaugaccu
2090420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 904gagaugaccu cuaugucuca
2090520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 905cuaugucuca
gaugcauucc
2090620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 906gaugcauucc auaaggcauu
2090720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 907auaaggcauu ucuugaggua
2090820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 908ucuugaggua
aaugaagaag
2090920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 909aaugaagaag gcagugaagc
2091020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 910gcagugaagc agcugcaagu
2091120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 911agcugcaagu
accgcuguug
2091220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 912accgcuguug ugauugcugg
2091320RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 913ugauugcugg ccguucgcua
2091420RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 914ccguucgcua
aaccccaaca
2091520RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 915aaccccaaca gggugacuuu
2091620RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 916gggugacuuu caaggccaac
2091720RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 917caaggccaac
aggccuuucc
2091820RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 918aggccuuucc ugguuuuuau
2091920RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 919ugguuuuuau aagagaaguu
2092020RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 920aagagaaguu
ccucugaaca
2092120RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 921ccucugaaca cuauuaucuu
2092220RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 922cuauuaucuu caugggcaga
2092320RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 923caugggcaga
guagccaacc
2092420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 924guagccaacc cuuguguuaa
2092520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 925cuuguguuaa guaaaauguu
2092620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 926guaaaauguu
cuuauucuuu
2092720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 927cuuauucuuu gcaccucuuc
2092820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 928gcaccucuuc cuauuuuugg
2092920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 929cuauuuuugg
uuugugaaca
2093020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 930uuugugaaca gaaguaaaaa
2093120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 931gaaguaaaaa uaaauacaaa
2093220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 932uaaauacaaa
cuacuuccau 20
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