POLYNUCLEOTIDE AGENTS TARGETING FACTOR XII (HAGEMAN FACTOR) (F12) AND METHODS OF USE THEREOF

Abstract

The invention relates to polynucleotide agents targeting Factor XII (F12) gene, and methods of using such polynucleotide agents to inhibit expression of Factor XII and to treat subjects having a Factor XII-associated disease, e.g., heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency.

Description

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 16/068,444, filed on Jan. 17, 2019, which is a 35 U.S.C. .sctn. 371 national stage filing of International Application No. PCT/US2017/012420, filed on Jan. 6, 2017, which in turn claims the benefit of priority to U.S. Provisional Patent Application No. 62/276,279, filed on Jan. 8, 2016. The entire contents of each of the foregoing applications are hereby 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 Oct. 29, 2020, is named 121301_05303_SL.txt and is 133,714 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Hereditary Angioedema (HAE) is a rare autosomal dominant disorder that causes recurrent edema and swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals and a nonpruritic rash in one-third of patients. Untreated HAE patients experience an average of one-to-two angioedema attacks per month, but the frequency and severity of episodes can vary significantly. Edema swelling is often disfiguring and disabling, results in frequent hospitalization, and patients may require psychiatric care to treat disease-associated anxiety. Abdominal attacks can cause severe pain, nausea and vomiting, and may lead to inappropriate surgeries. Furthermore, over half of HAE patients also experience life-threatening laryngeal edema during their lifetime that may require emergency tracheostomy to prevent asphyxiation. HAE affects an estimated 6,000 to 10,000 individuals of varying ethnic groups in the United States and causes significant economic harm to patients, accounting for 15,000 to 30,000 hospital visits and 20 to 100 sick days per year.

[0004] HAE results from a mutation of the C1 inhibitor (C1INH, SERPING1) gene that results in a deficiency of C1INH protein. Over 250 different C1INH mutations have been demonstrated to cause an HAE clinical presentation. These C1INH mutations are typically inherited genetically, however, up to 25% of HAE cases result from de novo mutation of C1INH. HAE type I is caused by C1INH mutations that result in lower levels of truncated or misfolded proteins that are inefficiently secreted, and accounts for approximately 85% of HAE cases. HAE type II constitutes about 15% of cases and is caused by mutations near the active site of C1INH that result in normal levels of dysfunctional C1INH protein. HAE type III, a rare third form the disease, occurs as a result of a gain-of-function mutation in coagulation factor XII (F12) (Hageman Factor).

[0005] C1 inhibitor is a serine protease inhibitor of the serpin family and a major inhibitor of proteases in the complement and contact activation pathways, as well as a minor inhibitor of fibrinolytic protease plasmin. These plasma proteolytic cascades are activated during an HAE attack, generating substances that increase vascular permeability. Studies have shown that the bradykinin peptide, which activates proinflammatory signaling pathways that dilate vessels and induces chemotaxis of neutrophils, is the primary substance that enhances vascular permeability in an HAE attack by binding to the bradykinin receptor on vascular endothelial cells. The formation of bradykinin in plasma generally requires interaction of three proteins, namely F12, prekallikrein and high-molecular-weight kininogen, collectively referred to as the "contact activation pathway" or the "Kallikrein-Kinin System." Prekallikrein and high-molecular-weight kininogen circulate as a bi-molecular complex. The contact activation pathway is initiated on binding of F12 to negatively charged surfaces (or macromolecules) which induces a conformational change in F12 resulting in active F12 (F12a). F12a cleaves prekallikrein to generate active kallikrein, which in turn reciprocally activates F12. The active kallikrein then digests high-molecular-weight kininogen to liberate bradykinin.

[0006] Typically, C1INH inhibits the autoactivation of F12, the ability of F12a to activate prekallilrein, the activation of high molecular weight kininogen by kallikrein, and the feedback activation of F12 by kallikrein. Consequently, mutations causing C1INH deficiency or F12 gain-of-function result in excess production of bradykinin and onset of HAE angioedema.

[0007] Currently, HAE may be treated with 17.alpha.-alkylated androgens prophylactically to reduce to probability of recurrent episodes, or with disease-specific therapeutics to treat acute attacks. About 70% of individuals with HAE are treated with androgens or remain untreated, and about 30% receive therapeutics. Androgens are unsuitable for short-term treatment of acute attacks because they take several days to become effective, and they can have significant side effects and may affect growth and development adversely. As a result, androgens are used only for long-term prophylaxis and are typically not administered to pregnant women or children. Furthermore, current therapeutics used to treat acute attacks must be administered intravenously numerous times per week or may cause side-effects that require drug administration and subsequent patient monitoring in a hospital, thereby limiting their regular prophylactic use to manage the disease long-term. Therefore, in the absence of regimens which be administered safely, effectively and by more convenient routes and regimens to treat acute angioedema attacks and prophylactically manage recurrent attacks in a large proportion of patients, including pregnant women and children, there is a need for alternative therapies for subjects suffering from HAE.

SUMMARY OF THE INVENTION

[0008] The present invention provides polynucleotide agents and compositions comprising such agents which target nucleic acids encoding a Factor XII (F12) gene and interfere with the normal function of the targeted nucleic acid. The Factor XII 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 Factor XII mRNA, e.g., a Factor XII-associated disease, such as a contact activation pathway-associated disease, such as hereditary angioedema (HAE), Flectcher Factor Deficiency, or essential hypertension using the polynucleotide agents and compositions of the invention.

[0009] Accordingly, in some aspects, the present invention provides antisense polynucleotide agents for inhibiting expression of Factor XII. The agents comprise 4 to 50 contiguous nucleotides, wherein at least one of the contiguous nucleotides is a modified nucleotide, and wherein the nucleotide sequence of the agent is at least 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs:1-4. In one embodiment, the equivalent region is a target region of SEQ ID NO:1, e.g., residues 21-74, 87-106, 143-183, 209-239, 254-273, 287-315, 320-350, 352-392, 395-414, 352-414, 430-470, 472-568, 595-680, 595-669, 682-701, 736-899, 793-843, 793-899, 804-899, 901-942, 736-942, 968-987, 990-1097, 990-1042, 1100-1197, 1100-1206, 1210-1252, 1210-1240, 1253-1272, 1298-1406, 1308-1406, 1430-1515, 1430-1482, 1541-1560, 1583-1602, 1683-1714, 1727-1768, 1737-1756, 1781-1976, 1816-1868, 1902-1967, 1891-1957, 1992-2043, or 1992-203 of SEQ ID NO:1. In certain embodiments, the target region of SEQ ID NO: 1 is any one of the target regions listed in Table 3. In certain embodiments, the target region of SEQ ID NO: 1 is one of the target regions selected from the group consisting of the regions targeted by antisense agent A-145669, A-145668, A-145667, A-145572, A-145567, A-145666, A-145670, A-145674, A-145660, A-145676, A-145539, A-145566, A-145527, A-145548, A-145549, A-145573, A-145661, A-145570, A-145597, A-145600, A-145658; A-145556, A-145656, A-145659, A-145671, A-145675, A-145519, A-145538, A-145619, A-145520, A-145594, A-145540, A-145547, A-145550, A-145551, A-145657, A-145568, A-145575, A-145565, A-145584, A-145616, A-145541, A-145542, A-145555, A-145571, A-145596, A-145599, A-145617, A-145655, A-145663, A-145532, A-145603, A-145665, A-145537, A-145612, A-145651, A-145586; A-145533, A-145569, A-145561, A-145604, A-145506, A-145664, A-145585, A-145595, A-145618, A-145613, A-145615, A-145677, A-145523, A-145552, A-145593, A-145598, A-145633, A-145543, A-145557, A-145624, A-145629, A-145646, A-145512, A-145536, A-145583, A-145625, A-145576, A-145623, A-145626; A-145562, A-145577, A-145588, A-145650, A-145534, A-145601, A-145662, A-145522, A-145525, A-145589, A-145607, A-145516, A-145529, A-145564, A-145605, A-145495, A-145496, A-145507, A-145614, A-145637, A-145497, A-145526, A-145591, A-145628, A-145647, A-145498, A-145501, A-145508, A-145560, A-145590, A-145611, A-145627, A-145652, A-145672, A-145513, A-145563, A-145581, A-145553, or A-145587. In certain embodiments, the target region of SEQ ID NO: 1 is one of the target regions selected from the group consisting of the regions targeted by antisense agent A-145669, A-145668, A-145667, A-145572, A-145567, A-145666, A-145670, A-145674, A-145660, A-145676, A-145539, A-145566, A-145527, A-145548, A-145549, A-145573, A-145661, A-145570, A-145597, A-145600, A-145658; A-145556, A-145656, A-145659, A-145671, A-145675, A-145519, A-145538, A-145619, A-145520, A-145594, A-145540, A-145547, A-145550, A-145551, A-145657, A-145568, A-145575, A-145565, A-145584, A-145616, A-145541, A-145542, A-145555, A-145571, A-145596, A-145599, A-145617, A-145655, A-145663, A-145532, A-145603, A-145665, A-145537, A-145612, A-145651, A-145586; A-145533, A-145569, A-145561, A-145604, A-145506, A-145664, A-145585, A-145595, A-145618, A-145613, A-145615, A-145677, A-145523, A-145552, A-145593, A-145598, A-145633, A-145543, A-145557, A-145624, A-145629, A-145646, A-145512, A-145536, A-145583, A-145625, A-145576, A-145623, or A-145626. In certain embodiments, the target region of SEQ ID NO: 1 is one of the target regions selected from the group consisting of the regions targeted by antisense agent A-145669, A-145668, A-145667, A-145572, A-145567, A-145666, A-145670, A-145674, A-145660, A-145676, A-145539, A-145566, A-145527, A-145548, A-145549, A-145573, A-145661, A-145570, A-145597, A-145600, A-145658; A-145556, A-145656, A-145659, A-145671, A-145675, A-145519, A-145538, A-145619, A-145520, A-145594, A-145540, A-145547, A-145550, A-145551, A-145657, A-145568, A-145575, A-145565, A-145584, A-145616, A-145541, A-145542, A-145555, A-145571, A-145596, A-145599, A-145617, A-145655, A-145663, A-145532, A-145603, A-145665, A-145537, A-145612, A-145651, or A-145586.

[0010] In another aspect, the present invention provides antisense polynucleotide agents for inhibiting expression of Factor XII, wherein the agent comprises at least 8 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences listed in Table 3. In certain embodiments, the agent comprises at least 8 contiguous nucleotides differing by no more than 3 nucleotides from any one of A-145669, A-145668, A-145667, A-145572, A-145567, A-145666, A-145670, A-145674, A-145660, A-145676, A-145539, A-145566, A-145527, A-145548, A-145549, A-145573, A-145661, A-145570, A-145597, A-145600, A-145658; A-145556, A-145656, A-145659, A-145671, A-145675, A-145519, A-145538, A-145619, A-145520, A-145594, A-145540, A-145547, A-145550, A-145551, A-145657, A-145568, A-145575, A-145565, A-145584, A-145616, A-145541, A-145542, A-145555, A-145571, A-145596, A-145599, A-145617, A-145655, A-145663, A-145532, A-145603, A-145665, A-145537, A-145612, A-145651, A-145586; A-145533, A-145569, A-145561, A-145604, A-145506, A-145664, A-145585, A-145595, A-145618, A-145613, A-145615, A-145677, A-145523, A-145552, A-145593, A-145598, A-145633, A-145543, A-145557, A-145624, A-145629, A-145646, A-145512, A-145536, A-145583, A-145625, A-145576, A-145623, A-145626; A-145562, A-145577, A-145588, A-145650, A-145534, A-145601, A-145662, A-145522, A-145525, A-145589, A-145607, A-145516, A-145529, A-145564, A-145605, A-145495, A-145496, A-145507, A-145614, A-145637, A-145497, A-145526, A-145591, A-145628, A-145647, A-145498, A-145501, A-145508, A-145560, A-145590, A-145611, A-145627, A-145652, A-145672, A-145513, A-145563, A-145581, A-145553, or A-145587.

[0011] In some embodiments, substantially all of the nucleotides of the antisense polynucleotide agents of the invention are modified nucleotides. In other embodiments, all of the nucleotides of the antisense polynucleotide agent are modified nucleotides.

[0012] The antisense polynucleotide agent may be about 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; 12 or 20 nucleotides in length.

[0013] In one embodiment, the modified nucleotide comprises a modified sugar moiety selected from the group consisting of a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, and a bicyclic sugar moiety.

[0014] 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.

[0015] In a further embodiment, n is 1 and R is CH.sub.3.

[0016] In another embodiment, the modified nucleotide is a 5-methylcytosine.

[0017] In one embodiment, the modified nucleotide comprises a modified internucleoside linkage, such as a phosphorothioate internucleoside linkage.

[0018] In one embodiment, an agent of the invention comprises a plurality of 2'-deoxynucleotides flanked on each side by at least one nucleotide having a modified sugar moiety.

[0019] 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.

[0020] 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.

[0021] In one embodiment, the 5'-wing segment is about 1 to 6 nucleotides in length, e.g., 2, 3, 4, or 5 nucleotides in length.

[0022] In one embodiment, the 3'-wing segment is about 1 to 6 nucleotides in length, e.g., 2, 3, 4, or 5 nucleotides in length.

[0023] In one embodiment, the gap segment is about 5 to 14 nucleotides in length, e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length.

[0024] In one aspect, the present invention provides antisense polynucleotide agents for inhibiting Factor XII expression, comprising a gap segment consisting of linked deoxynucleotides; a 5'-wing segment consisting of linked nucleotides; a 3'-wing segment consisting of linked nucleotides; wherein the gap segment is positioned between the 5'-wing segment and the 3'-wing segment and wherein each nucleotide of each wing segment comprises a modified sugar.

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

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

[0027] In yet another embodiment, the gap segment is ten 2'-deoxynucleotides in length and each of the wing segments is three nucleotides in length.

[0028] In another embodiment, the gap segment is ten 2'-deoxynucleotides in length and each of the wing segments is two nucleotides in length.

[0029] 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.

[0030] In some embodiments, the agents of the invention further comprise a ligand.

[0031] In one embodiment, the agent is conjugated to the ligand at the 3'-terminus.

[0032] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0033] In one embodiment the ligand is

##STR00001##

[0034] In one aspect, the present invention provides pharmaceutical compositions for inhibiting expression of a Factor XII gene comprising the agents of the invention.

[0035] In one embodiment, the agent is present in an unbuffered solution, such as saline or water.

[0036] In another embodiment, the agent is is present in a buffer solution, such as a buffer comprising acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.

[0037] In one embodiment, the buffer solution is phosphate buffered saline (PBS).

[0038] In another aspect, the present invention provides pharmaceutical composition comprising an agent of the invention and a lipid formulation, such as a lipid formulation comprising an LNP or a MC3.

[0039] In one aspect, the present invention provides methods of inhibiting Factor XII expression in a cell. The methods include contacting the cell with the agent of the invention or a pharmaceutical composition of the invention; and maintaining the cell for a time sufficient to obtain antisense inhibition of a Factor XII gene, thereby inhibiting expression of the Factor XII gene in the cell.

[0040] In some embodiments, the cell is within a subject.

[0041] In some embodiments, the subject is a human. In some embodiments, the subject is not human.

[0042] In one embodiment, the Factor XII expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or to below the level of detection of the assay.

[0043] In certain embodiments, the expression of the target is normalized, i.e., decreased to a level accepted as within the range of normal for an individual without such disorder. For example, the difference between a the expression level in a subject prior to treatment and normal expression level is decreased by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0044] In another aspect, the present invention provides methods of treating a subject having a disease or disorder that would benefit from reduction in Factor XII expression. The methods include administering to the subject a therapeutically effective amount of an agent of the invention or a pharmaceutical composition of the invention, thereby treating the subject.

[0045] In one embodiment, the administration of the antisense polynucleotide agent to the subject causes a decrease or normalization in at least one of elevated levels of bradykinin, prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, Fletcher Factor Deficiency, edema swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals, prodrome; laryngeal swelling; nonpruritic rash; nausea; vomiting; abdominal pain, or a decrease in Factor XII protein levels.

[0046] In one embodiment, the disorder is a Factor XII-associated disease.

[0047] In another embodiment, the Factor XII-associated disease is selected from heredity angioedema (HAE) (such as hereditary angioedema type I; hereditary angioedema type II; hereditary angioedema type III; or any other hereditary angioedema caused by elevated levels of bradykinin); prekallikrein deficiency, hypertension, e.g., malignant essential hypertension, end stage renal disease, or Fletcher Factor Deficiency.

[0048] In some embodiments, the Factor XII-associated disease is HAE.

[0049] In other embodiments, the Factor XII-associated disease is prekallikrein deficiency.

[0050] In other embodiments, the Factor XII-associated disease is hypertension, e.g., malignant essential hypertension.

[0051] In other embodiments, the Factor XII-associated disease is end stage renal disease.

[0052] In other embodiments, the Factor XII-associated disease is Fletcher Factor Deficiency.

[0053] In some embodiments the subject is human. In some embodiments, the subject is not human.

[0054] In some embodiments, the methods of the invention further include administering to the subject an androgen, such as danazol or oxandrolone, Berinert.RTM., Cinryze.TM., Rhuconest.RTM., Ecallantide, Firazyr.RTM., Kalbitor.RTM., or a combination of any of the foregoing to the subject.

[0055] In some embodiments, the methods of the invention further include determining Factor XII levels in the subject.

[0056] In some embodiments, the methods of the invention further include determining the level of bradykinin; prekallikrein, or blood pressure in the subject.

DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention provides polynucleotide agents, e.g., antisense polynucleotide agents, and compositions comprising such agents which target nucleic acids encoding Factor XII (e.g., mRNA encoding Factor XII as provided in, for example, any one of SEQ ID NOs:1-4). The polynucleotide agents bind to nucleic acids encoding Factor XII via, e.g., Watson-Crick base pairing, and interfere with the normal function of the targeted nucleic acid.

[0058] The antisense polynucleotide agents of the invention include a nucleotide sequence which is 4 to 50 nucleotides or less in length and which is 80% complementary to at least part of an mRNA transcript of a Factor XII gene. The use of these antisense polynucleotide agents enables the targeted inhibition of RNA expression or activity of a Factor XII gene in mammals.

[0059] The present inventors have demonstrated that antisense polynucleotide agents targeting Factor XII can mediate antisense inhibition in vitro resulting in significant inhibition of expression of a Factor XII gene. Thus, methods and compositions including these antisense polynucleotide agents are useful for treating a subject who would benefit by a reduction in the levels or activity of a Factor XII protein, such as a subject having a Factor XII-associated disease, such as heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency.

[0060] 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 Factor XII gene, e.g., a Factor XII-associated disease, such as heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency using the antisense polynucleotide agents and compositions of the invention.

[0061] The present invention also provides methods for preventing at least one symptom, e.g., elevated levels of bradykinin, prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, Fletcher Factor Deficiency, edema swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals, prodrome; laryngeal swelling; nonpruritic rash; nausea; vomiting; abdominal pain, in a subject having a disorder that would benefit from inhibiting or reducing the expression of a Factor XII gene, e.g., a Factor XII-associated disease, such as heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency. The present invention further provides compositions comprising antisense polynucleotide agents which effect antisense inhibition of a Factor XII gene. The Factor XII gene may be within a cell, e.g., a cell within a subject, such as a human.

[0062] The combination therapies of the present invention include administering to a subject having a Factor XII-associated disease, an antisense polynucleotide agent of the invention and an additional therapeutic, such as an androgen, such as danazol or oxandrolone, Berinert.RTM., Cinryze.TM., Rhuconest.RTM., Ecallantide, Firazyr.RTM., Kalbitor.RTM., and a combination of any of the foregoing.

[0063] The following detailed description discloses how to make and use antisense polynucleotide agents to inhibit the mRNA or protein expression of a Factor XII gene, as well as compositions, uses, and methods for treating subjects having diseases and disorders that would benefit from inhibition or reduction of the expression of this gene.

I. Definitions

[0064] 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.

[0065] 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.

[0066] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

[0067] The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise. For example, a nucleoside with a modified base or a modified sugar is understood to include the options of a nucleoside with a modified base, a nucleoside with a modified sugar, and a nucleoside with a modified base and a modified sugar.

[0068] The term "about" is used herein to mean within the typical ranges of tolerances in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means+10%. In certain embodiments, about means+5%. When about is present before a series of numbers or a range, it is understood that "about" can modify each of the numbers in the series or range.

[0069] The term "at least" prior to a number or series of numbers is understood to include the number adjacent to the term "at least", and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 17 nucleotides of a 20 nucleotide nucleic acid molecule" means that 17, 18, 19, or 20 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range.

[0070] As used herein, "no more than" or "less than" is understood as the value adjacent to the phrase and logical lower values or intergers, as logical from context, to zero. For example, a duplex with mismatches to a target site of "no more than 2 nucleotides" has a 2, 1, or 0 mismatches. When "no more than" is present before a series of numbers or a range, it is understood that "no more than" can modify each of the numbers in the series or range.

[0071] As used herein, "up to" as in "up to 10" is understood as up to and including 10, i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0072] Ranges provided herein are understood to include all individual interger values and all subranges within the ranges.

[0073] As used herein, "Factor XII (Hageman Factor)," used interchangeably with the terms "coagulation factor XII," "FXII," "F12," active F12," and "F12a," refers to the naturally occurring gene that encodes the zymogen form of F12a. F12 a is an enzyme (EC 3.4.21.38) of the serine protease (or serine endopeptidase) class that cleaves prekallikrein to form kallikrein, which subsequently releases bradykinin from high-molecular weight kininogen and activates F12. The amino acid and complete coding sequences of the reference sequence of the F12 gene may be found in, for example, GenBank Accession No. GI:145275212 (RefSeq Accession No. NM_000505; SEQ ID NO:1). Mammalian orthologs of the human F12 gene may be found in, for example, GenBank Accession Nos. GI:544441267 (RefSeq Accession No. XM_005558647, cynomolgus monkey; SEQ ID NO:2); GI:805299477 (RefSeq Accession No. NM_021489, mouse; SEQ ID NO:3); and GI:62078740 (RefSeq Accession No. NM_001014006, rat; SEQ ID NO:4).

[0074] Additional examples of Factor XII mRNA sequences are readily available using publicly available databases, e.g., GenBank. Additional information on the human Factor XII gene can be found, for example, at www.ncbi.nlm.nih.gov/gene/2161 which is incorporated herein by reference in the version available on the filing date of the instant application.

[0075] The term"Factor XII," as used herein, also refers to naturally occurring DNA sequence variations of the Factor XII gene, such as a single nucleotide polymorphism in the Factor XII gene. Numerous SNPs within the Factor XII gene have been identified and may be found at, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=2161, which is incorporated herein by reference in the version available on the filing date of the instant application)

[0076] 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 Factor XII (e.g., mRNA encoding Factor XII 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."

[0077] 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.

[0078] In some embodiments, antisense inhibition refers to "inhibiting the expression" of target nucleic acid levels 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 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.

[0079] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a Factor XII gene, including mRNA that is a product of RNA processing of a primary transcription product.

[0080] As used herein, "target nucleic acid" refers to a nucleic acid molecule to which an antisense polynucleotide agent specifically hybridizes.

[0081] 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.

[0082] A target sequence may be about 4-50 nucleotides in length, e.g., about 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 Factor XII gene. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

[0083] 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.

[0084] 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 Factor XII). For example, a polynucleotide is complementary to at least a part of a Factor XII mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding Factor XII.

[0085] As used herein, the term "region of complementarity" refers to the region of the antisense polynucletiode agent that is substantially complementary to a sequence, for example a target sequence, e.g., a Factor XII 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'- or 3'-terminus of the antisense polynucleotide.

[0086] 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.

[0087] 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.

[0088] "Complementary" sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs 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.

[0089] 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.

[0090] "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.

[0091] A "nucleoside" is a base-sugar combination. The "nucleobase" (also known as "base") portion of the nucleoside is normally a heterocyclic base moiety. "Nucleotides" are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. "Polynucleotides," also referred to as "oligonucleotides," are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the polynucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the polynucleotide.

[0092] 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.

[0093] As used herein, the term "modified nucleotide" refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, 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 polynucleiotde 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.

[0094] 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 Factor XII expression; a human at risk for a disease, disorder, or condition that would benefit from reduction in Factor XII expression; or a human having a disease, disorder or condition that would benefit from reduction in Factor XII expression.

[0095] As used herein, the terms "treating" or "treatment" refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms associated with heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0096] The term "lower" or "reduce" and the like in the context of the level of a Factor XII 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 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; or a similar level of reduction in the difference between a disease level and a normal level of a marker in an appropriate control subject. In certain embodiments, the decrease is down to a level accepted as within the range of normal for an individual without such disorder which can also be referred to as a normalization of a level. For example, lowering cholesterol to 180 mg/dl or lower would be considered to be within the range of normal for a subject. A subject having a cholesterol level of 230 mg/dl with a cholesterol level decreased to 210 mg/dl would have a cholesterol level that was decreased by 40% (230-210/230-180=20/50=40% reduction). In certain embodiments, the reduction is the normalization of the level of a sign or symptom of a disease, a reduction in the difference between the subject level of a sign of the disease and the normal level of the sign for the disease (e.g., upper level of normal, lower level of normal, average of upper and lower level of normal). For example, reduction can be understood as normalization of blood pressure, decreasing an elevated blood pressure or increasing a low blood pressure to reduce the difference from a normal reading.

[0097] As used herein, the term "Factor XII-associated disease" is a disease or disorder that is caused by, or associated with abnormal signaling in the contact activation pathway, e.g., abnormal contact activation pathway gene expression (i.e KLKB1 gene expression, F12 gene expression, and/or KNG1 gene expression) or contact activation pathway protein production (i.e., KLKB1 protein production, F12 protein production, and/or KNG1 protein production). Such diseases are typically include a disease, disorder or condition that would benefit from reduction in contact activation pathway gene expression and/or activity. Non-limiting examples of contact activation pathway-associated diseases include, for example, diseases and conditions including heredity angioedema (HAE) (such as hereditary angioedema type I; hereditary angioedema type II; hereditary angioedema type III; or any other hereditary angioedema caused by elevated levels of bradykinin), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, Fletcher Factor Deficiency, edema swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals, prodrome; laryngeal swelling; nonpruritic rash; nausea; vomiting; abdominal pain.

[0098] In one embodiment, a Factor XII-associated disease is HAE. In another embodiment, a Factor XII-associated disease is prekallikrein deficiency. In another embodiment, a Factor XII-associated disease is hypertension, e.g., malignant essential hypertension. In another embodiment, a Factor XII-associated disease is end stage renal disease. In another embodiment, a Factor XII-associated disease is Fletcher Factor deficiency.

II. Polynucleotide Agents of the Invention

[0099] The present invention provides polynucleotide agents, e.g., antisense polynucleotide agents, and compositions comprising such agents, which target a Factor XII gene and inhibit the expression of the Factor XII gene. In one embodiment, the polynucleotide agents, e.g., antisense polynucleotide agents, inhibit the expression of a Factor XII gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having a Factor XII-associated disease, e.g., heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency.

[0100] The antisense polynucleotde 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 Factor XII gene. The region of complementarity may be about 50 nucleotides or less in length (e.g., 22-12, 20-14, 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 Factor XII gene, the antisense polynucleotide agent inhibits the expression of the Factor XII gene (e.g., a human, a primate, a non-primate, or a bird Factor XII gene) by at least 20% 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. In preferred embodiments, the inhibition of expression is determined using the qPCR method provided in Example 2 herein.

[0101] 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 Factor XII gene.

[0102] Accordingly, in one aspect, an antisense polynucleotide agent of the invention specifically hybridizes to a target nucleic acid molecule, such as the mRNA encoding Factor XII, 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.

[0103] 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 Factor XII 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 Factor XII mRNA.

[0104] 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 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 at least 85%, 90%, 95%, or 100% complementary.

[0105] In some embodiments, an antisense polynucleotide agent comprises a contiguous nucleotide sequence which is fully complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1-4 (or a fragment of any one of SEQ ID NOs:1-4). For example, the nucleotide sequence of an antisense polynucleotide agent is fully complementary over its entire length to the equivalent region of nucleotides 1-20 of GI:145275212 (SEQ ID NO:1) (see, e.g., Table 3).

[0106] An antisense polynucleotide agent may comprise a contiguous nucleotide sequence of about 4 to 50 nucleotides in length, or any subrange falling within that range, e.g., about 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.

[0107] In some embodiments, an antisense polynucleotide agent may comprise a contiguous nucleotide sequence of no more than 22 nucleotides, e.g., no more than any of 21 nucleotides, 20 nucleotides, 19 nucleotides, no more than 18 nucleotides, 17 nucleotides, 16 nucleotides, 15 nucleotides, or 14 nucleotides. In other embodiments, the antisense polynucleotide agents of the invention are 20 nucleotides in length. In other embodiments, the antisense polynucleotide agents of the invention are 14 nucleotides in length. In certain embodiments, the antisense polynucleotide agents of the invention are at least 12 nucleotides in length.

[0108] In one aspect, an antisense polynucleotide agents of the invention include a sequence selected from the group of sequences provided in Table 3. It will be understood that, although some of the sequences in Table 3 are described as modified or conjugated sequences, an antisense polynucleotide agent of the invention, may also comprise any one of the sequences set forth in Table 3 that is un-modified, un-conjugated, or modified or conjugated differently than described therein.

[0109] By virtue of the nature of the nucleotide sequences provided in Table 3, antisense polynucleotide agents of the invention may include one of the sequences of Table 3 minus only a few nucleotides on one or both ends and yet remain similarly effective as compared to the antisense polynucleotide agents described above. Hence, antisense polynucleotide agents having a sequence of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides derived from one of the sequences of Table 3 and differing in their ability to inhibit the expression of a Factor XII gene by not more than 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.

[0110] In addition, the antisense polynucleotide agents provided in Table 3 identify a region(s) in a Factor XII transcript that is susceptible to antisense inhibition (e.g., the regions encompassed by the start and end positions of the nucleotide sequence of any one of the antisense agents selected from the group consisting of A-145669, A-145668, A-145667, A-145572, A-145567, A-145666, A-145670, A-145674, A-145660, A-145676, A-145539, A-145566, A-145527, A-145548, A-145549, A-145573, A-145661, A-145570, A-145597, A-145600, A-145658; A-145556, A-145656, A-145659, A-145671, A-145675, A-145519, A-145538, A-145619, A-145520, A-145594, A-145540, A-145547, A-145550, A-145551, A-145657, A-145568, A-145575, A-145565, A-145584, A-145616, A-145541, A-145542, A-145555, A-145571, A-145596, A-145599, A-145617, A-145655, A-145663, A-145532, A-145603, A-145665, A-145537, A-145612, A-145651, A-145586; A-145533, A-145569, A-145561, A-145604, A-145506, A-145664, A-145585, A-145595, A-145618, A-145613, A-145615, A-145677, A-145523, A-145552, A-145593, A-145598, A-145633, A-145543, A-145557, A-145624, A-145629, A-145646, A-145512, A-145536, A-145583, A-145625, A-145576, A-145623, A-145626; A-145562, A-145577, A-145588, A-145650, A-145534, A-145601, A-145662, A-145522, A-145525, A-145589, A-145607, A-145516, A-145529, A-145564, A-145605, A-145495, A-145496, A-145507, A-145614, A-145637, A-145497, A-145526, A-145591, A-145628, A-145647, A-145498, A-145501, A-145508, A-145560, A-145590, A-145611, A-145627, A-145652, A-145672, A-145513, A-145563, A-145581, A-145553, and A-145587 in Table 3). As such, the present invention further features antisense polynucleotide agents that target within one of these sites. As used herein, an antisense polynucleotide agent is said to target within a particular site of an RNA transcript if the antisense polynucleotide agent promotes antisense inhibition of the target at that site. Such an antisense polynucleotide agent will generally include at least 14 contiguous nucleotides from one of the sequences provided in Table 3 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a Factor XII gene.

[0111] While a target sequence is generally 4-50 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing antisense inhibition of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a "window" or "mask" of a given size (as a non-limiting example, 20 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence "window" progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an antisense polynucleotide agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Table 3 represent effective target sequences, it is contemplated that further optimization of antisense inhibition efficiency can be achieved by progressively "walking the window" one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

[0112] Further, it is contemplated that for any sequence identified, e.g., in Table 3, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of antisense polynucleotide agents based on those target sequences in an inhibition assay as known in the art 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 or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

III. Modified Antisense Polynucleotide Agents of the Invention

[0113] In some embodiments, the nucleotides of an antisense polynucleotide agent of the invention is un-modified, and does not comprise, e.g., chemical modifications or conjugations known in the art and described herein. In another embodiment, at least one of the nucleotides of an antisense polynucleotide agent of the invention is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an antisense polynucleotide agent of the invention are modified. In other embodiments of the invention, all of the nucleotides of an antisense polynucleotide agent of the invention are modified. Antisense polynucleotide agents of the invention in which "substantially all of the nucleotides are modified" are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0114] The nucleic acids featured in the invention can be synthesized 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 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; or backbone modifications, including modification or replacement of the phosphodiester linkages.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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..n, OCH.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 10.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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).

[0127] 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.

[0128] 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.

[0129] 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).

[0130] 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."

[0131] 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).

[0132] 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."

[0133] 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).

[0134] 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).

[0135] 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.

[0136] 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.

[0137] One or more of the nucleotides of an iRNA of the invention may also include a hydroxymethyl substituted nucleotide. A "hydroxymethyl substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also referred to as an "unlocked nucleic acid" ("UNA") modification. Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

[0138] Additional modification which may potentially stabilize the ends of antisense polynucleotide agents can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-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 US20120142101.

[0139] Any of the antisense polynucleotide agents of the invention may be optionally conjugated with a GalNAc derivative ligand, as described below.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] In certain specific embodiments, the antisense polynucleotide agent for use in the methods of the invention is an agent selected from the group of agents listed in Table 3. These agents may further comprise a ligand, as described below.

[0144] A. Antisense Polynucleotide Agents Comprising Motifs

[0145] In certain embodiments of the invention, at least one of the contiguous nucleotides of the antisense polynucleotide agents of the invention may be a modified nucleotide. In one embodiment, the modified nucleotide comprises one or more modified sugars. In other embodiments, the modified nucleotide comprises one or more modified nucleobases. In yet other embodiments, the modified nucleotide comprises one or more modified internucleoside linkages. In some embodiments, the modifications (sugar modifications, nucleobase modifications, 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.

[0146] 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, 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.

[0147] 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.

[0148] 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.

[0149] 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).

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] In some embodiments of the invention, antisense polynucleotide agents targeting Factor XII include a 5-10-5 gapmer motif. In other embodiments of the invention, antisense polynucleotide agents targeting Factor XII include a 4-10-4 gapmer motif. In another embodiment of the invention, antisense polynucleotide agents targeting Factor XII include a 3-10-3 gapmer motif. In yet other embodiments of the invention, antisense polynucleotide agents targeting Factor XII include a 2-10-2 gapmer motif.

[0156] The 5'-wing or 3'-wing of a gapmer may independently include 1-6 modified nucleotides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleotides.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] The 5'-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleotides.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] The 3'-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleotides.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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.

[0182] In another embodiment, an antisense polynucleotide agent targeting a Factor XII 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.

[0183] In another embodiment, an antisense polynucleotide agent targeting a Factor XII 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.

[0184] In another embodiment, an antisense polynucleotide agent targeting a Factor XII 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.

[0185] 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 Factor XII gene is a 5-methylcytosine.

[0186] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 some embodiments, the agent further comprises a ligand.

[0187] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 some embodiments, the agent further comprises a ligand.

[0188] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 internucleoitde linkage of the agent is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0189] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0190] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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.

[0191] In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0192] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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.

[0193] In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0194] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 internucleoitde linkage of the agent is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0195] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0196] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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.

[0197] In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0198] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0199] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 internucleoitde linkage of the agent is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0200] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0201] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 internucleotde linkage of the agent is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0202] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0203] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 internucleoitde linkage of the agent is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0204] In one embodiment, an antisense polynucleotide agent targeting a Factor XII 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 some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0205] Further gapmer designs suitable for use in the agents, compositions, and methods of the invention are disclosed in, for example, U.S. Pat. Nos. 7,687,617 and 8,580,756; U.S. Patent Publication Nos. 20060128646, 20090209748, 20140128586, 20140128591, 20100210712, and 20080015162A1; and International Publication No. WO 2013/159108, the entire content of each of which are incorporated herein by reference.

IV. Antisense Polynucleotide Agents Conjugated to Ligands

[0206] Another modification of the polynucleotide agents of the invention involves chemically linking to the agent one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense polynucleotide agent. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Am. 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., FMBO 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).

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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]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.

[0211] 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-icB.

[0212] 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, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0213] 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 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] A. Lipid Conjugates

[0219] 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, or (c) can be used to adjust binding to a serum protein, e.g., HSA.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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).

[0224] B. Cell Permeation Agents

[0225] 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.

[0226] 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 5-50 amino acids long, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0227] 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: 5). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 6) 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: 7) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 8) 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 antisens epolynucleotide 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 5 amino acids to 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.

[0228] An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0229] 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).

[0230] C. Carbohydrate Conjugates

[0231] 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 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).

[0232] 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##

[0233] 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##

[0234] 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.

[0235] 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 or a cell permeation peptide.

[0236] D. Linkers

[0237] 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.

[0238] 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 1-24 atoms or any subrange within that range, e.g., 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

[0239] 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 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least 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).

[0240] 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.

[0241] 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.

[0242] 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.

[0243] Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

[0244] 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 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 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).

[0245] i. Redox Cleavable Linking Groups

[0246] 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 10% in the blood. In other embodiments, useful candidate compounds are degraded at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 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.

[0247] ii. Phosphate-Based Cleavable Linking Groups

[0248] 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.

[0249] iii. Acid Cleavable Linking Groups

[0250] 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 6.5 or lower (e.g., 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.

[0251] iv. Ester-Based Linking Groups

[0252] 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.

[0253] v. Peptide-Based Cleaving Groups

[0254] In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (--C(O)NH--). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula--NHCHRAC(O)NHCHRBC(O)--, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

[0255] 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##

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

[0256] In certain embodiments of the compositions and methods of the invention, a ligand is one or more GaNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

[0257] In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXII)-(XXXV):

##STR00012##

wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P.sup.2A, P.sup.2B, P.sup.3A, P.sup.3B, P.sup.4A, P.sup.4B, P.sup.5AT.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--

##STR00013##

or heterocyclyl;

[0258] 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 GaNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; andRa is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXVI):

##STR00014##

[0259] wherein L.sup.5A, L.sup.5B and L.sup.5C represent a monosaccharide, such as GaNAc derivative.

[0260] Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas I, VII, XI, X, and XIII.

[0261] 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.

[0262] 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.

[0263] "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, 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.

[0264] 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., EAMBO 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 an Antisense Polynucleotide Agent of the Invention

[0265] The delivery of an antisense polynucleotide agent of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a Factor XII-associated disease) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an antisense polynucleotide agent of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an antisense polynucleotide agent to a subject.

[0266] 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.

[0267] 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).

[0268] 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.

[0269] 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

[0270] The present invention also includes pharmaceutical compositions and formulations which include the antisense polynucleotide agents of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an antisense polynucleotide agent, as described herein, and a pharmaceutically acceptable carrier.

[0271] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, 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.

[0272] 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 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.

[0273] The pharmaceutical compositions containing the antisense polynucleotide agents are useful for treating a disease or disorder associated with the expression or activity of a Factor XII gene, e.g. a Factor XII-associated disease. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by subcutaneous (SC) or intravenous (IV) delivery. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a Factor XII gene. In general, a suitable dose of an antisense polynucleotide agent of the invention will be in the range of 0.001 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the antisense polynucleotide agent can be administered at 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

[0274] 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 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0275] In another embodiment, the antisense polynucleotide agent is administered at a dose of about 0.1 to 50 mg/kg, 0.25 to 50 mg/kg, 0.5 to 50 mg/kg, 0.75 to 50 mg/kg, 1 to 50 mg/mg, 1.5 to 50 mg/kb, 2 to 50 mg/kg, 2.5 to 50 mg/kg, 3 to 50 mg/kg, 3.5 to 50 mg/kg, 4 to 50 mg/kg, 4.5 to 50 mg/kg, 5 to 50 mg/kg, 7.5 to 50 mg/kg, 10 to 50 mg/kg, 15 to 50 mg/kg, 20 to 50 mg/kg, 20 to 50 mg/kg, 25 to 50 mg/kg, 25 to 50 mg/kg, 30 to 50 mg/kg, 35 to 50 mg/kg, 40 to 50 mg/kg, 45 to 50 mg/kg, 0.1 to 45 mg/kg, 0.25 to 45 mg/kg, 0.5 to 45 mg/kg, 0.75 to 45 mg/kg, 1 to 45 mg/mg, 1.5 to 45 mg/kb, 2 to 45 mg/kg, 2.5 to 45 mg/kg, 3 to 45 mg/kg, 3.5 to 45 mg/kg, 4 to 45 mg/kg, 4.5 to 45 mg/kg, 5 to 45 mg/kg, 7.5 to 45 mg/kg, 10 to 45 mg/kg, 15 to 45 mg/kg, 20 to 45 mg/kg, 20 to 45 mg/kg, 25 to 45 mg/kg, 25 to 45 mg/kg, 30 to 45 mg/kg, 35 to 45 mg/kg, 40 to 45 mg/kg, 0.1 to 40 mg/kg, 0.25 to 40 mg/kg, 0.5 to 40 mg/kg, 0.75 to 40 mg/kg, 1 to 40 mg/mg, 1.5 to 40 mg/kb, 2 to 40 mg/kg, 2.5 to 40 mg/kg, 3 to 40 mg/kg, 3.5 to 40 mg/kg, 4 to 40 mg/kg, 4.5 to 40 mg/kg, 5 to 40 mg/kg, 7.5 to 40 mg/kg, 10 to 40 mg/kg, 15 to 40 mg/kg, 20 to 40 mg/kg, 20 to 40 mg/kg, 25 to 40 mg/kg, 25 to 40 mg/kg, 30 to 40 mg/kg, 35 to 40 mg/kg, 0.1 to 30 mg/kg, 0.25 to 30 mg/kg, 0.5 to 30 mg/kg, 0.75 to 30 mg/kg, 1 to 30 mg/mg, 1.5 to 30 mg/kb, 2 to 30 mg/kg, 2.5 to 30 mg/kg, 3 to 30 mg/kg, 3.5 to 30 mg/kg, 4 to 30 mg/kg, 4.5 to 30 mg/kg, 5 to 30 mg/kg, 7.5 to 30 mg/kg, 10 to 30 mg/kg, 15 to 30 mg/kg, 20 to 30 mg/kg, 20 to 30 mg/kg, 25 to 30 mg/kg, 0.1 to 20 mg/kg, 0.25 to 20 mg/kg, 0.5 to 20 mg/kg, 0.75 to 20 mg/kg, 1 to 20 mg/mg, 1.5 to 20 mg/kb, 2 to 20 mg/kg, 2.5 to 20 mg/kg, 3 to 20 mg/kg, 3.5 to 20 mg/kg, 4 to 20 mg/kg, 4.5 to 20 mg/kg, 5 to 20 mg/kg, 7.5 to 20 mg/kg, 10 to 20 mg/kg, or 15 to 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0276] 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 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0277] In another embodiment, the antisense polynucleotide agent is administered at a dose of about 0.5 to 50 mg/kg, 0.75 to 50 mg/kg, 1 to 50 mg/mg, 1.5 to 50 mg/kgb, 2 to 50 mg/kg, 2.5 to 50 mg/kg, 3 to 50 mg/kg, 3.5 to 50 mg/kg, 4 to 50 mg/kg, 4.5 to 50 mg/kg, 5 to 50 mg/kg, 7.5 to 50 mg/kg, 10 to 50 mg/kg, 15 to 50 mg/kg, 20 to 50 mg/kg, 20 to 50 mg/kg, 25 to 50 mg/kg, 25 to 50 mg/kg, 30 to 50 mg/kg, 35 to 50 mg/kg, 40 to 50 mg/kg, 45 to 50 mg/kg, 0.5 to 45 mg/kg, 0.75 to 45 mg/kg, 1 to 45 mg/mg, 1.5 to 45 mg/kb, 2 to 45 mg/kg, 2.5 to 45 mg/kg, 3 to 45 mg/kg, 3.5 to 45 mg/kg, 4 to 45 mg/kg, 4.5 to 45 mg/kg, 5 to 45 mg/kg, 7.5 to 45 mg/kg, 10 to 45 mg/kg, 15 to 45 mg/kg, 20 to 45 mg/kg, 20 to 45 mg/kg, 25 to 45 mg/kg, 25 to 45 mg/kg, 30 to 45 mg/kg, 35 to 45 mg/kg, 40 to 45 mg/kg, 0.5 to 40 mg/kg, 0.75 to 40 mg/kg, 1 to 40 mg/mg, 1.5 to 40 mg/kb, 2 to 40 mg/kg, 2.5 to 40 mg/kg, 3 to 40 mg/kg, 3.5 to 40 mg/kg, 4 to 40 mg/kg, 4.5 to 40 mg/kg, 5 to 40 mg/kg, 7.5 to 40 mg/kg, 10 to 40 mg/kg, 15 to 40 mg/kg, 20 to 40 mg/kg, 20 to 40 mg/kg, 25 to 40 mg/kg, 25 to 40 mg/kg, 30 to 40 mg/kg, 35 to 40 mg/kg, 0.5 to 30 mg/kg, 0.75 to 30 mg/kg, 1 to 30 mg/mg, 1.5 to 30 mg/kb, 2 to 30 mg/kg, 2.5 to 30 mg/kg, 3 to 30 mg/kg, 3.5 to 30 mg/kg, 4 to 30 mg/kg, 4.5 to 30 mg/kg, 5 to 30 mg/kg, 7.5 to 30 mg/kg, 10 to 30 mg/kg, 15 to 30 mg/kg, 20 to 30 mg/kg, 20 to 30 mg/kg, 25 to 30 mg/kg, 0.5 to 20 mg/kg, 0.75 to 20 mg/kg, 1 to 20 mg/kg, 1.5 to 20 mg/kg, 2 to 20 mg/kg, 2.5 to 20 mg/kg, 3 to 20 mg/kg, 3.5 to 20 mg/kg, 4 to 20 mg/kg, 4.5 to 20 mg/kg, 5 to 20 mg/kg, 7.5 to 20 mg/kg, 10 to 20 mg/kg, or 15 to 20 mg/kg. In one embodiment, the antisense polynucleotide agent is administered at a dose of 10 mg/kg to 30 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0278] For example, subjects can be administered, e.g., subcutaneously or intravenously, a single therapeutic amount of antisense polynucleotide agent, such as about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 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 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0279] 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, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 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 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.

[0280] 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 of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 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 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.

[0281] The pharmaceutical composition can be administered by intravenous infusion over a period of time, such as over about a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, 25, 26, 27, 28, or 29 minute period. In certain embodiments, the infusion is administered over about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 hours. 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.

[0282] 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.

[0283] 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.

[0284] 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 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.

[0285] 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 Factor XII. 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 can be selected based on the specific disease or condition to be treated.

[0286] 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.

[0287] 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).

[0288] 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-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.

[0289] A. Antisense Polynucleotide Agent Formulations Comprising Membranous Molecular Assemblies

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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.

[0294] 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).

[0295] 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).

[0296] 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 or phosphatidylcholine or cholesterol.

[0297] 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.

[0298] 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).

[0299] 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).

[0300] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

[0301] 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.

[0302] 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.

[0303] 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).

[0304] 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.

[0305] 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).

[0306] 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.

[0307] 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 agenst to epidermal cells and also to enhance the penetration of antisense polynucleotide agenst 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).

[0308] 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 distearatel 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.

[0309] Liposomes that include antisense polynucleotide agent can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include antisense polynucleotide agents can be delivered, for example, subcutaneously by infection in order to deliver antisense polynucleotide agents to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

[0310] Other formulations amenable to the present invention are described in WO2009088892, WO2009086558, WO2009132131, and WO2008042973.

[0311] 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.

[0312] 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).

[0313] 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 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.

[0314] 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.

[0315] 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.

[0316] 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.

[0317] 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).

[0318] 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.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] Phenol or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] B. Lipid Particles

[0327] 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.

[0328] 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 50 nm to 150 nm, more typically 60 nm to 130 nm, more typically 70 nm to 110 nm, most typically 70 nm to 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.

[0329] Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

[0330] 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 1:1 to 50:1, from 1:1 to 25:1, from 3:1 to 15:1, from 4:1 to 10:1, from 5:1 to 9:1, or 6:1 to 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

[0331] 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 20 mol % to 50 mol % or 40 mol % of the total lipid present in the particle.

[0332] 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.

[0333] 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.

[0334] 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 5 mol % to 90 mol %, 10 mol %, or 58 mol % if cholesterol is included, of the total lipid present in the particle.

[0335] 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 20 mol % or 2 mol % of the total lipid present in the particle.

[0336] In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., 10 mol % to 60 mol % or 48 mol % of the total lipid present in the particle.

[0337] 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., LNPO1 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 35-45% and the final sodium acetate concentration is 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 pH 7, e.g., pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, or pH 7.4.

##STR00015##

[0338] LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

[0339] 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 Lipid:santisense polynucleotide agent Ionizable/Cationic Lipid ratio SNALP- 1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/ 1 dimethylaminopropane PEG-cDMA (DLinDMA) (57.1/7.1/34.4/1.4) lipid:santisense polynucleotide agent ~ 7:1 2-XTC 2,2-Dilinoleyl-4- XTC/DPPC/Cholesterol/PEG-cDMA dimethylaminoethyl- 57.1/7.1/34.4/1.4 [1,3]-dioxolane (XTC) lipid:santisense polynucleotide agent ~ 7:1 LNP05 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl- 57.5/7.5/31.5/3.5 [1,3]-dioxolane (XTC) lipid:santisense polynucleotide agent ~ 6:1 LNP06 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl- 57.5/7.5/31.5/3.5 [1,3]-thoxolane (XTC) lipid:santisense polynucleotide agent ~ 11:1 LNP07 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl- 60/7.5/31/1.5, [1,3]-dioxolane (XTC) lipid:santisense polynucleotide agent ~ 6:1 LNP08 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl- 60/7.5/31/1.5, [1,3]-dioxolane (XTC) lipid:santisense polynucleotide agent ~ 11:1 LNP09 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl- 50/10/38.5/1.5 [1,3]-dioxolane (XTC) Lipid:santisense polynucleotide agent 10:1 LNP10 (3aR, 5s, 6aS)-N,N- ALN100/DSPC/Cholesterol/ dimethyl-2,2-di((9Z, PEG-DMG 12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH- Lipid:santisense polynucleotide agent cyclopenta[d][1,3] 10:1 dioxol-5-amine (ALN100) LNP11 (6Z, 9Z, 28Z, 31Z)- MC-3/DSPC/Cholesterol/PEG-DMG heptatriaconta-6,9,28, 50/10/38.5/1.5 31-tetraen-19-yl 4- Lipid:santisense polynucleotide agent (dimethylamino) 10:1 butanoate (MC3) LNP12 1,1'-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino) 50/10/38.5/1.5 ethyl)(2-hydroxy- Lipid:santisense polynucleotide agent dodecyl)amino)ethyl) 10:1 piperazin-1-yl)ethyl- azanediyl)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, which is hereby incorporated by reference. XTC comprising formulations are described in WO2010008537, which is hereby incorporated by reference. MC3 comprising formulations are described 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 in WO201054406, which is hereby incorporated by reference. C12-200 comprising formulations are described in WO2010129709, which is hereby incorporated by reference.

[0340] 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 or esters or salts thereof, bile acids 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 Pubn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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 or dextran. The suspension can also contain stabilizers.

[0345] C. Additional Formulations

[0346] i. Emulsions

[0347] The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 .mu.m in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

[0348] 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).

[0349] 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).

[0350] 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.

[0351] 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).

[0352] 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.

[0353] 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.

[0354] 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.

[0355] ii. Microemulsions

[0356] 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).

[0357] 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.

[0358] 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.

[0359] 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. Erp. 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.

[0360] 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.

[0361] iii. Microparticles

[0362] 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.

[0363] iv. Penetration Enhancers

[0364] 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.

[0365] 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.

[0366] 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).

[0367] 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).

[0368] 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).

[0369] 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 (enaminesxsee 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).

[0370] 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).

[0371] 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.), FreeStylem MAX (Invitrogen; Carlsbad, Calif.), Lipofectaminem 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectaminem (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectaminem (Invitrogen; Carlsbad, Calif.), Optifect.TM. (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam.RTM. Reagent (Promega; Madison, Wis.), TransFast.TM. Transfection Reagent (Promega; Madison, Wis.), Tfx.TM.-20 Reagent (Promega; Madison, Wis.), Tfx.TM.-50 Reagent (Promega; Madison, Wis.), DreamFect.TM. (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass' 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.

[0372] 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.

[0373] v. Carriers

[0374] 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.

[0375] vi. Excipients

[0376] 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).

[0377] 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.

[0378] 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.

[0379] 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.

[0380] vii. Other Components

[0381] 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 or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0382] Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension can also contain stabilizers.

[0383] 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 lmited to an anti-inflammatory agent, anti-steatosis agent, anti-viral, 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.

[0384] 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.

[0385] 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 EDso 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 ICso (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.

[0386] In addition to their administration, as discussed above, the antisense polynucleotide agents featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by Factor XII expression. In any event, the administering physician can adjust the amount and timing of antisense polynucleotide agent administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VII. Methods for Inhibiting Factor XII Expression

[0387] The present invention provides methods of inhibiting expression of Factor XII in a cell. The methods include contacting a cell with an antisense polynucleotide agent of the invention in an amount effective to inhibit expression of the Factor XII in the cell, thereby inhibiting expression of the Factor XII in the cell.

[0388] 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.

[0389] The term "inhibiting," as used herein, is used interchangeably with "reducing," "silencing," "downregulating" and other similar terms, and includes any level of inhibition.

[0390] The phrase "inhibiting expression of a Factor XII" is intended to refer to inhibition of expression of any Factor XII gene (such as, e.g., a mouse Factor XII gene, arat Factor XII gene, a monkey Factor XII gene, or a human Factor XII gene) as well as variants or mutants of a Factor XII gene. Thus, the Factor XII gene may be a wild-type Factor XII gene, a mutant Factor XII gene, or a transgenic Factor XII gene in the context of a genetically manipulated cell, group of cells, or organism.

[0391] "Inhibiting expression of a Factor XII gene" includes any level of inhibition of a Factor XII gene, e.g., at least partial suppression of the expression of a Factor XII gene. The expression of the Factor XII gene may be assessed based on the level, or the change in the level, of any variable associated with Factor XII gene expression, e.g., Factor XII mRNA level, Factor XII protein level, or for example, bradykinin elevation, prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, Fletcher Factor Deficiency, edema swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals, prodrome; laryngeal swelling; nonpruritic rash; nausea; vomiting; abdominal pain may also be measured to assess Factor XII expression. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.

[0392] Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with Factor XII 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).

[0393] In some embodiments of the methods of the invention, expression of a Factor XII gene is inhibited by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, at least 70%, 75%, 80%, 85%, 90%, 95%, or to below the level of detection of the assay method; or a similar level of reduction in the difference between a disease level and a normal level in an appropriate control subject. In preferred embodiments, the level of expression is detected using the in vitro method provided in the Examples.

[0394] Inhibition of the expression of a Factor XII 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 Factor XII 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 Factor XII gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s)). In preferred embodiments, the inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells, using the following formula:

(mRNA in control cells)-(mRNA in treated cells)/(mRNA in control cells)100%

[0395] Alternatively, inhibition of the expression of a Factor XII gene may be assessed in terms of a reduction of a parameter that is functionally linked to Factor XII gene expression, e.g., Factor XII protein expression, bradykinin level, prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, Fletcher Factor Deficiency, edema swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals, prodrome; laryngeal swelling; nonpruritic rash; nausea; vomiting; abdominal pain. Factor XII gene silencing may be determined in any cell expressing Factor XII, either constitutively or by genetic engineering, and by any assay known in the art. The liver is a major site of Factor XII expression.

[0396] Inhibition of the expression of a Factor XII protein may be manifested by a reduction in the level of the Factor XII 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 inhibiton 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.

[0397] A control cell or group of cells that may be used to assess the inhibition of the expression of a Factor XII 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.

[0398] The level of Factor XII 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 Factor XII in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the Factor XII 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.

[0399] In one embodiment, the level of expression of Factor XII 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 Factor XII. 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.

[0400] 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 Factor XII 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 Factor XII mRNA.

[0401] An alternative method for determining the level of expression of Factor XII in a sample involves the process of nucleic acid amplification 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 Factor XII is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan.TM. System).

[0402] The expression levels of Factor XII 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 Factor XII expression level may also comprise using nucleic acid probes in solution.

[0403] In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of a qPCR methods is described and exemplified in the Examples presented herein.

[0404] The level of Factor XII 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.

[0405] 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.

[0406] 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 Factor XII may be assessed using measurements of the level or change in the level of Factor XII mRNA or Factor XII 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.

[0407] 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.

[0408] 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 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.

[0409] 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 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 or are known in the art.

VIII. Methods for Treating a Factor XII-Associated Disorder

[0410] The present invention also provides therapeutic and prophylactic methods which include administering to a subject having a Factor XII-associated disease, e.g., heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency, an antisense polynucleotide agent or pharmaceutical compositions comprising an antisense polynucleotide agent of the invention.

[0411] In one aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in Factor XII expression, e.g., a Factor XII-associated disease, e.g., heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency. 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 Factor XII gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a Factor XII gene, thereby treating the subject having a disorder that would benefit from reduction in Factor XII expression.

[0412] In another aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in Factor XII expression, e.g., a Factor XII-associated disease, e.g., heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency, which include administering to the subject, e.g., a human, a therapeutically effective amount of an antisense polynucleotide agent targeting a Factor XII gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a Factor XII gene, and an additional therapeutic agent, such as an androgen, such as danazol or oxandrolone, Berinert.RTM., Cinryze.TM., Rhuconest.RTM., Ecallantide, Firazyr.RTM., Kalbitor.RTM., and a combination of any of the foregoing, thereby treating the subject having a disorder that would benefit from reduction in Factor XII expression.

[0413] "Therapeutically effective amount," as used herein, is intended to include the amount of an antisense polynucleotide agent, that, when administered to a subject having a Factor XII-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective amount" may vary depending on the antisense polynucleotide agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

[0414] A "therapeutically effective amount" also includes an amount of an antisense 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.

[0415] 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 or inhibition of Factor XII expression.

[0416] In another aspect, the present invention provides uses of a therapeutically effective amount of an antisense polynucleotide agent of the invention and an additional therapeutic agent, such as an androgen, such as danazol or oxandrolone, Berinert.RTM., Cinryzem, Rhuconest.RTM., Ecallantide, Firazyr.RTM., Kalbitor.RTM., and a combination of any of the foregoing, for treating a subject, e.g., a subject that would benefit from a reduction or inhibition of Factor XII expression.

[0417] In yet another aspect, the present invention provides use of an antisense polynucleotide agent of the invention targeting a Factor XII gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a Factor XII gene in the manufacture of a medicament for treating a subject, e.g., a subject that would benefit from a reduction or inhibition of Factor XII expression, such as a subject having a disorder that would benefit from reduction in Factor XII expression, e.g., a Factor XII-associated disease, e.g., heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency.

[0418] In another aspect, the present invention provides uses of an antisense polynucleotide agent of the invention targeting a Factor XII gene or a pharmaceutical composition comprising an antisense polynucleotide agent targeting a Factor XII gene in the manufacture of a medicament for use in combination with an additional therapeutic agent, such as an androgen, such as danazol or oxandrolone, Berinert.RTM., Cinryze.TM., Rhuconest.RTM., Ecallantide, Firazyr.RTM., Kalbitor.RTM., and a combination of any of the foregoing, for treating a subject, e.g., a subject that would benefit from a reduction or inhibition of Factor XII expression, e.g., a Factor XII-associated disease, e.g., heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency.

[0419] In yet another aspect, the invention provides uses of an antisense polynucleotide agent of the invention, and an additional therapeutic agent, such as an androgen, such as danazol or oxandrolone, Berinert.RTM., Cinryze.TM., Rhuconest.RTM., Ecallantide, Firazyr.RTM., Kalbitor.RTM., and a combination of any of the foregoing, for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction or inhibition of Factor XII expression, such as a Factor XII-associated disease, e.g., heredity angioedema (HAE), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, or Fletcher Factor Deficiency.

[0420] 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 or inhibition of Factor XII expression, such as a a Factor XII-associated disease, e.g., heredity angioedema (HAE) (such as hereditary angioedema type I; hereditary angioedema type II; hereditary angioedema type III; or any other hereditary angioedema caused by elevated levels of bradykinin), prekallikrein deficiency, malignant essential hypertension, hypertension, end stage renal disease, Fletcher Factor Deficiency, edema swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals, prodrome; laryngeal swelling; nonpruritic rash; nausea; vomiting; abdominal pain.

[0421] In one embodiment, an antisense polynucleotide agent targeting Factor XII is administered to a subject having a Factor XII-associated disease such that Factor XII levels, e.g., in a cell, tissue, blood, urine or other tissue or fluid of the subject are reduced as compared to an appropriate control by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or to a level below the level of detection of the assay method as compared to an appropriate control. In certain embodiments, the Factor XII level a similar level of reduction in the difference between a disease level and a normal level in an appropriate control subject.

[0422] The additional therapeutic may be an androgen, such as danazol or oxandrolone, Berinert.RTM., Cinryze.TM., Rhuconest.RTM., Ecallantide, Firazyr.RTM., Kalbitor.RTM., and a combination of any of the foregoing.

[0423] The additional therapeutic, e.g., an androgen, such as danazol or oxandrolone, Berinert.RTM., Cinryze.TM., Rhuconest.RTM., Ecallantide, Firazyr.RTM., Kalbitor.RTM., and a combination of any of the foregoing, may be administered to the subject at the same time as the antisense polynucleotide agent targeting Factor XII or at a different time.

[0424] Moreover, the additional therapeutic, e.g., an androgen, such as danazol or oxandrolone, Berinert.RTM., Cinryze.TM., Rhuconest.RTM., Ecallantide, Firazyr.RTM., Kalbitor.RTM., and a combination of any of the foregoing, may be administered to the subject in the same formulation as the antisense polynucleotide agent targeting Factor XII or in a different formulation as the antisense polynucleotide agent targeting Factor XII.

[0425] The methods and uses of the invention include administering a composition described herein such that expression of the target Factor XII gene is decreased, such as for at least 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 80 hours. In one embodiment, expression of the target Factor XII gene is decreased for an extended duration, e.g., at least two, three, four, five, six, seven days or more, e.g., one week, two weeks, three weeks, or four weeks, or longer.

[0426] 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, or markers of such diseases or disorders in a patient with a Factor XII-associated disease. By "reduction" in this context is meant a statistically significant decrease in such level as compared to an appropriate control. The reduction can be, for example, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or to below the level of detection of the assay used for detection; or a similar level of reduction in the difference between a disease level and a normal level in an appropriate control subject. In certain embodiments, the reduction is the normalization of the level of a sign or symptom of a disease, a reduction in the difference between the subject level of a sign of the disease and the normal level of the sign for the disease (e.g., upper level of normal, lower level of normal, average of upper and lower level of normal). For example, reduction can be understood as normalization of blood pressure, decreasing an elevated blood pressure or increasing a low blood pressure to reduce the difference from a normal reading.

[0427] Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, reduction in symptom severity, e.g., severity of edema and swelling, nonpruritic rash, nausea, and vomiting; reduction in pain e.g., abdominal pain, and the 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 HAE can be determined by assessing severe swelling of the arms, legs, hands, feet, face, tongue and larynx, abdomen, nausea, vomiting, and nonpriuric rash. Elevanted levels of bradykinin peptide are observed during HAE attacks or episdes. 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 Factor XII or pharmaceutical composition thereof, "effective against" a Factor XII-associated disease indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as improvement of symptoms, a cure, a reduction in disease, extension of life, or other effect generally recognized as positive by medical doctors familiar with treating a Factor XII-associated disease and the related causes.

[0428] A treatment 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.

[0429] 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 criteria based on the disease state. 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.

[0430] Subjects can be administered a therapeutic amount of antisense polynucleotide agent, such as about 0.01 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 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0431] 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 5 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.05 mg/kg to 5 mg/kg, 0.05 mg/kg to 10 mg/kg, 0.1 mg/kg to 5 mg/kg, 0.1 mg/kg to 10 mg/kg, 0.2 mg/kg to 5 mg/kg, 0.2 mg/kg to 10 mg/kg, 0.3 mg/kg to 5 mg/kg, 0.3 mg/kg to 10 mg/kg, 0.4 mg/kg to 5 mg/kg, 0.4 mg/kg to 10 mg/kg, 0.5 mg/kg to 5 mg/kg, 0.5 mg/kg to 10 mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 10 mg/kg, 1.5 mg/kg to 5 mg/kg, 1.5 mg/kg to 10 mg/kg, 2 mg/kg to 2.5 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 5 mg/kg, 3 mg/kg to 10 mg/kg, 3.5 mg/kg to 5 mg/kg, 4 mg/kg to 5 mg/kg, 4.5 mg/kg to 5 mg/kg, 4 mg/kg to 10 mg/kg, 4.5 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 5.5 mg/kg to 10 mg/kg, 6 mg/kg to 10 mg/kg, 6.5 mg/kg to 10 mg/kg, 7 mg/kg to 10 mg/kg, 7.5 mg/kg to 10 mg/kg, 8 mg/kg to 10 mg/kg, 8.5 mg/kg to 10 mg/kg, 9 mg/kg to 10 mg/kg, or 9.5 mg/kg to 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0432] 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.

[0433] 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 50 mg/kg, 0.25 to 50 mg/kg, 0.5 to 50 mg/kg, 0.75 to 50 mg/kg, 1 to 50 mg/mg, 1.5 to 50 mg/kb, 2 to 50 mg/kg, 2.5 to 50 mg/kg, 3 to 50 mg/kg, 3.5 to 50 mg/kg, 4 to 50 mg/kg, 4.5 to 50 mg/kg, 5 to 50 mg/kg, 7.5 to 50 mg/kg, 10 to 50 mg/kg, 15 to 50 mg/kg, 20 to 50 mg/kg, 20 to 50 mg/kg, 25 to 50 mg/kg, 25 to 50 mg/kg, 30 to 50 mg/kg, 35 to 50 mg/kg, 40 to 50 mg/kg, 45 to 50 mg/kg, 0.1 to 45 mg/kg, 0.25 to 45 mg/kg, 0.5 to 45 mg/kg, 0.75 to 45 mg/kg, 1 to 45 mg/mg, 1.5 to 45 mg/kb, 2 to 45 mg/kg, 2.5 to 45 mg/kg, 3 to 45 mg/kg, 3.5 to 45 mg/kg, 4 to 45 mg/kg, 4.5 to 45 mg/kg, 5 to 45 mg/kg, 7.5 to 45 mg/kg, 10 to 45 mg/kg, 15 to 45 mg/kg, 20 to 45 mg/kg, 20 to 45 mg/kg, 25 to 45 mg/kg, 25 to 45 mg/kg, 30 to 45 mg/kg, 35 to 45 mg/kg, 40 to 45 mg/kg, 0.1 to 40 mg/kg, 0.25 to 40 mg/kg, 0.5 to 40 mg/kg, 0.75 to 40 mg/kg, 1 to 40 mg/mg, 1.5 to 40 mg/kb, 2 to 40 mg/kg, 2.5 to 40 mg/kg, 3 to 40 mg/kg, 3.5 to 40 mg/kg, 4 to 40 mg/kg, 4.5 to 40 mg/kg, 5 to 40 mg/kg, 7.5 to 40 mg/kg, 10 to 40 mg/kg, 15 to 40 mg/kg, 20 to 40 mg/kg, 20 to 40 mg/kg, 25 to 40 mg/kg, 25 to 40 mg/kg, 30 to 40 mg/kg, 35 to 40 mg/kg, 0.1 to 30 mg/kg, 0.25 to 30 mg/kg, 0.5 to 30 mg/kg, 0.75 to 30 mg/kg, 1 to 30 mg/mg, 1.5 to 30 mg/kb, 2 to 30 mg/kg, 2.5 to 30 mg/kg, 3 to 30 mg/kg, 3.5 to 30 mg/kg, 4 to 30 mg/kg, 4.5 to 30 mg/kg, 5 to 30 mg/kg, 7.5 to 30 mg/kg, 10 to 30 mg/kg, 15 to 30 mg/kg, 20 to 30 mg/kg, 20 to 30 mg/kg, 25 to 30 mg/kg, 0.1 to 20 mg/kg, 0.25 to 20 mg/kg, 0.5 to 20 mg/kg, 0.75 to 20 mg/kg, 1 to 20 mg/mg, 1.5 to 20 mg/kb, 2 to 20 mg/kg, 2.5 to 20 mg/kg, 3 to 20 mg/kg, 3.5 to 20 mg/kg, 4 to 20 mg/kg, 4.5 to 20 mg/kg, 5 to 20 mg/kg, 7.5 to 20 mg/kg, 10 to 20 mg/kg, or 15 to 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 10 to 30 mg/kg of antisense polynucleotide agent. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0434] 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 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0435] 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 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.

[0436] Administration of the antisense polynucleotide agent can reduce Factor XII levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or to below the level of detection of the assay method used; or a similar level of reduction in the difference between a disease level and a normal level in an appropriate control subject.

[0437] 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.

[0438] Owing to the inhibitory effects on Factor XII expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.

[0439] 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.

[0440] Alternatively, an antisense polynucleotide agent of the invention may be administered as a pharmaceutical composition, such as an antisense polynucleotide agent liposomal formulation.

[0441] Subjects that would benefit from a reduction or inhibition of Factor XII gene expression are those having a Factor XII-associated disease or disorder as described herein. In one embodiment, a subject having a Factor XII-associated disease has heredity angioedema (HAE). In another embodiment, a subject having a Factor XII-associated disease has prekallikrein deficiency. In another embodiment, a subject having a Factor XII-associated disease has hypertension, e.g., malignant essential hypertension. In yet another embodiment, a subject having a Factor XII-associated disease has end stage renal disease. In one embodiment, a subject having a Factor XII-associated disease has Fletcher Factor Deficiency.

[0442] The invention further provides methods and uses of an antisense polynucleotide agent or a pharmaceutical composition thereof (including methods and uses of an antisense polynucleotide agent or a pharmaceutical composition comprising an antisense polynucleotide agent and an androgen, such as danazol or oxandrolone, Berinert.RTM., Cinryze.TM., Rhuconest.RTM., Ecallantide, Firazyr@, Kalbitor.RTM., and a combination of any of the foregoing for treating a subject that would benefit from reduction or inhibition of Factor XII expression, e.g., a subject having a Factor XII-associated disease, in combination with other pharmaceuticals or other therapeutic methods, e.g., with known pharmaceuticals 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 Factor XII is administered in combination with, e.g., an agent useful in treating a Factor XII-associated disease as described elsewhere herein.

[0443] The antisense polynucleotide agent and an additional therapeutic agent or treatment may be administered at the same time 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 or by another method known in the art or described herein.

[0444] The present invention also provides methods of using an antisense polynucleotide agent of the invention or a composition containing an antisense polynucleotide agent of the invention to reduce or inhibit Factor XII expression in a cell. In other aspects, the present invention provides an antisense polynucleotide agent of the invention or a composition comprising an antisense polynucleotide agent of the invention for use in reducing or inhibiting Factor XII expression in a cell. In yet other aspects, use of an antisense polynucleotide agent of the invention or a composition comprising an antisense polynucleotide agent of the invention for the manufacture of a medicament for reducing or inhibiting Factor XII expression in a cell are provided.

[0445] 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 Factor XII gene, thereby inhibiting expression of the Factor XII gene in the cell.

[0446] Reduction in gene expression can be assessed by any methods known in the art and as provided herein.

[0447] 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 Polynucleotide Synthesis

[0448] A set of antisense polynucleotide agents targeting the human F12 gene (coagulation factor XII) (human NCBI refseqID: NM_000505; NCBI GeneID: 2161) were designed using custom R and Python scripts. The rationale and method for the set of designs is as follows: the predicted efficacy for every potential 19mer from position 1 through the end of the mRNA was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct ASO designs targeting a large number of vertebrate genes. Starting from position 1 a set of s was created by systematically picking a design whose 5' base began every 11 bases along the entire length of the mRNA. Predicted efficacy was used to allow for the substitution of a neighboring, predicted-to-be-more-potent design where the neighboring was 1 base either toward the 5' or 3' end of the mRNA. Low complexity designs were removed by filtering with a Shannon entropy index greater than 1.35.

[0449] The antisense polynucleotides targeting Factor XII were synthesized using standard synthesis methods well known in the art.

[0450] A detailed list of antisense polynucleotide agents targeting Factor XII is shown in Table 3.

Example 2. In Vitro Screening

[0451] Human hepatocarcinoma cells, Huh-7 cells, were obtained from American Type Culture Collection (Rockville, Md., cat. No. PTA-8561) and cultured in Dulbecco's MEM (Biochrom GmbH, Berlin #FG0435), supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, cat. No. S0115), 200 .mu.M L-Glutamin (Biochrom GmbH, Berlin, Germany, cat. No. K0283) and Penicillin 100 U/ml, Streptomycin 100 mg/ml (Biochrom GmbH, Berlin, Germany, cat. No. A2213), at 37.degree. C. in an atmosphere with 5% CO.sub.2 in a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany).

[0452] Transfection of Huh-7 cells with an antisense polynucleotide agent was performed directly after seeding 20,000 cells/well in a 96-well plate, and was carried out with Lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) as described by the manufacturer. Transfections were performed in quadruplicates and cells were transfected with theantisense polynucleotide agents at a concentration of 5 nM.

[0453] For measurement of FXII mRNA cells were harvested 24 hours after transfection and lysed at 53.degree. C. following procedures recommended by the manufacturer of the Quantigene II Kit for FXII and Quantigene I Explore Kit for GAPDH (Affymetrix/eBioscience, Frankfurt, Germany, cat. No. 15735 or QG0004, respectively) bDNA. Afterwards, 50 .mu.l of the lysates were incubated with probesets specific to human FXII and 10 .mu.l of the lysates for human GAPDH and processed according to the manufacturer's protocol for QuantiGene. Chemoluminescence was measured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relative light units) and values obtained with the human FXII probeset were normalized to the respective human GAPDH values for each well and then related to the mean of three unrelated control antisense polynucleotide agents.

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) 5'-methyl-deoxycytidine-3'-phosphate (5MdC)s 5'-methyl-deoxycytidine-3'-phosphorothioate

TABLE-US-00003 TABLE 3 Antisense polynucleotide agents targeting Factor XII. SEQ Target SEQ Oligo Unmodified Sequence ID site on ID Name (5' to 3') NO: SEQ ID 1 Modified Sequence (5' to 3') NO: A-145494 AGCUGCCTATCCAGGAGUCC 9 11-30 asgscsusgs(m5dCs)(m5dCs)dTsdAsdTs 194 (m5dCs)(m5dCs)dAsdGsdGsasgsuscsc A-145495 CCGUUGGTCCAGCTGCCUAU 10 21-40 cscsgsususdGsdGsdTs(m5dCs)(m5dCs)dAsdGs 195 (m5dCs)dTsdGscscsusasu A-145496 AUGGCATCCGTCCGTUGGUC 11 32-51 asusgsgscsdAsdTs(m5dCs)(m5dCs)dGsdTs 196 (m5dCs)(m5dCs)dGsdTsusgsgsusc A-145497 AGCAGAGCCCTCATGGCAUC 12 44-63 asgscsasgsdAsdGs(m5dCs)(m5dCs)(m5dCs)dT 197 s(m5dCs)dAsdTsdGsgscsasusc A-145498 ACCCCAGGAGCAGCAGAGCC 13 55-74 ascscscscsdAsdGsdGsdAsdGs(m5dCs)dAsdGs 198 (m5dCs)dAsgsasgscsc A-145499 ACCAGCAGGAACCCCAGGAG 14 65-84 ascscsasgs(m5dCs)dAsdGsdGsdAsdAs(m5dCs) 199 (m5dCs)(m5dCs)(m5dCs)asgsgsasg A-145500 ACUCCAAGCTCACCAGCAGG 15 76-95 ascsuscscsdAsdAsdGs(m5dCs)dTs(m5dCs)dAs 200 (m5dCs)(m5dCs)dAsgscsasgsg A-145501 CGAAAGTGTTGACTCCAAGC 16 87-106 csgsasasasdGsdTsdGsdTsdTsdGsdAs(m5dCs)d 201 Ts(m5dCs)csasasgsc A-145502 CCAAGGTGGAATCGAAAGUG 17 99-118 cscsasasgsdGsdTsdGsdGsdAsdAsdTs(m5dCs)d 202 GsdAsasasgsusg A-145503 UGGGGGCTTCCCAAGGUGGA 18 109-128 usgsgsgsgsdGs(m5dCs)dTsdTs(m5dCs) 203 (m5dCs)(m5dCs)dAsdAsdGsgsusgsgsa A-145504 ACUUATGCTCCTTGGGGGCU 19 121-140 ascsususasdTsdGs(m5dCs)dTs(m5dCs) 204 (m5dCs)dTsdTsdGsdGsgsgsgscsu A-145505 CUUCAGCTTTGTACTUAUGC 20 133-152 csususcsasdGs(m5dCs)dTsdTsdTsdGsdTsdAs 205 (m5dCs)dTsusasusgsc A-145506 ACUGUGTGCTCTTCAGCUUU 21 143-162 ascsusgsusdGsdTsdGs(m5dCs)dTs(m5dCs)dTs 206 dTs(m5dCs)dAsgscsususu A-145507 ACAGUGAGAACGACTGUGUG 22 155-174 ascsasgsusdGsdAsdGsdAsdAs(m5dCs)dGsdAs 207 (m5dCs)dTsgsusgsusg A-145508 UCCCCGGTGACAGTGAGAAC 23 164-183 uscscscscsdGsdGsdTsdGsdAs(m5dCs)dAsdGsd 208 TsdGsasgsasasc A-145509 AGUGGCAGGGCTCCCCGGUG 24 175-194 asgsusgsgs(m5dCs)dAsdGsdGsdGs(m5dCs)dTs 209 (m5dCs)(m5dCs)(m5dCs)csgsgsusg A-145510 ACUGGAAGGGGAAGTGGCAG 25 187-206 ascsusgsgsdAsdAsdGsdGsdGsdGsdAsdAsdGsdT 210 sgsgscsasg A-145511 GCUGCCGGTGGTACTGGAAG 26 199-218 gscsusgscs(m5dCs)dGsdGsdTsdGsdGsdTsdAs 211 (m5dCs)dTsgsgsasasg A-145512 UUGUGGTACAGCTGCCGGUG 27 209-228 ususgsusgsdGsdTsdAs(m5dCs)dAsdGs(m5dCs) 212 dTsdGs(m5dCs)csgsgsusg A-145513 UGUGGGTACATTTGTGGUAC 28 220-239 usgsusgsgsdGsdTsdAs(m5dCs)dAsdTsdTsdTsd 213 GsdTsgsgsusasc A-145514 UGGCCGGCCCTTGTGGGUAC 29 231-250 usgsgscscsdGsdGs(m5dCs)(m5dCs)(m5dCs)dT 214 sdTsdGsdTsdGsgsgsusasc A-145515 GGCUGAGGGCCTGGCCGGCC 30 242-261 gsgscsusgsdAsdGsdGsdGs(m5dCs)(m5dCs)dTs 215 dGsdGs(m5dCs)csgsgscsc A-145516 GUAGCACACCAGGGCUGAGG 31 254-273 gsusasgscsdAs(m5dCs)dAs(m5dCs)(m5dCs)dA 216 sdGsdGsdGs(m5dCs)usgsasgsg A-145517 AGUUGGGGGTGGTAGCACAC 32 265-284 asgsususgsdGsdGsdGsdGsdTsdGsdGsdTsdAsdG 217 scsascsasc A-145518 CCUGATCAAAGTTGGGGGUG 33 274-293 cscsusgsasdTs(m5dCs)dAsdAsdAsdGsdTsdTsd 218 GsdGsgsgsgsusg A-145519 CCCCATCGCTGGTCCUGAUC 34 287-306 cscscscsasdTs(m5dCs)dGs(m5dCs)dTsdGsdGs 219 dTs(m5dCs)(m5dCs)usgsasusc A-145520 AAACAGTATCCCCATCGCUG 35 296-315 asasascsasdGsdTsdAsdTs(m5dCs)(m5dCs) 220 (m5dCs)(m5dCs)dAsdTscsgscsusg A-145521 UCUUGGGCTCCAAACAGUAU 36 307-326 uscsususgsdGsdGs(m5dCs)dTs(m5dCs) 221 (m5dCs)dAsdAsdAs(m5dCs)asgsusasu A-145522 UGGUCTTTCACTTTCUUGGG 37 320-339 usgsgsuscsdTsdTsdTs(m5dCs)dAs(m5dCs)dTs 222 dTsdTs(m5dCs)ususgsgsg A-145523 GUUUGCTGCAGTGGTCUUUC 38 331-350 gsusususgs(m5dCs)dTsdGs(m5dCs)dAsdGsdTs 223 dGsdGsdTscsusususc A-145524 AGGGGCTGTGTTTGCUGCAG 39 340-359 asgsgsgsgs(m5dCs)dTsdGsdTsdGsdTsdTsdTsd 224 Gs(m5dCs)usgscsasg A-145525 CUCCUTTCTGGCAGGGGCUG 40 352-371 csuscscsusdTsdTs(m5dCs)dTsdGsdGs(m5dCs) 225 dAsdGsdGsgsgscsusg A-145526 ACACAGGTCCCTCCTUUCUG 41 362-381 ascsascsasdGsdGsdTs(m5dCs)(m5dCs) 226 (m5dCs)dTs(m5dCs)(m5dCs)dTsususcsusg A-145527 UUGGCATGTTCACACAGGUC 42 373-392 ususgsgscsdAsdTsdGsdTsdTs(m5dCs)dAs 227 (m5dCs)dAs(m5dCs)asgsgsusc A-145528 AGUGGGGGCCGCTTGGCAUG 43 385-404 asgsusgsgsdGsdGsdGs(m5dCs)(m5dCs)dGs 228 (m5dCs)dTsdTsdGsgscsasusg A-145529 GGACAGAGACAGTGGGGGCC 44 395-414 gsgsascsasdGsdAsdGsdAs(m5dCs)dAsdGsdTsd 229 GsdGsgsgsgscsc A-145530 UGAGGTGTTGTGGACAGAGA 45 406-425 usgsasgsgsdTsdGsdTsdTsdGsdTsdGsdGsdAs 230 (m5dCs)asgsasgsa A-145531 AGUGGTTTCCAGTGAGGUGU 46 418-437 asgsusgsgsdTsdTsdTs(m5dCs)(m5dCs)dAsdGs 231 dTsdGsdAsgsgsusgsu A-145532 UCUCUTTCTGGCAGTGGUUU 47 430-449 uscsuscsusdTsdTs(m5dCs)dTsdGsdGs(m5dCs) 232 dAsdGsdTsgsgsususu A-145533 CAAAGCACTTCTCTTUCUGG 48 439-458 csasasasgs(m5dCs)dAs(m5dCs)dTsdTs 233 (m5dCs)dTs(m5dCs)dTsdTsuscsusgsg A-145534 GAAGCTGAGGCTCAAAGCAC 49 451-470 gsasasgscsdTsdGsdAsdGsdGs(m5dCs)dTs 234 (m5dCs)dAsdAsasgscsasc A-145535 GGAAAAACCGGAGAAGCUGA 50 463-482 gsgsasasasdAsdAs(m5dCs)(m5dCs)dGsdGsdAs 235 dGsdAsdAsgscsusgsa A-145536 CAUUCTTGTGGAAAAACCGG 51 472-491 csasususcsdTsdTsdGsdTsdGsdGsdAsdAsdAsdA 236 sascscsgsg A-145537 AUACCATATCTCATTCUUGU 52 483-502 asusascscsdAsdTsdAsdTs(m5dCs)dTs(m5dCs) 237 dAsdTsdTscsususgsu A-145538 UGCUCAGTTCTATACCAUAU 53 494-513 usgscsuscsdAsdGsdTsdTs(m5dCs)dTsdAsdTsd 238 As(m5dCs)csasusasu A-145539 GGCCACAGCTGCTTGCUCAG 54 507-526 gsgscscsas(m5dCs)dAsdGs(m5dCs)dTsdGs 239 (m5dCs)dTsdTsdGscsuscsasg A-145540 ACUGGCATCTGGCCACAGCU 55 517-536 ascsusgsgs(m5dCs)dAsdTs(m5dCs)dTsdGsdGs 240 (m5dCs)(m5dCs)dAscsasgscsu A-145541 AGGACCCTTGCACTGGCAUC 56 528-547 asgsgsascs(m5dCs)(m5dCs)dTsdTsdGs 241 (m5dCs)dAs(m5dCs)dTsdGsgscsasusc A-145542 AGUGGGCATCAGGACCCUUG 57 538-557 asgsusgsgsdGs(m5dCs)dAsdTs(m5dCs)dAsdGs 242 dGsdAs(m5dCs)cscsususg A-145543 CAGCCGCTGGCAGTGGGCAU 58 549-568 csasgscscsdGs(m5dCs)dTsdGsdGs(m5dCs)dAs 243 dGsdTsdGsgsgscsasu A-145544 AGGCCTGGCTGGCCAGCCGC 59 562-581 asgsgscscsdTsdGsdGs(m5dCs)dTsdGsdGs 244 (m5dCs)(m5dCs)dAsgscscsgsc A-145545 UGGUGCGGCAGGCCTGGCUG 60 571-590 usgsgsusgs(m5dCs)dGsdGs(m5dCs)dAsdGsdGs 245 (m5dCs)(m5dCs)dTsgsgscsusg A-145546 GGAGGCACGGGTTGGUGCGG 61 583-602 gsgsasgsgs(m5dCs)dAs(m5dCs)dGsdGsdGsdTs 246 dTsdGsdGsusgscsgsg A-145547 AGCGACCCCCATGGAGGCAC 62 595-614 asgscsgsas(m5dCs)(m5dCs)(m5dCs)(m5dCs) 247 (m5dCs)dAsdTsdGsdGsdAsgsgscsasc A-145548 ACCUCTAGGCAGCGACCCCC 63 605-624 ascscsuscsdTsdAsdGsdGs(m5dCs)dAsdGs 248 (m5dCs)dGsdAscscscscsc A-145549 GGUGGCCCTCCACCTCUAGG 64 616-635 gsgsusgsgs(m5dCs)(m5dCs)(m5dCs)dTs 249 (m5dCs)(m5dCs)dAs(m5dCs)(m5dCs)dTscsusa sgsg A-145550 UGGCACAGGCGGTGGCCCUC 65 626-645 usgsgscsas(m5dCs)dAsdGsdGs(m5dCs)dGsdGs 250 dTsdGsdGscscscsusc A-145551 CCCACCGGGCAGTGGCACAG 66 638-657 cscscsascs(m5dCs)dGsdGsdGs(m5dCs)dAsdGs 251 dTsdGsdGscsascsasg A-145552 GCUCCGGTGTAGCCCACCGG 67 650-669 gscsuscscsdGsdGsdTsdGsdTsdAsdGs(m5dCs) 252 (m5dCs)(m5dCs)ascscsgsg A-145553 CGUCGCAGAAGGCTCCGGUG 68 661-680 csgsuscsgs(m5dCs)dAsdGsdAsdAsdGsdGs 253 (m5dCs)dTs(m5dCs)csgsgsusg A-145554 UGGUGTCCACGTCGCAGAAG 69 670-689 usgsgsusgsdTs(m5dCs)(m5dCs)dAs(m5dCs)dG 254

sdTs(m5dCs)dGs(m5dCs)asgsasasg A-145555 AGCAGCTTGCCTTGGUGUCC 70 682-701 asgscsasgs(m5dCs)dTsdTsdGs(m5dCs) 255 (m5dCs)dTsdTsdGsdGsusgsuscsc A-145556 CGGCCATCATAGCAGCUUGC 71 692-711 csgsgscscsdAsdTs(m5dCs)dAsdTsdAsdGs 256 (m5dCs)dAsdGscsususgsc A-145557 AGCUGAGCCCGCGGCCAUCA 72 703-722 asgscsusgsdAsdGs(m5dCs)(m5dCs)(m5dCs)dG 257 s(m5dCs)dGsdGs(m5dCs)csasuscsa A-145558 GCCAGGCCGCGGTAGCUGAG 73 716-735 gscscsasgsdGs(m5dCs)(m5dCs)dGs(m5dCs)dG 258 sdGsdTsdAsdGscsusgsasg A-145559 CGUGGTCCTGGCCAGGCCGC 74 726-745 csgsusgsgsdTs(m5dCs)(m5dCs)dTsdGsdGs 259 (m5dCs)(m5dCs)dAsdGsgscscsgsc A-145560 CACCCGAGAGCGTGGUCCUG 75 736-755 csascscscsdGsdAsdGsdAsdGs(m5dCs)dGsdTsd 260 GsdGsuscscsusg A-145561 GCUGACAGGGCGCACCCGAG 76 748-767 gscsusgsas(m5dCs)dAsdGsdGsdGs(m5dCs)dGs 261 (m5dCs)dAs(m5dCs)cscsgsasg A-145562 CGAGGCCCACGGCTGACAGG 77 759-778 csgsasgsgs(m5dCs)(m5dCs)(m5dCs)dAs 262 (m5dCs)dGsdGs(m5dCs)dTsdGsascsasgsg A-145563 AGGUGGCCTCCGAGGCCCAC 78 769-788 asgsgsusgsdGs(m5dCs)(m5dCs)dTs(m5dCs) 263 (m5dCs)dGsdAsdGsdGscscscsasc A-145564 GUCACGTTCCGGTAGGUGGC 79 782-801 gsuscsascsdGsdTsdTs(m5dCs)(m5dCs)dGsdGs 264 dTsdAsdGsgsusgsgsc A-145565 CUUGCTCGGCAGTCACGUUC 80 793-812 csususgscsdTs(m5dCs)dGsdGs(m5dCs)dAsdGs 265 dTs(m5dCs)dAscsgsususc A-145566 CCAGUTCCGCGCTTGCUCGG 81 804-823 cscsasgsusdTs(m5dCs)(m5dCs)dGs(m5dCs)dG 266 s(m5dCs)dTsdTsdGscsuscsgsg A-145567 CGCCCAGTCCCCAGTUCCGC 82 814-833 csgscscscsdAsdGsdTs(m5dCs)(m5dCs) 267 (m5dCs)(m5dCs)dAsdGsdTsuscscsgsc A-145568 AAGGCGTGGCCGCCCAGUCC 83 824-843 asasgsgscsdGsdTsdGsdGs(m5dCs)(m5dCs)dGs 268 (m5dCs)(m5dCs)(m5dCs)asgsuscsc A-145569 GGGUUCCGGCAGAAGGCGUG 84 836-855 gsgsgsusus(m5dCs)(m5dCs)dGsdGs(m5dCs)dA 269 sdGsdAsdAsdGsgscsgsusg A-145570 UGUCGTTGTCCGGGTUCCGG 85 847-866 usgsuscsgsdTsdTsdGsdTs(m5dCs)(m5dCs)dGs 270 dGsdGsdTsuscscsgsg A-145571 ACCACGGGCGGATGTCGUUG 86 859-878 ascscsascsdGsdGsdGs(m5dCs)dGsdGsdAsdTsd 271 GsdTscsgsususg A-145572 AGCACGAAGCACCACGGGCG 87 869-888 asgscsascsdGsdAsdAsdGs(m5dCs)dAs(m5dCs) 272 (m5dCs)dAs(m5dCs)gsgsgscsg A-145573 GGUCGCGGTTCAGCACGAAG 88 880-899 gsgsuscsgs(m5dCs)dGsdGsdTsdTs(m5dCs)dAs 273 dGs(m5dCs)dAscsgsasasg A-145574 CAGCUCAGCCGGTCGCGGUU 89 890-909 csasgscsus(m5dCs)dAsdGs(m5dCs)(m5dCs)dG 274 sdGsdTs(m5dCs)dGscsgsgsusu A-145575 CGCAGTACTCCCAGCUCAGC 90 901-920 csgscsasgsdTsdAs(m5dCs)dTs(m5dCs) 275 (m5dCs)(m5dCs)dAsdGs(m5dCs)uscsasgsc A-145576 ACUGUGCCAGGTCGCAGUAC 91 913-932 ascsusgsusdGs(m5dCs)(m5dCs)dAsdGsdGsdTs 276 (m5dCs)dGs(m5dCs)asgsusasc A-145577 GGGGUCTGGCACTGTGCCAG 92 923-942 gsgsgsgsus(m5dCs)dTsdGsdGs(m5dCs)dAs 277 (m5dCs)dTsdGsdTsgscscsasg A-145578 GCCGCCTGGGTTGGGGUCUG 93 935-954 gscscsgscs(m5dCs)dTsdGsdGsdGsdTsdTsdGsd 278 GsdGsgsuscsusg A-145579 GGGGUCGGAGGCGCCGCCUG 94 947-966 gsgsgsgsus(m5dCs)dGsdGsdAsdGsdGs(m5dCs) 279 dGs(m5dCs)(m5dCs)gscscsusg A-145580 AGGGGACACCGGGGTCGGAG 95 957-976 asgsgsgsgsdAs(m5dCs)dAs(m5dCs)(m5dCs)dG 280 sdGsdGsdGsdTscsgsgsasg A-145581 ACAUGAAGCCTAGGGGACAC 96 968-987 ascsasusgsdAsdAsdGs(m5dCs)(m5dCs)dTsdAs 281 dGsdGsdGsgsascsasc A-145582 GGCAUGAGTGGGACAUGAAG 97 980-999 gsgscsasusdGsdAsdGsdTsdGsdGsdGsdAs 282 (m5dCs)dAsusgsasasg A-145583 CGGCUGCGCGGGCATGAGUG 98 990-1009 csgsgscsusdGs(m5dCs)dGs(m5dCs)dGsdGsdGs 283 (m5dCs)dAsdTsgsasgsusg A-145584 UUCGGCGGTGCCGGCUGCGC 99 1001-1020 ususcsgsgs(m5dCs)dGsdGsdTsdGs(m5dCs) 284 (m5dCs)dGsdGs(m5dCs)usgscsgsc A-145585 GGGCUGAGGCTTCGGCGGUG 100 1011-1030 gsgsgscsusdGsdAsdGsdGs(m5dCs)dTsdTs 285 (m5dCs)dGsdGscsgsgsusg A-145586 GGUCCGGGTCGTGGGCUGAG 101 1023-1042 gsgsuscscsdGsdGsdGsdTs(m5dCs)dGsdTsdGsd 286 GsdGscsusgsasg A-145587 ACUGAGGCGGGGTCCGGGUC 102 1033-1052 ascsusgsasdGsdGs(m5dCs)dGsdGsdGsdGsdTs 287 (m5dCs)(m5dCs)gsgsgsusc A-145588 CCGGGGTCTGGGACTGAGGC 103 1045-1064 cscsgsgsgsdGsdTs(m5dCs)dTsdGsdGsdGsdAs 288 (m5dCs)dTsgsasgsgsc A-145589 GGCAAGGCTCCCGGGGUCUG 104 1055-1074 gsgscsasasdGsdGs(m5dCs)dTs(m5dCs) 289 (m5dCs)(m5dCs)dGsdGsdGsgsuscsusg A-145590 UCCCGCTTCGCCGGCAAGGC 105 1067-1086 uscscscsgs(m5dCs)dTsdTs(m5dCs)dGs 290 (m5dCs)(m5dCs)dGsdGs(m5dCs)asasgsgsc A-145591 AAGGCGGCTGCTCCCGCUUC 106 1078-1097 asasgsgscsdGsdGs(m5dCs)dTsdGs(m5dCs)dTs 291 (m5dCs)(m5dCs)(m5dCs)gscsususc A-145592 CUGGUCAGGGAAGGCGGCUG 107 1088-1107 csusgsgsus(m5dCs)dAsdGsdGsdGsdAsdAsdGsd 292 Gs(m5dCs)gsgscsusg A-145593 AGUGGGCCGTTCCTGGUCAG 108 1100-1119 asgsusgsgsdGs(m5dCs)(m5dCs)dGsdTsdTs 293 (m5dCs)(m5dCs)dTsdGsgsuscsasg A-145594 CCCGCAGCTCAGTGGGCCGU 109 1110-1129 cscscsgscsdAsdGs(m5dCs)dTs(m5dCs)dAsdGs 294 dTsdGsdGsgscscsgsu A-145595 UGCGGAGCCGCTGCCCGCAG 110 1123-1142 usgscsgsgsdAsdGs(m5dCs)(m5dCs)dGs 295 (m5dCs)dTsdGs(m5dCs)(m5dCs)csgscsasg A-145596 ACAGACTCTTGCGGAGCCGC 111 1132-1151 ascsasgsas(m5dCs)dTs(m5dCs)dTsdTsdGs 296 (m5dCs)dGsdGsdAsgscscsgsc A-145597 GGGUCATCGAAGACAGACUC 112 1144-1163 gsgsgsuscsdAsdTs(m5dCs)dGsdAsdAsdGsdAs 297 (m5dCs)dAsgsascsusc A-145598 CCAACGACGCGGGTCAUCGA 113 1154-1173 cscsasascsdGsdAs(m5dCs)dGs(m5dCs)dGsdGs 298 dGsdTs(m5dCs)asuscsgsa A-145599 GCCACCAGCCCGCCAACGAC 114 1166-1185 gscscsascs(m5dCs)dAsdGs(m5dCs)(m5dCs) 299 (m5dCs)dGs(m5dCs)(m5dCs)dAsascsgsasc A-145600 GCCCCGCGTAGCGCCACCAG 115 1178-1197 gscscscscsdGs(m5dCs)dGsdTsdAsdGs(m5dCs) 300 dGs(m5dCs)(m5dCs)ascscsasg A-145601 UAGGGGTGCGCCCCGCGUAG 116 1187-1206 usasgsgsgsdGsdTsdGs(m5dCs)dGs(m5dCs) 301 (m5dCs)(m5dCs)(m5dCs)dGscsgsusasg A-145602 AGCGCGGCGATGTAGGGGUG 117 1199-1218 asgscsgscsdGsdGs(m5dCs)dGsdAsdTsdGsdTsd 302 AsdGsgsgsgsusg A-145603 GGCCCCAGTACAGCGCGGCG 118 1210-1229 gsgscscscs(m5dCs)dAsdGsdTsdAs(m5dCs)dAs 303 dGs(m5dCs)dGscsgsgscsg A-145604 GCAGAAACTGTGGCCCCAGU 119 1221-1240 gscsasgsasdAsdAs(m5dCs)dTsdGsdTsdGsdGs 304 (m5dCs)(m5dCs)cscsasgsu A-145605 GAGGCTGCCGGCGCAGAAAC 120 1233-1252 gsasgsgscsdTsdGs(m5dCs)(m5dCs)dGsdGs 305 (m5dCs)dGs(m5dCs)dAsgsasasasc A-145606 GGGGGCGATGAGGCTGCCGG 121 1242-1261 gsgsgsgsgs(m5dCs)dGsdAsdTsdGsdAsdGsdGs 306 (m5dCs)dTsgscscsgsg A-145607 AGCACCCAGCAGGGGGCGAU 122 1253-1272 asgscsascs(m5dCs)(m5dCs)dAsdGs(m5dCs)dA 307 sdGsdGsdGsdGsgscsgsasu A-145608 GAGCGGCCGTCAGCACCCAG 123 1264-1283 gsasgscsgsdGs(m5dCs)(m5dCs)dGsdTs 308 (m5dCs)dAsdGs(m5dCs)dAscscscsasg A-145609 CCUGCAGGCAGTGAGCGGCC 124 1276-1295 cscsusgscsdAsdGsdGs(m5dCs)dAsdGsdTsdGsd 309 AsdGscsgsgscsc A-145610 GUGCGGGCCGGTCCTGCAGG 125 1288-1307 gsusgscsgsdGsdGs(m5dCs)(m5dCs)dGsdGsdTs 310 (m5dCs)(m5dCs)dTsgscsasgsg A-145611 AGAUCCTCGGGTGCGGGCCG 126 1298-1317 asgsasuscs(m5dCs)dTs(m5dCs)dGsdGsdGsdTs 311 dGs(m5dCs)dGsgsgscscsg A-145612 CACCACCGTCAGATCCUCGG 127 1308-1327 csascscsas(m5dCs)(m5dCs)dGsdTs(m5dCs)dA 312 sdGsdAsdTs(m5dCs)csuscsgsg A-145613 GUUCCTGGCCGAGCACCACC 128 1321-1340 gsususcscsdTsdGsdGs(m5dCs)(m5dCs)dGsdAs 313 dGs(m5dCs)dAscscsascsc A-145614 UGGUUACGGCGTTCCUGGCC 129 1331-1350 usgsgsususdAs(m5dCs)dGsdGs(m5dCs)dGsdTs 314 dTs(m5dCs)(m5dCs)usgsgscsc A-145615 GCUCACAGCTGTGGTUACGG 130 1342-1361 gscsuscsas(m5dCs)dAsdGs(m5dCs)dTsdGsdTs 315 dGsdGsdTsusascsgsg A-145616 ACGUCTGGCACGGCTCACAG 131 1354-1373 ascsgsuscsdTsdGsdGs(m5dCs)dAs(m5dCs)dGs 316 dGs(m5dCs)dTscsascsasg A-145617 GCACGGCCAACGTCTGGCAC 132 1363-1382

gscsascsgsdGs(m5dCs)(m5dCs)dAsdAs 317 (m5dCs)dGsdTs(m5dCs)dTsgsgscsasc A-145618 AGCGGTAGGAGCGCACGGCC 133 1375-1394 asgscsgsgsdTsdAsdGsdGsdAsdGs(m5dCs)dGs 318 (m5dCs)dAscsgsgscsc A-145619 AGGCCTCGTGCAAGCGGUAG 134 1387-1406 asgsgscscsdTs(m5dCs)dGsdTsdGs(m5dCs)dAs 319 dAsdGs(m5dCs)gsgsusasg A-145620 ACGGGCGAGAAGGCCUCGUG 135 1397-1416 ascsgsgsgs(m5dCs)dGsdAsdGsdAsdAsdGsdGs 320 (m5dCs)(m5dCs)uscsgsusg A-145621 GCUGGTAGCTGACGGGCGAG 136 1408-1427 gscsusgsgsdTsdAsdGs(m5dCs)dTsdGsdAs 321 (m5dCs)dGsdGsgscsgsasg A-145622 AGCCAGGTCGTGCTGGUAGC 137 1419-1438 asgscscsasdGsdGsdTs(m5dCs)dGsdTsdGs 322 (m5dCs)dTsdGsgsusasgsc A-145623 AGGCGCAACAGAGCCAGGUC 138 1430-1449 asgsgscsgs(m5dCs)dAsdAs(m5dCs)dAsdGsdAs 323 dGs(m5dCs)(m5dCs)asgsgsusc A-145624 CAUCCTCCTGAAGGCGCAAC 139 1441-1460 csasuscscsdTs(m5dCs)(m5dCs)dTsdGsdAsdAs 324 dGsdGs(m5dCs)gscsasasc A-145625 AGCUGCCGTCCGCATCCUCC 140 1453-1472 asgscsusgs(m5dCs)(m5dCs)dGsdTs(m5dCs) 325 (m5dCs)dGs(m5dCs)dAsdTscscsuscsc A-145626 AGGAGCGCGCAGCTGCCGUC 141 1463-1482 asgsgsasgs(m5dCs)dGs(m5dCs)dGs(m5dCs)dA 326 sdGs(m5dCs)dTsdGscscsgsusc A-145627 ACGUAAGGCGACAGGAGCGC 142 1475-1494 ascsgsusasdAsdGsdGs(m5dCs)dGsdAs(m5dCs) 327 dAsdGsdGsasgscsgsc A-145628 ACACCGGCTGAACGTAAGGC 143 1486-1505 ascsascscsdGsdGs(m5dCs)dTsdGsdAsdAs 328 (m5dCs)dGsdTsasasgsgsc A-145629 CUUGGCAGGCACACCGGCUG 144 1496-1515 csususgsgs(m5dCs)dAsdGsdGs(m5dCs)dAs 329 (m5dCs)dAs(m5dCs)(m5dCs)gsgscsusg A-145630 CGCGCGGCGCCGCTTGGCAG 145 1508-1527 csgscsgscsdGsdGs(m5dCs)dGs(m5dCs) 330 (m5dCs)dGs(m5dCs)dTsdTsgsgscsasg A-145631 UCGGAGGGTCGCGCGGCGCC 146 1517-1536 uscsgsgsasdGsdGsdGsdTs(m5dCs)dGs(m5dCs) 331 dGs(m5dCs)dGsgscsgscsc A-145632 AGAGCGTGGTCTCGGAGGGU 147 1528-1547 asgsasgscsdGsdTsdGsdGsdTs(m5dCs)dTs 332 (m5dCs)dGsdGsasgsgsgsu A-145633 CCGGCCACCTGGCAGAGCGU 148 1541-1560 cscsgsgscs(m5dCs)dAs(m5dCs)(m5dCs)dTsdG 333 sdGs(m5dCs)dAsdGsasgscsgsu A-145634 UGGCCCCAGCCGGCCACCUG 149 1550-1569 usgsgscscs(m5dCs)(m5dCs)dAsdGs(m5dCs) 334 (m5dCs)dGsdGs(m5dCs)(m5dCs)ascscsusg A-145635 CCUCGAACTGGTGGCCCCAG 150 1561-1580 cscsuscsgsdAsdAs(m5dCs)dTsdGsdGsdTsdGsd 335 Gs(m5dCs)cscscsasg A-145636 AUUCCTCCGCCCCCTCGAAC 151 1573-1592 asususcscsdTs(m5dCs)(m5dCs)dGs(m5dCs) 336 (m5dCs)(m5dCs)(m5dCs)(m5dCs)dTscsgsasa sc A-145637 AAGCUGGCATATTCCUCCGC 152 1583-1602 asasgscsusdGsdGs(m5dCs)dAsdTsdAsdTsdTs 337 (m5dCs)(m5dCs)uscscsgsc A-145638 CGCCUCCTGCAGGAAGCUGG 153 1596-1615 csgscscsus(m5dCs)(m5dCs)dTsdGs(m5dCs)dA 338 sdGsdGsdAsdAsgscsusgsg A-145639 AACGGTACCTGCGCCUCCUG 154 1607-1626 asascsgsgsdTsdAs(m5dCs)(m5dCs)dTsdGs 339 (m5dCs)dGs(m5dCs)(m5dCs)uscscsusg A-145640 AGGGAGAGGAACGGTACCUG 155 1616-1635 asgsgsgsasdGsdAsdGsdGsdAsdAs(m5dCs)dGsd 340 GsdTsascscsusg A-145641 AGCAGCGCTCCAGGGAGAGG 156 1627-1646 asgscsasgs(m5dCs)dGs(m5dCs)dTs(m5dCs) 341 (m5dCs)dAsdGsdGsdGsasgsasgsg A-145642 CGUCCGGGGCTGAGCAGCGC 157 1639-1658 csgsuscscsdGsdGsdGsdGs(m5dCs)dTsdGsdAsd 342 Gs(m5dCs)asgscsgsc A-145643 AGGAUCCGTGCACGTCCGGG 158 1651-1670 asgsgsasus(m5dCs)(m5dCs)dGsdTsdGs 343 (m5dCs)dAs(m5dCs)dGsdTscscsgsgsg A-145644 GGGGAGGATGGAGGAUCCGU 159 1662-1681 gsgsgsgsasdGsdGsdAsdTsdGsdGsdAsdGsdGsdA 344 suscscsgsu A-145645 GAGCATGCCGGGGAGGAUGG 160 1671-1690 gsasgscsasdTsdGs(m5dCs)(m5dCs)dGsdGsdGs 345 dGsdAsdGsgsasusgsg A-145646 GAACCCTGCGCAGAGCAUGC 161 1683-1702 gsasascscs(m5dCs)dTsdGs(m5dCs)dGs 346 (m5dCs)dAsdGsdAsdGscsasusgsc A-145647 GCCGCCCTCGAGGAACCCUG 162 1695-1714 gscscsgscs(m5dCs)(m5dCs)dTs(m5dCs)dGsdA 347 sdGsdGsdAsdAscscscsusg A-145648 ACGCATCGGTGCCGCCCUCG 163 1705-1724 ascsgscsasdTs(m5dCs)dGsdGsdTsdGs(m5dCs) 348 (m5dCs)dGs(m5dCs)cscsuscsg A-145649 AAUCACCCTGGCACGCAUCG 164 1717-1736 asasuscsas(m5dCs)(m5dCs)(m5dCs)dTsdGsdG 349 s(m5dCs)dAs(m5dCs)dGscsasuscsg A-145650 GGGCCTCCGGAATCACCCUG 165 1727-1746 gsgsgscscsdTs(m5dCs)(m5dCs)dGsdGsdAsdAs 350 dTs(m5dCs)dAscscscsusg A-145651 ACACACCAGCGGGCCUCCGG 166 1737-1756 ascsascsas(m5dCs)(m5dCs)dAsdGs(m5dCs)dG 351 sdGsdGs(m5dCs)(m5dCs)uscscsgsg A-145652 AGCUUGGTCCTCACACACCA 167 1749-1768 asgscsususdGsdGsdTs(m5dCs)(m5dCs)dTs 352 (m5dCs)dAs(m5dCs)dAscsascscsa A-145653 CCGGCGCTCTGCAGCUUGGU 168 1761-1780 cscsgsgscsdGs(m5dCs)dTs(m5dCs)dTsdGs 353 (m5dCs)dAsdGs(m5dCs)ususgsgsu A-145654 CAGGGTGAGCCGGCGCUCUG 169 1770-1789 csasgsgsgsdTsdGsdAsdGs(m5dCs)(m5dCs)dGs 354 dGs(m5dCs)dGscsuscsusg A-145655 AUGAUGCCTTGCAGGGUGAG 170 1781-1800 asusgsasusdGs(m5dCs)(m5dCs)dTsdTsdGs 355 (m5dCs)dAsdGsdGsgsusgsasg A-145656 AUCCCCAGCTGATGAUGCCU 171 1792-1811 asuscscscs(m5dCs)dAsdGs(m5dCs)dTsdGsdAs 356 dTsdGsdAsusgscscsu A-145657 ACCACAGCCCGATCCCCAGC 172 1803-1822 ascscsascsdAsdGs(m5dCs)(m5dCs)(m5dCs)dG 357 sdAsdTs(m5dCs)(m5dCs)cscsasgsc A-145658 GCUUGTTGCGGTCACCACAG 173 1816-1835 gscsususgsdTsdTsdGs(m5dCs)dGsdGsdTs 358 (m5dCs)dAs(m5dCs)csascsasg A-145659 GUAGACGCCTGGCTTGUUGC 174 1827-1846 gsusasgsas(m5dCs)dGs(m5dCs)(m5dCs)dTsdG 359 sdGs(m5dCs)dTsdTsgsususgsc A-145660 CACAUCGGTGTAGACGCCUG 175 1836-1855 csascsasus(m5dCs)dGsdGsdTsdGsdTsdAsdGsd 360 As(m5dCs)gscscsusg A-145661 CCAGGTAGTAGGCCACAUCG 176 1849-1868 cscsasgsgsdTsdAsdGsdTsdAsdGsdGs(m5dCs) 361 (m5dCs)dAscsasuscsg A-145662 GGAUCCAGGCCAGGTAGUAG 177 1858-1877 gsgsasuscs(m5dCs)dAsdGsdGs(m5dCs) 362 (m5dCs)dAsdGsdGsdTsasgsusasg A-145663 GGUGUGCTCCCGGATCCAGG 178 1869-1888 gsgsusgsusdGs(m5dCs)dTs(m5dCs)(m5dCs) 363 (m5dCs)dGsdGsdAsdTscscsasgsg A-145664 AAUCAGGAAACGGTGUGCUC 179 1880-1899 asasuscsasdGsdGsdAsdAsdAs(m5dCs)dGsdGsd 364 TsdGsusgscsusc A-145665 AGUCCCTGAGCAATCAGGAA 180 1891-1910 asgsuscscs(m5dCs)dTsdGsdAsdGs(m5dCs)dAs 365 dAsdTs(m5dCs)asgsgsasa A-145666 AGGGAAAGATGAGTCCCUGA 181 1902-1921 asgsgsgsasdAsdAsdGsdAsdTsdGsdAsdGsdTs 366 (m5dCs)cscsusgsa A-145667 GAAUCACCAAGGAGGGAAAG 182 1914-1933 gsasasuscsdAs(m5dCs)(m5dCs)dAsdAsdGsdGs 367 dAsdGsdGsgsasasasg A-145668 UCUCACTGCGGAATCACCAA 183 1924-1943 uscsuscsas(m5dCs)dTsdGs(m5dCs)dGsdGsdAs 368 dAsdTs(m5dCs)ascscsasa A-145669 CCCCAGCCACTCTCTCACUG 184 1936-1955 cscscscsasdGs(m5dCs)(m5dCs)dAs(m5dCs)dT 369 s(m5dCs)dTs(m5dCs)dTscsascsusg A-145670 UUGCCTTCCATGCCCCAGCC 185 1948-1967 ususgscscsdTsdTs(m5dCs)(m5dCs)dAsdTsdGs 370 (m5dCs)(m5dCs)(m5dCs)csasgscsc A-145671 GACACAATCTTGCCTUCCAU 186 1957-1976 gsascsascsdAsdAsdTs(m5dCs)dTsdTsdGs 371 (m5dCs)(m5dCs)dTsuscscsasu A-145672 CUGGGGGAATGGGACACAAU 187 1969-1988 csusgsgsgsdGsdGsdAsdAsdTsdGsdGsdGsdAs 372 (m5dCs)ascsasasu A-145673 AGCUGGCCGCACTGGGGGAA 188 1980-1999 asgscsusgsdGs(m5dCs)(m5dCs)dGs(m5dCs)dA 373 s(m5dCs)dTsdGsdGsgsgsgsasa A-145674 AUCCUGGCGCGGAGCUGGCC 189 1992-2011 asuscscsusdGsdGs(m5dCs)dGs(m5dCs)dGsdGs 374 dAsdGs(m5dCs)usgsgscsc A-145675 GUUCCTGCGCCATCCUGGCG 190 2003-2022 gsususcscsdTsdGs(m5dCs)dGs(m5dCs) 375 (m5dCs)dAsdTs(m5dCs)(m5dCs)usgsgscsg A-145676 CACUUTATTGAGTTCCUGCG 191 2014-2033 csascsususdTsdAsdTsdTsdGsdAsdGsdTsdTs 376 (m5dCs)csusgscsg A-145677 AUUUUCAAAGCACTTUAUUG 192 2024-2043 asusususus(m5dCs)dAsdAsdAsdGs(m5dCs)dAs 377 (m5dCs)dTsdTsusasususg A-145678 UUUUUCTCAGCATTTUCAAA 193 2035-2054 ususususus(m5dCs)dTs(m5dCs)dAsdGs 378 (m5dCs)dAsdTsdTsdTsuscsasasa

TABLE-US-00004 TABLE 4 Factor XII single dose screen in primary human cells Oligo % Residual Target site on Name mRNA SD SEQ ID 1 A-145494 77% 7% 11-30 A-145495 54% 7% 21-40 A-145496 54% 6% 32-51 A-145497 56% 6% 44-63 A-145498 57% 7% 55-74 A-145499 65% 13% 65-84 A-145500 66% 3% 76-95 A-145501 57% 6% 87-106 A-145502 76% 8% 99-118 A-145503 71% 11% 109-128 A-145504 94% 14% 121-140 A-145505 73% 10% 133-152 A-145506 42% 4% 143-162 A-145507 54% 11% 155-174 A-145508 57% 8% 164-183 A-145509 66% 9% 175-194 A-145510 81% 7% 187-206 A-145511 65% 6% 199-218 A-145512 48% 3% 209-228 A-145513 58% 13% 220-239 A-145514 66% 13% 231-250 A-145515 69% 9% 242-261 A-145516 53% 13% 254-273 A-145517 66% 11% 265-284 A-145518 66% 13% 274-293 A-145519 31% 5% 287-306 A-145520 32% 6% 296-315 A-145521 69% 5% 307-326 A-145522 52% 4% 320-339 A-145523 45% 6% 331-350 A-145524 63% 11% 340-359 A-145525 52% 5% 352-371 A-145526 56% 6% 362-381 A-145527 28% 4% 373-392 A-145528 60% 15% 385-404 A-145529 53% 6% 395-414 A-145530 69% 13% 406-425 A-145531 60% 6% 418-437 A-145532 37% 7% 430-449 A-145533 40% 3% 439-458 A-145534 51% 3% 451-470 A-145535 70% 11% 463-482 A-145536 48% 5% 472-491 A-145537 38% 9% 483-502 A-145538 31% 2% 494-513 A-145539 27% 5% 507-526 A-145540 33% 4% 517-536 A-145541 36% 5% 528-547 A-145542 36% 5% 538-557 A-145543 46% 4% 549-568 A-145544 96% 11% 562-581 A-145545 104% 3% 571-590 A-145546 70% 5% 583-602 A-145547 33% 11% 595-614 A-145548 28% 4% 605-624 A-145549 28% 3% 616-635 A-145550 33% 5% 626-645 A-145551 33% 4% 638-657 A-145552 45% 8% 650-669 A-145553 59% 5% 661-680 A-145554 71% 9% 670-689 A-145555 36% 4% 682-701 A-145556 30% 4% 692-711 A-145557 46% 11% 703-722 A-145558 68% 15% 716-735 A-145559 64% 11% 726-745 A-145560 57% 5% 736-755 A-145561 41% 8% 748-767 A-145562 50% 6% 759-778 A-145563 58% 12% 769-788 A-145564 53% 3% 782-801 A-145565 35% 4% 793-812 A-145566 27% 6% 804-823 A-145567 22% 4% 814-833 A-145568 34% 9% 824-843 A-145569 40% 10% 836-855 A-145570 29% 9% 847-866 A-145571 36% 11% 859-878 A-145572 20% 2% 869-888 A-145573 28% 5% 880-899 A-145574 61% 10% 890-909 A-145575 34% 11% 901-920 A-145576 49% 4% 913-932 A-145577 50% 7% 923-942 A-145578 61% 7% 935-954 A-145579 71% 16% 947-966 A-145580 62% 10% 957-976 A-145581 58% 8% 968-987 A-145582 67% 5% 980-999 A-145583 48% 8% 990-1009 A-145584 35% 4% 1001-1020 A-145585 43% 4% 1011-1030 A-145586 39% 3% 1023-1042 A-145587 59% 12% 1033-1052 A-145588 50% 4% 1045-1064 A-145589 52% 12% 1055-1074 A-145590 57% 5% 1067-1086 A-145591 56% 8% 1078-1097 A-145592 70% 19% 1088-1107 A-145593 45% 9% 1100-1119 A-145594 32% 3% 1110-1129 A-145595 43% 8% 1123-1142 A-145596 36% 5% 1132-1151 A-145597 29% 3% 1144-1163 A-145598 45% 5% 1154-1173 A-145599 36% 6% 1166-1185 A-145600 29% 3% 1178-1197 A-145601 51% 8% 1187-1206 A-145602 69% 10% 1199-1218 A-145603 37% 5% 1210-1229 A-145604 41% 15% 1221-1240 A-145605 53% 16% 1233-1252 A-145606 68% 18% 1242-1261 A-145607 52% 11% 1253-1272 A-145608 66% 21% 1264-1283 A-145609 69% 25% 1276-1295 A-145610 64% 14% 1288-1307 A-145611 57% 10% 1298-1317 A-145612 38% 11% 1308-1327 A-145613 44% 5% 1321-1340 A-145614 54% 11% 1331-1350 A-145615 44% 7% 1342-1361 A-145616 35% 4% 1354-1373 A-145617 36% 7% 1363-1382 A-145618 43% 8% 1375-1394 A-145619 31% 3% 1387-1406 A-145620 64% 27% 1397-1416 A-145621 75% 12% 1408-1427 A-145622 71% 27% 1419-1438 A-145623 49% 8% 1430-1449 A-145624 47% 6% 1441-1460 A-145625 48% 11% 1453-1472 A-145626 49% 12% 1463-1482 A-145627 57% 14% 1475-1494 A-145628 56% 9% 1486-1505 A-145629 47% 10% 1496-1515 A-145630 64% 10% 1508-1527 A-145631 60% 11% 1517-1536 A-145632 62% 6% 1528-1547 A-145633 45% 14% 1541-1560 A-145634 61% 9% 1550-1569 A-145635 66% 17% 1561-1580 A-145636 79% 12% 1573-1592 A-145637 55% 8% 1583-1602 A-145638 64% 7% 1596-1615 A-145639 65% 3% 1607-1626 A-145640 78% 10% 1616-1635 A-145641 92% 9% 1627-1646 A-145642 67% 8% 1639-1658 A-145643 61% 9% 1651-1670 A-145644 62% 10% 1662-1681 A-145645 83% 13% 1671-1690 A-145646 47% 3% 1683-1702 A-145647 56% 10% 1695-1714 A-145648 86% 9% 1705-1724 A-145649 79% 9% 1717-1736 A-145650 50% 5% 1727-1746 A-145651 38% 3% 1737-1756 A-145652 57% 9% 1749-1768 A-145653 69% 9% 1761-1780 A-145654 80% 34% 1770-1789 A-145655 36% 6% 1781-1800 A-145656 30% 3% 1792-1811 A-145657 33% 4% 1803-1822 A-145658 29% 6% 1816-1835 A-145659 30% 7% 1827-1846 A-145660 25% 4% 1836-1855 A-145661 28% 4% 1849-1868 A-145662 51% 7% 1858-1877 A-145663 36% 3% 1869-1888 A-145664 42% 3% 1880-1899 A-145665 37% 6% 1891-1910 A-145666 22% 5% 1902-1921 A-145667 19% 5% 1914-1933 A-145668 15% 6% 1924-1943 A-145669 13% 3% 1936-1955 A-145670 23% 8% 1948-1967 A-145671 30% 5% 1957-1976 A-145672 57% 14% 1969-1988 A-145673 66% 18% 1980-1999 A-145674 24% 4% 1992-2011 A-145675 30% 6% 2003-2022 A-145676 25% 3% 2014-2033 A-145677 44% 9% 2024-2043 A-145678 70% 14% 2035-2054

Sequence CWU 1

1

37812060DNAHomo sapiens 1ctattgatct ggactcctgg ataggcagct ggaccaacgg acggatgcca tgagggctct 60gctgctcctg gggttcctgc tggtgagctt ggagtcaaca ctttcgattc caccttggga 120agcccccaag gagcataagt acaaagctga agagcacaca gtcgttctca ctgtcaccgg 180ggagccctgc cacttcccct tccagtacca ccggcagctg taccacaaat gtacccacaa 240gggccggcca ggccctcagc cctggtgtgc taccaccccc aactttgatc aggaccagcg 300atggggatac tgtttggagc ccaagaaagt gaaagaccac tgcagcaaac acagcccctg 360ccagaaagga gggacctgtg tgaacatgcc aagcggcccc cactgtctct gtccacaaca 420cctcactgga aaccactgcc agaaagagaa gtgctttgag cctcagcttc tccggttttt 480ccacaagaat gagatatggt atagaactga gcaagcagct gtggccagat gccagtgcaa 540gggtcctgat gcccactgcc agcggctggc cagccaggcc tgccgcacca acccgtgcct 600ccatgggggt cgctgcctag aggtggaggg ccaccgcctg tgccactgcc cggtgggcta 660caccggagcc ttctgcgacg tggacaccaa ggcaagctgc tatgatggcc gcgggctcag 720ctaccgcggc ctggccagga ccacgctctc gggtgcgccc tgtcagccgt gggcctcgga 780ggccacctac cggaacgtga ctgccgagca agcgcggaac tggggactgg gcggccacgc 840cttctgccgg aacccggaca acgacatccg cccgtggtgc ttcgtgctga accgcgaccg 900gctgagctgg gagtactgcg acctggcaca gtgccagacc ccaacccagg cggcgcctcc 960gaccccggtg tcccctaggc ttcatgtccc actcatgccc gcgcagccgg caccgccgaa 1020gcctcagccc acgacccgga ccccgcctca gtcccagacc ccgggagcct tgccggcgaa 1080gcgggagcag ccgccttccc tgaccaggaa cggcccactg agctgcgggc agcggctccg 1140caagagtctg tcttcgatga cccgcgtcgt tggcgggctg gtggcgctac gcggggcgca 1200cccctacatc gccgcgctgt actggggcca cagtttctgc gccggcagcc tcatcgcccc 1260ctgctgggtg ctgacggccg ctcactgcct gcaggaccgg cccgcacccg aggatctgac 1320ggtggtgctc ggccaggaac gccgtaacca cagctgtgag ccgtgccaga cgttggccgt 1380gcgctcctac cgcttgcacg aggccttctc gcccgtcagc taccagcacg acctggctct 1440gttgcgcctt caggaggatg cggacggcag ctgcgcgctc ctgtcgcctt acgttcagcc 1500ggtgtgcctg ccaagcggcg ccgcgcgacc ctccgagacc acgctctgcc aggtggccgg 1560ctggggccac cagttcgagg gggcggagga atatgccagc ttcctgcagg aggcgcaggt 1620accgttcctc tccctggagc gctgctcagc cccggacgtg cacggatcct ccatcctccc 1680cggcatgctc tgcgcagggt tcctcgaggg cggcaccgat gcgtgccagg gtgattccgg 1740aggcccgctg gtgtgtgagg accaagctgc agagcgccgg ctcaccctgc aaggcatcat 1800cagctgggga tcgggctgtg gtgaccgcaa caagccaggc gtctacaccg atgtggccta 1860ctacctggcc tggatccggg agcacaccgt ttcctgattg ctcagggact catctttccc 1920tccttggtga ttccgcagtg agagagtggc tggggcatgg aaggcaagat tgtgtcccat 1980tcccccagtg cggccagctc cgcgccagga tggcgcagga actcaataaa gtgctttgaa 2040aatgctgaga aaaaaaaaaa 206022198DNAMacaca fascicularis 2ttcctgactc aagtccagag cttccctacc ttgcttctaa ggtgaccaag gaagtcagtc 60cacttggctt tccataaaca gcctgtgccc caccaggctc aggggggcag cttgaccaat 120ccctatttcc aagacctttg gccaacccta ttgatctgga ctcctgggca gacagttgga 180ccaacggaca gacgccatga gggctctgct gctcatgggg ttcctgctgg tgagcctgga 240gtcaacactt tcgattccac cttggaaagc gcccaaggag cataagtaca aagctgaaga 300gcacacagtc gttctcactg tcaccgggga gccctgccac ttccccttcc agtaccaccg 360gcggctgtac cacaaatgta cccacaaggg ccggccaggc cctcagacct ggtgtgctac 420cacccctaac tttgatgagg accagcgctg gggatactgt gtggagccca agaaagtgaa 480agaccactgc agtaaacaca gcccctgcca gaaagggggg acctgtgtga acacgctgag 540tggcacccac tgtctctgtc cacaacacct cactgggaac cactgccaga gagagaagtg 600ctttgagcct cagcttctcc ggtttttcca cgagaatgag atatggtata gatctgagca 660agcagctgtg gccagatgcc agtgcaaggg tcctgatgcc cactgccagc ggctggccag 720ccaggcctgc cgcaccaacc cgtgcctcca tgggggtcgc tgcctagagg tggagggcca 780ccgcctgtgc cactgccccg tgggctacac cggacccttc tgcgacttgg acaccaaggc 840gagctgctat gatggccgcg ggctcagcta ccgcggcctg gccaggacca cgctctcggg 900tgcgccctgt cagccgtgga cctcggaggc cacctactgg aacgtgacgg ccgagcaagc 960gcggaactgg ggactgggcg gccacgcctt ctgccggaac ccggacaacg acatccgccc 1020gtggtgcttc gtgctgatcg gcgacaggct aagctgggag tactgcgacg tggcacagtg 1080ccaggcccca acccaggcgg cgcctccgac gccggtgtcc cctgggcttc atgtcccact 1140catgcccccg cagccggcac cgccgaagct tcagcccacg acccggaccc cgcctcagtc 1200ccagaccccg ggagccttgc cagtgaagcg ggagcagccg cctcgcctga cccggaacgg 1260ctcagtgagc tgcgggcagc ggctccgcaa gagtctgtct tcgatgaccc gcgtcgttgg 1320cgggctggtg gcgctacgcg gggcgcaccc ctacatcgcc gcgctgtact ggggccacag 1380tttctgcgcc ggcagcctca tcgccccctg ctgggtgctg acggccgctc actgcctgca 1440ggaccggccg gcacccgagg atctgacggt agtactcggc caggaacgcc ataaccacag 1500ctgtgaacag tgccagactc tggccgtgcg ctcctaccgc ttgcacgagg ccttctcgcc 1560cgacagctac cagcacgacc tggctctgtt gcgccttcag gaggatgcgg acggcagctg 1620cgcgctcctg tcgccttacg ttcagccggt gtgcctgcca agcggcgccg cgcgaccctc 1680cgagcccgcg ctctgccagg tggctggctg gggccaccag ttcgaggggg cggaggaata 1740ttccagcttc ctgcaggagg cgcaggtacc cttcctctcc ctggagagct gctcagcacc 1800ggaggtgcac ggagcctcca tcctccccgg catgctctgc gcagggttcc tcgagggcgg 1860caccgatgca tgccagggtg attccggagg cccgctggtg tgtgaggacc aagccgcaga 1920gcgccggctc accctgcaag gcatcatcag ctggggatcg ggctgtggtg accgcaacaa 1980gccaggcgtc tacaccgatg tggcctacta cctggcctgg atccgggagc acaccgcttc 2040ctgattgctc agggactcat ctttccctcc tcggtgattc cgcagtggga gactggctgg 2100ggcatggaag gcaaaattgt gtcccattcc cccaatgcgg ccagctccgc gccaggatgg 2160cgcaggaact caataaagtg ctttgaaaat gctgagaa 219831971DNAMus musculus 3actcctgggc aggcagcggg gccatcggca gacgccatga cggctctgtt gttcctgggg 60tctctgctga tgagtctgga tctgacactt tcggctccac catggaaaga ctccaagaaa 120tttaaggacg cacctgatgg gcccacagtg gttctcactg tggatgggag gctctgccat 180tttccctttc agtaccaccg tcagctacac cacaaatgca tccacaaaag gcggccaggc 240tcccgcccct ggtgtgctac cacccccaac tttgatgaag atcagcaatg gggatactgc 300ttggagccca agaaagtgaa agaccattgc agcaaacaca acccgtgcca caaaggaggg 360acatgtatca acacccccaa tgggccacac tgtctctgcc ctgaacacct cactgggaaa 420cattgccaga aagagaaatg ctttgagcct cagcttctca agttcttcca cgagaatgag 480ctatggttta gaacggggcc aggaggtgtg gccaggtgcg agtgcaaagg ttctgaggct 540cactgcaagc cggtggccag ccaggcctgc agcatcaatc cgtgccttaa tgggggcagc 600tgcctcctcg tggaggacca cccactgtgc cgttgcccta caggctacac tggatatttt 660tgcgacttgg acctttgggc gacctgctat gaaggcaggg ggctcagcta ccggggccag 720gctggaacta cgcaatcggg tgcgccatgt cagcggtgga ccgtggaggc cacctaccgg 780aacatgactg agaagcaagc gctaagctgg ggcctgggcc accacgcatt ttgccggaac 840ccagataatg acacacgtcc atggtgcttc gtctggagtg gcgacaggct gagctgggac 900tattgcggcc tggagcagtg ccagacgcca acgtttgcac ctctagttgt ccctgagagt 960caggaggagt ccccgtccca ggcaccatct ctgtcccatg caccaaatga ctcgaccgat 1020catcagactt ctctgtccaa gaccaacacg atgggctgcg gacagaggtt ccgcaaggga 1080ctgtcctcgt tcatgcgcgt ggtgggcgga ctagtggctc tgcctgggtc gcacccctac 1140atcgctgcac tgtactgggg taacaacttc tgcgcgggca gtctcatcgc cccctgttgg 1200gtgctgaccg cggctcactg cctgcagaat cggccagcgc ccgaggaact gacagtggta 1260cttggtcaag atcgccacaa ccagagctgc gagtggtgcc agactctggc tgtgcgctcc 1320taccgccttc acgagggctt ctcctccatc acctaccagc acgacttggc tctgctgcgc 1380ctgcaggaaa gcaaaaccaa cagttgcgcg atcctgtcac ctcacgttca gcctgtgtgt 1440ctacccagcg gcgcggcccc accctctgag acagtgctct gcgaggtggc cggctggggt 1500caccagttcg agggggctga agaatactcc accttcctgc aggaggcaca ggttcccttt 1560atcgccctgg atcgctgctc caactctaac gtgcacggag acgccattct ccctgggatg 1620ctttgcgctg gcttcttgga gggaggcacc gatgcctgcc agggtgactc cgggggccct 1680ctggtgtgtg aggaaggaac tgcagaacat cagctcaccc tgcgcggagt catcagctgg 1740ggctccggct gtggtgaccg caacaagccc ggagtctaca cagacgtggc caactacctg 1800gcttggatcc agaagcatat tgcttcataa ctaaccaggc tttatccttc cctccttgtg 1860tgctccttgg gatgggacga tgaatgtggc atgctgggtc acagtgaagc tagtgccccg 1920acactggggg cacagaaact caataaagtg ctttgaaaac gttcctcaga a 197142128DNARattus norvegicus 4caggaaccca tctcggtact ctgcttccac cagccttgcc ctgctcacga gggttcgacg 60gcgctgttct tctgggcttt ctgtgaagtc tgtgctcctc aggcccagga gggagcttaa 120ccaatctcca cctctgaggt ttctgagacc tttgcccaca tctattgatc cttactctgg 180ggcaggcagc tgggccattg gcggacgcca tgacggctct gttgttcctg gggtctctgc 240tgatgagtct ggacttgaca ctttcggcgc caccgtggaa gtccaaggag ttcaaggacg 300gagctggcga tccctctgtg gttctcactg tggacgggaa gctctgccac tttccctttc 360agtaccaccg tcgcctgtac cacaaatgca tccacaaagg acagccaggc tccaggccct 420ggtgtgctac cacccccaac tttgacgagg accagcaatg gggatactgc ttggagccca 480agaaagtgaa agaccattgc agcaaacaca gcccctgcca caaaggaggg acgtgtgtca 540acacccccaa cggcccgcac tgtctctgcc ctgaacacct caccgggaaa cattgccaga 600gagagaaatg ctttgagtct cagctcctca agttcttcca tgagaatgag atatggttta 660gaactgggcc aggaggtgtg gccaggtgcc agtgcaaagg tcctcaggct gtttgcaagc 720tgctgaccag tcaggtttgc agggtcaatc cgtgccttaa tggaggcacc tgcctcctcg 780tggaggacca ccgactgtgc cactgccctg caggctatgc cggacctttt tgcgacttag 840accttaaggc gacttgctac gaagacaggg gtctcagcta ccggggccag gctaaaacta 900ctctgtcggg tgcaccatgt cagcggtggg cctcggaggc cacctaccgg aacatgactg 960agacgcaagc tctaagctgg ggcctgggcc accacgcatt ctgccggaac ccagataatg 1020acacacgtcc atggtgctac gtctggagtg gcgacaggct gagctgggac tactgcgacc 1080tggaacagtg ccagatgcca acgctcacat ctccggtttc ccctgagagt cacgacatgc 1140tgaagccccg gcctcccata ttgcagatgc ctcagttccc gtctctgtcc gatgcactag 1200acaactcgac ccgtaatcag aatgttgtgt ccaggaccag tacggtggtc tgcggacaga 1260ggtttcgcaa gcgactgtcc tcgctcaggc gcgtggtggg cggactagtg gctctgcctg 1320gatcgcatcc ctacatcgct gcactgtact ggggcgacag cttctgcgca ggcagtctca 1380tcgacccctg ctgggtgctg accgctgctc actgcttgca gaaacggcca gcgcccgagg 1440aactgacagt ggtacttggt caagatcgcc ataaccagag ctgcgagagg tgccagactc 1500tggctgtgca ctcctaccgc cttcacgagg gcttctcttc caaaacctac cagcatgatt 1560tggctctgct gcgcctgcgg gggaggaaaa acagctgcgc gatcttgtcg cctcatgtcc 1620agccggtgtg tctgcccagc agcgcggccc caccctctga gacagtgctc tgcgaggtgg 1680ccggctgggg tcatcagttc gagggggctg aagaatacgc cacctttctg caggaggcac 1740aggtaccctt catctccctg gatcgctgct ccagctctaa cgtgcacgga gacgccatcc 1800tgcctgggat gctttgtgct ggcttcttgg agggaggcgc cgatgcctgt cagggtgact 1860ccgggggtcc tctggtatgt gatgaaggag ttacagagcg tcagctcacc ctgcgaggag 1920tcatcagctg gggctccggc tgtggtgacc ggaacaagcc cggggtctac actgacgtgg 1980ccaattacct ggattggatc caggagcata ctgctttcta agtaaccagg gtcggtcctt 2040gcgaagctag tggctgggcc ccagggacac agaaactcaa taaagtgctt tgaaaacgtt 2100aaaaaaaaaa aaaaaaaaaa aaaaaaaa 2128516PRTUnknownDescription of Unknown RFGF peptide 5Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15611PRTUnknownDescription of Unknown RFGF analogue peptide 6Ala Ala Leu Leu Pro Val Leu Leu Ala Ala Pro1 5 10713PRTHuman immunodeficiency virus 7Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln1 5 10816PRTDrosophila sp. 8Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10 15920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 9agcugcctat ccaggagucc 201020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 10ccguuggtcc agctgccuau 201120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 11auggcatccg tccgtugguc 201220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 12agcagagccc tcatggcauc 201320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 13accccaggag cagcagagcc 201420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 14accagcagga accccaggag 201520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 15acuccaagct caccagcagg 201620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 16cgaaagtgtt gactccaagc 201720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 17ccaaggtgga atcgaaagug 201820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 18ugggggcttc ccaaggugga 201920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 19acuuatgctc cttgggggcu 202020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 20cuucagcttt gtactuaugc 202120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 21acugugtgct cttcagcuuu 202220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 22acagugagaa cgactgugug 202320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 23uccccggtga cagtgagaac 202420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 24aguggcaggg ctccccggug 202520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 25acuggaaggg gaagtggcag 202620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 26gcugccggtg gtactggaag 202720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 27uuguggtaca gctgccggug 202820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 28ugugggtaca tttgtgguac 202920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 29uggccggccc ttgtggguac 203020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 30ggcugagggc ctggccggcc 203120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 31guagcacacc agggcugagg 203220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 32aguugggggt ggtagcacac 203320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 33ccugatcaaa gttgggggug 203420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 34ccccatcgct ggtccugauc 203520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 35aaacagtatc cccatcgcug 203620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 36ucuugggctc caaacaguau 203720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic

oligonucleotide 37ugguctttca ctttcuuggg 203820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 38guuugctgca gtggtcuuuc 203920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 39aggggctgtg tttgcugcag 204020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 40cuccuttctg gcaggggcug 204120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 41acacaggtcc ctcctuucug 204220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 42uuggcatgtt cacacagguc 204320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 43agugggggcc gcttggcaug 204420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 44ggacagagac agtgggggcc 204520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 45ugaggtgttg tggacagaga 204620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 46aguggtttcc agtgaggugu 204720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 47ucucuttctg gcagtgguuu 204820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 48caaagcactt ctcttucugg 204920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 49gaagctgagg ctcaaagcac 205020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 50ggaaaaaccg gagaagcuga 205120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 51cauucttgtg gaaaaaccgg 205220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 52auaccatatc tcattcuugu 205320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 53ugcucagttc tataccauau 205420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 54ggccacagct gcttgcucag 205520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 55acuggcatct ggccacagcu 205620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 56aggacccttg cactggcauc 205720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 57agugggcatc aggacccuug 205820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 58cagccgctgg cagtgggcau 205920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 59aggcctggct ggccagccgc 206020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 60uggugcggca ggcctggcug 206120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 61ggaggcacgg gttggugcgg 206220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 62agcgaccccc atggaggcac 206320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 63accuctaggc agcgaccccc 206420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 64gguggccctc cacctcuagg 206520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 65uggcacaggc ggtggcccuc 206620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 66cccaccgggc agtggcacag 206720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 67gcuccggtgt agcccaccgg 206820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 68cgucgcagaa ggctccggug 206920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 69uggugtccac gtcgcagaag 207020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 70agcagcttgc cttggugucc 207120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 71cggccatcat agcagcuugc 207220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 72agcugagccc gcggccauca 207320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 73gccaggccgc ggtagcugag 207420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 74cguggtcctg gccaggccgc 207520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 75cacccgagag cgtgguccug 207620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 76gcugacaggg cgcacccgag 207720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 77cgaggcccac ggctgacagg 207820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 78agguggcctc cgaggcccac 207920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 79gucacgttcc ggtagguggc 208020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 80cuugctcggc agtcacguuc 208120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 81ccagutccgc gcttgcucgg 208220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 82cgcccagtcc ccagtuccgc 208320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 83aaggcgtggc cgcccagucc 208420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 84ggguuccggc agaaggcgug 208520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 85ugucgttgtc cgggtuccgg 208620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 86accacgggcg gatgtcguug 208720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 87agcacgaagc accacgggcg 208820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 88ggucgcggtt cagcacgaag 208920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 89cagcucagcc ggtcgcgguu 209020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 90cgcagtactc ccagcucagc 209120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 91acugugccag gtcgcaguac 209220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 92gggguctggc actgtgccag 209320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 93gccgcctggg ttggggucug 209420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 94ggggucggag gcgccgccug 209520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 95aggggacacc ggggtcggag 209620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 96acaugaagcc taggggacac 209720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 97ggcaugagtg ggacaugaag 209820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 98cggcugcgcg ggcatgagug 209920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 99uucggcggtg ccggcugcgc 2010020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 100gggcugaggc ttcggcggug 2010120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 101gguccgggtc gtgggcugag 2010220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 102acugaggcgg ggtccggguc 2010320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 103ccggggtctg ggactgaggc 2010420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 104ggcaaggctc ccggggucug 2010520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 105ucccgcttcg ccggcaaggc 2010620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 106aaggcggctg ctcccgcuuc 2010720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 107cuggucaggg aaggcggcug 2010820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 108agugggccgt tcctggucag 2010920DNAArtificial SequenceDescription of Artificial Sequence Synthetic

oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 109cccgcagctc agtgggccgu 2011020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 110ugcggagccg ctgcccgcag 2011120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 111acagactctt gcggagccgc 2011220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 112gggucatcga agacagacuc 2011320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 113ccaacgacgc gggtcaucga 2011420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 114gccaccagcc cgccaacgac 2011520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 115gccccgcgta gcgccaccag 2011620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 116uaggggtgcg ccccgcguag 2011720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 117agcgcggcga tgtaggggug 2011820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 118ggccccagta cagcgcggcg 2011920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 119gcagaaactg tggccccagu 2012020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 120gaggctgccg gcgcagaaac 2012120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 121gggggcgatg aggctgccgg 2012220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 122agcacccagc agggggcgau 2012320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 123gagcggccgt cagcacccag 2012420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 124ccugcaggca gtgagcggcc 2012520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 125gugcgggccg gtcctgcagg 2012620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 126agaucctcgg gtgcgggccg 2012720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 127caccaccgtc agatccucgg 2012820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 128guucctggcc gagcaccacc 2012920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 129ugguuacggc gttccuggcc 2013020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 130gcucacagct gtggtuacgg 2013120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 131acguctggca cggctcacag 2013220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 132gcacggccaa cgtctggcac 2013320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 133agcggtagga gcgcacggcc 2013420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 134aggcctcgtg caagcgguag 2013520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 135acgggcgaga aggccucgug 2013620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 136gcuggtagct gacgggcgag 2013720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 137agccaggtcg tgctgguagc 2013820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 138aggcgcaaca gagccagguc 2013920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 139caucctcctg aaggcgcaac 2014020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 140agcugccgtc cgcatccucc 2014120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 141aggagcgcgc agctgccguc 2014220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 142acguaaggcg acaggagcgc 2014320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 143acaccggctg aacgtaaggc 2014420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 144cuuggcaggc acaccggcug 2014520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 145cgcgcggcgc cgcttggcag 2014620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 146ucggagggtc gcgcggcgcc 2014720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 147agagcgtggt ctcggagggu 2014820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 148ccggccacct ggcagagcgu 2014920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 149uggccccagc cggccaccug 2015020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 150ccucgaactg gtggccccag 2015120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 151auucctccgc cccctcgaac 2015220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 152aagcuggcat attccuccgc 2015320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 153cgccucctgc aggaagcugg 2015420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 154aacggtacct gcgccuccug 2015520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 155agggagagga acggtaccug 2015620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 156agcagcgctc cagggagagg 2015720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 157cguccggggc tgagcagcgc 2015820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 158aggauccgtg cacgtccggg 2015920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 159ggggaggatg gaggauccgu 2016020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 160gagcatgccg gggaggaugg 2016120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 161gaaccctgcg cagagcaugc 2016220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 162gccgccctcg aggaacccug 2016320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 163acgcatcggt gccgcccucg 2016420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 164aaucaccctg gcacgcaucg 2016520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 165gggcctccgg aatcacccug 2016620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 166acacaccagc gggccuccgg 2016720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 167agcuuggtcc tcacacacca 2016820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 168ccggcgctct gcagcuuggu 2016920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 169cagggtgagc cggcgcucug 2017020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 170augaugcctt gcagggugag 2017120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 171auccccagct gatgaugccu 2017220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 172accacagccc gatccccagc 2017320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 173gcuugttgcg gtcaccacag 2017420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 174guagacgcct ggcttguugc 2017520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 175cacaucggtg tagacgccug 2017620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 176ccaggtagta ggccacaucg 2017720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 177ggauccaggc caggtaguag 2017820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 178ggugugctcc cggatccagg 2017920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 179aaucaggaaa cggtgugcuc 2018020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 180aguccctgag caatcaggaa 2018120DNAArtificial

SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 181agggaaagat gagtcccuga 2018220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 182gaaucaccaa ggagggaaag 2018320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 183ucucactgcg gaatcaccaa 2018420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 184ccccagccac tctctcacug 2018520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 185uugccttcca tgccccagcc 2018620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 186gacacaatct tgcctuccau 2018720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 187cugggggaat gggacacaau 2018820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 188agcuggccgc actgggggaa 2018920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 189auccuggcgc ggagcuggcc 2019020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 190guucctgcgc catccuggcg 2019120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 191cacuutattg agttccugcg 2019220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 192auuuucaaag cacttuauug 2019320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 193uuuuuctcag catttucaaa 2019420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 194agcugcctat ccaggagucc 2019520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 195ccguuggtcc agctgccuau 2019620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 196auggcatccg tccgtugguc 2019720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 197agcagagccc tcatggcauc 2019820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 198accccaggag cagcagagcc 2019920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 199accagcagga accccaggag 2020020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 200acuccaagct caccagcagg 2020120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 201cgaaagtgtt gactccaagc 2020220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 202ccaaggtgga atcgaaagug 2020320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 203ugggggcttc ccaaggugga 2020420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 204acuuatgctc cttgggggcu 2020520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 205cuucagcttt gtactuaugc 2020620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 206acugugtgct cttcagcuuu 2020720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 207acagugagaa cgactgugug 2020820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 208uccccggtga cagtgagaac 2020920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 209aguggcaggg ctccccggug 2021020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 210acuggaaggg gaagtggcag 2021120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 211gcugccggtg gtactggaag 2021220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 212uuguggtaca gctgccggug 2021320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 213ugugggtaca tttgtgguac 2021420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 214uggccggccc ttgtggguac 2021520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 215ggcugagggc ctggccggcc 2021620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 216guagcacacc agggcugagg 2021720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 217aguugggggt ggtagcacac 2021820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 218ccugatcaaa gttgggggug 2021920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 219ccccatcgct ggtccugauc 2022020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 220aaacagtatc cccatcgcug 2022120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 221ucuugggctc caaacaguau 2022220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 222ugguctttca ctttcuuggg 2022320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 223guuugctgca gtggtcuuuc 2022420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 224aggggctgtg tttgcugcag 2022520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 225cuccuttctg gcaggggcug 2022620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 226acacaggtcc ctcctuucug 2022720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 227uuggcatgtt cacacagguc 2022820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 228agugggggcc gcttggcaug 2022920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 229ggacagagac agtgggggcc 2023020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 230ugaggtgttg tggacagaga 2023120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 231aguggtttcc agtgaggugu 2023220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 232ucucuttctg gcagtgguuu 2023320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 233caaagcactt ctcttucugg 2023420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 234gaagctgagg ctcaaagcac 2023520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 235ggaaaaaccg gagaagcuga 2023620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 236cauucttgtg gaaaaaccgg 2023720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 237auaccatatc tcattcuugu 2023820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 238ugcucagttc tataccauau 2023920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 239ggccacagct gcttgcucag 2024020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 240acuggcatct ggccacagcu 2024120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 241aggacccttg cactggcauc 2024220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 242agugggcatc aggacccuug 2024320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 243cagccgctgg cagtgggcau 2024420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 244aggcctggct ggccagccgc 2024520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 245uggugcggca ggcctggcug 2024620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 246ggaggcacgg gttggugcgg 2024720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 247agcgaccccc atggaggcac 2024820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 248accuctaggc agcgaccccc 2024920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 249gguggccctc cacctcuagg 2025020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 250uggcacaggc ggtggcccuc 2025120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 251cccaccgggc agtggcacag 2025220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 252gcuccggtgt agcccaccgg

2025320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 253cgucgcagaa ggctccggug 2025420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 254uggugtccac gtcgcagaag 2025520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 255agcagcttgc cttggugucc 2025620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 256cggccatcat agcagcuugc 2025720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 257agcugagccc gcggccauca 2025820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 258gccaggccgc ggtagcugag 2025920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 259cguggtcctg gccaggccgc 2026020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 260cacccgagag cgtgguccug 2026120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 261gcugacaggg cgcacccgag 2026220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 262cgaggcccac ggctgacagg 2026320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 263agguggcctc cgaggcccac 2026420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 264gucacgttcc ggtagguggc 2026520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 265cuugctcggc agtcacguuc 2026620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 266ccagutccgc gcttgcucgg 2026720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 267cgcccagtcc ccagtuccgc 2026820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 268aaggcgtggc cgcccagucc 2026920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 269ggguuccggc agaaggcgug 2027020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 270ugucgttgtc cgggtuccgg 2027120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 271accacgggcg gatgtcguug 2027220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 272agcacgaagc accacgggcg 2027320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 273ggucgcggtt cagcacgaag 2027420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 274cagcucagcc ggtcgcgguu 2027520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 275cgcagtactc ccagcucagc 2027620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 276acugugccag gtcgcaguac 2027720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 277gggguctggc actgtgccag 2027820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 278gccgcctggg ttggggucug 2027920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 279ggggucggag gcgccgccug 2028020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 280aggggacacc ggggtcggag 2028120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 281acaugaagcc taggggacac 2028220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 282ggcaugagtg ggacaugaag 2028320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 283cggcugcgcg ggcatgagug 2028420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 284uucggcggtg ccggcugcgc 2028520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 285gggcugaggc ttcggcggug 2028620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 286gguccgggtc gtgggcugag 2028720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 287acugaggcgg ggtccggguc 2028820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 288ccggggtctg ggactgaggc 2028920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 289ggcaaggctc ccggggucug 2029020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 290ucccgcttcg ccggcaaggc 2029120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 291aaggcggctg ctcccgcuuc 2029220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 292cuggucaggg aaggcggcug 2029320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 293agugggccgt tcctggucag 2029420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 294cccgcagctc agtgggccgu 2029520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 295ugcggagccg ctgcccgcag 2029620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 296acagactctt gcggagccgc 2029720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 297gggucatcga agacagacuc 2029820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 298ccaacgacgc gggtcaucga 2029920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 299gccaccagcc cgccaacgac 2030020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 300gccccgcgta gcgccaccag 2030120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 301uaggggtgcg ccccgcguag 2030220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 302agcgcggcga tgtaggggug 2030320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 303ggccccagta cagcgcggcg 2030420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 304gcagaaactg tggccccagu 2030520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 305gaggctgccg gcgcagaaac 2030620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 306gggggcgatg aggctgccgg 2030720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 307agcacccagc agggggcgau 2030820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 308gagcggccgt cagcacccag 2030920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 309ccugcaggca gtgagcggcc 2031020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 310gugcgggccg gtcctgcagg 2031120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 311agaucctcgg gtgcgggccg 2031220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 312caccaccgtc agatccucgg 2031320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 313guucctggcc gagcaccacc 2031420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 314ugguuacggc gttccuggcc 2031520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 315gcucacagct gtggtuacgg 2031620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 316acguctggca cggctcacag 2031720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 317gcacggccaa cgtctggcac 2031820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 318agcggtagga gcgcacggcc 2031920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 319aggcctcgtg caagcgguag 2032020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 320acgggcgaga aggccucgug 2032120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 321gcuggtagct gacgggcgag 2032220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 322agccaggtcg tgctgguagc 2032320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 323aggcgcaaca gagccagguc 2032420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA

Molecule Synthetic oligonucleotide 324caucctcctg aaggcgcaac 2032520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 325agcugccgtc cgcatccucc 2032620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 326aggagcgcgc agctgccguc 2032720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 327acguaaggcg acaggagcgc 2032820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 328acaccggctg aacgtaaggc 2032920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 329cuuggcaggc acaccggcug 2033020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 330cgcgcggcgc cgcttggcag 2033120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 331ucggagggtc gcgcggcgcc 2033220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 332agagcgtggt ctcggagggu 2033320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 333ccggccacct ggcagagcgu 2033420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 334uggccccagc cggccaccug 2033520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 335ccucgaactg gtggccccag 2033620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 336auucctccgc cccctcgaac 2033720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 337aagcuggcat attccuccgc 2033820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 338cgccucctgc aggaagcugg 2033920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 339aacggtacct gcgccuccug 2034020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 340agggagagga acggtaccug 2034120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 341agcagcgctc cagggagagg 2034220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 342cguccggggc tgagcagcgc 2034320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 343aggauccgtg cacgtccggg 2034420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 344ggggaggatg gaggauccgu 2034520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 345gagcatgccg gggaggaugg 2034620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 346gaaccctgcg cagagcaugc 2034720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 347gccgccctcg aggaacccug 2034820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 348acgcatcggt gccgcccucg 2034920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 349aaucaccctg gcacgcaucg 2035020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 350gggcctccgg aatcacccug 2035120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 351acacaccagc gggccuccgg 2035220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 352agcuuggtcc tcacacacca 2035320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 353ccggcgctct gcagcuuggu 2035420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 354cagggtgagc cggcgcucug 2035520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 355augaugcctt gcagggugag 2035620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 356auccccagct gatgaugccu 2035720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 357accacagccc gatccccagc 2035820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 358gcuugttgcg gtcaccacag 2035920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 359guagacgcct ggcttguugc 2036020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 360cacaucggtg tagacgccug 2036120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 361ccaggtagta ggccacaucg 2036220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 362ggauccaggc caggtaguag 2036320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 363ggugugctcc cggatccagg 2036420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 364aaucaggaaa cggtgugcuc 2036520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 365aguccctgag caatcaggaa 2036620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 366agggaaagat gagtcccuga 2036720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 367gaaucaccaa ggagggaaag 2036820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 368ucucactgcg gaatcaccaa 2036920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 369ccccagccac tctctcacug 2037020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 370uugccttcca tgccccagcc 2037120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 371gacacaatct tgcctuccau 2037220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 372cugggggaat gggacacaau 2037320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 373agcuggccgc actgggggaa 2037420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 374auccuggcgc ggagcuggcc 2037520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 375guucctgcgc catccuggcg 2037620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 376cacuutattg agttccugcg 2037720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 377auuuucaaag cacttuauug 2037820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 378uuuuuctcag catttucaaa 20

Claims

1. An antisense polynucleotide agent for inhibiting expression of a Factor XII (F12) gene, wherein the agent comprises 4 to 50 contiguous nucleotides, wherein at least one of the contiguous nucleotides is a modified nucleotide, and wherein the nucleotide sequence of the agent is 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs:1-4.

2. The agent of claim 1, wherein the agent comprises 4 to 50 contiguous nucleotides, wherein at least one of the contiguous nucleotides is a modified nucleotide, and wherein the nucleotide sequence of the agent is 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of residues 21-74, 87-106, 143-183, 209-239, 254-273, 287-315, 320-350, 352-392, 395-414, 352-414, 430-470, 472-568, 595-680, 595-669, 682-701, 736-899, 793-843, 793-899, 804-899, 901-942, 736-942, 968-987, 990-1097, 990-1042, 1100-1197, 1100-1206, 1210-1252, 1210-1240, 1253-1272, 1298-1406, 1308-1406, 1430-1515, 1430-1482, 1541-1560, 1583-1602, 1683-1714, 1727-1768, 1737-1756, 1781-1976, 1816-1868, 1902-1967, 1891-1957, 1992-2043, and 1992-2033 of SEQ ID NO:1.

3.-6. (canceled)

7. The agent of claim 1, wherein substantially all of the nucleotides of the antisense polynucleotide agent are modified nucleotides.

8. The agent of claim 1, wherein all of the nucleotides of the antisense polynucleotide agent are modified nucleotides.

9. 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: 14 to 20 nucleotides in length: 14 nucleotides in length: or 20 nucleotides in length.

10.-16. (canceled)

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

18. The agent of claim 17, wherein 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.

19. (canceled)

20. (canceled)

21. The agent of claim 17, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

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

23. (canceled)

24. The agent of claim 22, wherein the modified sugar moiety is selected from the group consisting of a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, and a bicyclic sugar moiety.

25. The agent of claim 22, wherein the 5'-wing segment is 1 to 6 nucleotides in length: 2 nucleotides in length: 3 nucleotides in length; 4 nucleotides in length: or 5 nucleotides in length.

26. The agent of claim 22, wherein the 3'-wing segment is 1 to 6 nucleotides in length: 2 nucleotides in length: 3 nucleotides in length: 4 nucleotides in length: or 5 nucleotides in length.

27. The agent of claim 22, wherein the gap segment is 5 to 14 nucleotides in length: or 10 nucleotides in length.

28.-42. (canceled)

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

44. The agent of claim 43, wherein the antisense polynucleotide agent is conjugated to the ligand at the 3'-terminus.

45. The agent of claim 43, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

46. The agent of claim 45, wherein the ligand is ##STR00016##

47. A pharmaceutical composition for inhibiting expression of a Factor XII gene, comprising the agent of claim 1.

48.-55. (canceled)

56. A method of inhibiting expression of a Factor XII (F12) gene in a cell, the method comprising: (a) contacting the cell with the agent of claim 1; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain antisense inhibition of a Factor XII (F12) gene, thereby inhibiting expression of the Factor XII gene in the cell.

57.-59. (canceled)

60. A method of treating a subject having a disease or disorder that would benefit from reduction in expression of a Factor XII (F12) gene, the method comprising administering to the subject a therapeutically effective amount of the agent of claim 1, thereby treating the subject.

61.-77. (canceled)