A composition comprising factor XII (Hagemann factor) (F12), kallikrein B, plasma (Fletcher factor) 1 (KLKB1), and kininogen 1 (KNG1) iRNA, and a method for using the same.

Abstract

The present invention provides compositions and methods for inhibiting thrombosis in subjects at risk of thrombosis, such as those with a genetic, acquired, or environmental risk of thrombosis. [Solution] The present invention provides RNAi agents, such as double-stranded RNAi agents, that target kallikrein B, plasma (Fletcher factor) 1 (KLKB1) gene, factor XII (Hageman factor (F12) gene, or kininogen 1 (KNG1) gene, as well as methods for inhibiting the expression of the KLKB1 gene, F12 gene, and / or KNG1 gene using such RNAi agents, and methods for treating subjects with hereditary angioedema (HAE) and / or contact activation pathway-related disorders.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 157,890, filed on 6 May 2015, U.S. Provisional Patent Application No. 62 / 260,887, filed on 30 November 2015, and U.S. Provisional Patent Application No. 62 / 266,958, filed on 14 December 2015. The contents of each of the aforementioned applications are incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on 3 May 2016, is named 121301-03120_SL.txt and has a size of 721,827 bytes. [Background technology]

[0003] The blood coagulation system is essential for hemostasis and responds to vascular trauma by the local formation of blood clots composed of fibrin mesh and activated platelets. Blood coagulation, thrombin production, and fibrin formation can be initiated by two distinct pathways, referred to as the extrinsic and intrinsic pathways.

[0004] The exogenous pathway involves the binding of plasma factor VIIa (FVIIa) to extravascular tissue factor (TF) at the site of vascular trauma.

[0005] The endogenous pathway is initiated by surface-dependent activation of plasma factor XII (F12) to F12a in a process called contact activation. Contact activation involves two other proteins circulating as a bimolecular complex: prekallikrein and high molecular weight kininogen. In summary, these three proteins—FXII, prekallikrein, and HK—constitute the "contact activation pathway," also known as the "kallikrein-kinin system." When the contact activation pathway is initiated by F12 binding to a negatively charged surface (or macromolecule), a conformational change in F12 occurs, leading to the formation of active F12 (F12a). F12a cleaves prekallikrein to produce active kallikrein (α-kallikrein), which then reciprocally activates F12, generating further F12a. Next, active kallikrein digests high molecular weight kininogen, releasing bradykinin. The F12a produced by contact activation also activates factor XI (F11) into F11a, triggering a series of proteolytic cleavage events, which ultimately lead to thrombin production and fibrin clot formation.

[0006] Interestingly, this contact system has been shown to be unnecessary for hemostasis. Most humans and other animals lacking contact-activated protein are asymptomatic, and homozygous F12 deficiency has not been associated with any disease or disorder. However, the contact system has been shown to play an important role in thrombotic diseases, as pharmacological inhibition of F12a or removal of F12 or high molecular weight kininogen genes can protect mice from experimentally induced thrombosis in various models.

[0007] In healthy individuals, a homeostatic balance exists between procoagulant, anticoagulant, and fibrinolytic capacities. However, numerous genetic, acquired, and environmental factors can disrupt this balance, leading to a favorable effect on coagulation, resulting in thrombosis (pathological thrombus formation) and potentially life-threatening events. For example, vein thrombus formation can lead to deep vein thrombosis (DVT), while arterial or ventricular thrombus formation can lead to myocardial infarction or stroke. Thrombi can obstruct blood flow at the site of formation or break off to form emboli, blocking distant vessels (e.g., pulmonary embolism or embolic stroke).

[0008] Acquired / environmental factors that can lead to pathological contact activation and contact-mediated thrombosis include various dental, surgical, and medical settings such as atrial fibrillation, cancer treatment, immobilization, central venous catheterization, implants, and extracorporeal oxygenation. As a result of such medical and surgical settings, tissue damage releases tissue factor, exposing various contact pathway triggers (DNA, RNA, phosphate, collagen, and laminin) that activate contact pathways and lead to thrombosis.

[0009] Hereditary angioedema (HAE) is a genetic disorder that disrupts the homeostatic balance between procoagulant, anticoagulant, and fibrinolytic activity. HAE is a rare autosomal dominant disorder, causing recurrent edema and swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals (genetials), as well as non-pruritic rashes, in one-third of patients. Untreated HAE patients experience an average of 1-2 angioedema episodes per month, although the frequency and severity of episodes can vary greatly. Edema and swelling are often disfiguring and disrupt daily life, leading to repeated hospitalizations, and patients may require psychiatric care to address disease-related anxiety. Abdominal episodes cause severe pain, nausea, and vomiting, sometimes leading to inappropriate surgery. Furthermore, more than half of HAE patients will experience life-threatening laryngeal edema at some point in their lives, which may require emergency tracheostomy to prevent suffocation. In the United States, an estimated 6,000 to 10,000 people from various racial groups suffer from HAE, resulting in 15,000 to 30,000 medical consultations per year and serious economic losses for patients, ranging from 20 to 100 days of sick leave.

[0010] HAE is caused by mutations in the C1 inhibitor (C1INH, SERPING1) gene, resulting in a deficiency of the C1INH protein. More than 250 different C1INH mutations have been demonstrated to cause HAE clinical symptoms. These C1INH mutations are typically inherited, however, up to 25% of HAE cases are caused by de novo mutations in C1INH. HAE type I, which accounts for about 85% of HAE cases, is caused by C1INH mutations that result in low levels of inefficiently secreted truncated or misfolded proteins. HAE type II, which accounts for about 15% of cases, is caused by mutations near the C1INH active site, resulting in normal levels of dysfunctional C1INH protein. In addition, HAE type III is a rare third form of the disease, caused by gain-of-function mutations in coagulation factor XII (F12) (Hagemann factor).

[0011] C1 inhibitors are serine protease inhibitors of the serpine family, major inhibitors of proteases in complement and contact activation pathways, and minor inhibitors of the fibrinolytic protease plasmin. During HAE attacks, these plasma proteolytic cascades are activated, producing substances that increase vascular permeability, such as bradykinin. Studies have shown that bradykinin peptides, which activate pro-inflammatory signaling pathways that dilate blood vessels and induce neutrophil chemotaxis, are the main substances that enhance vascular permeability during HAE attacks by binding to bradykinin receptors on vascular endothelial cells.

[0012] Typically, C1INH inhibits F12 autoactivation, F12a's ability to activate prekallilrein, kallikrein-mediated activation of high molecular weight kininogen, and kallikrein-mediated feedback activation of F12. Consequently, C1INH deficiency or mutations that cause gain-of-function F12 lead to bradykinin overproduction and the development of HAE angioedema.

[0013] Currently, HAEs can be treated prophylactically with 17α-alkylated androgens to reduce the likelihood of recurrent episodes, or with disease-specific therapeutic agents for the treatment of acute attacks. Approximately 70% of people with HAEs are treated with androgens or remain untreated, and about 30% receive therapeutic agents. Androgens are unsuitable for the short-term treatment of acute attacks because they take several days to become effective, and they can have severe side effects that can adversely affect growth and development. As a result, androgens are used only for long-term prophylaxis and are generally not administered to pregnant women or children. Furthermore, current therapeutic agents used to treat acute attacks must be administered intravenously several times a week or cause side effects that require drug administration and subsequent patient monitoring under hospitalization, which may limit their prophylactic use for long-term disease management. Therefore, there is no regimen that is safe, effective, and administered via a simpler route, nor is there one that treats acute angioedema attacks and prophylactically manages recurrent attacks in the majority of patients, including pregnant women and children. Alternative therapies are needed for those suffering from HAE. [Overview of the Initiative] [Problems that the invention aims to solve]

[0014] Therefore, in this technical field, there is a need for compositions and methods to inhibit thrombosis in subjects at risk of thrombosis, such as those who have a genetic, acquired, or environmental risk of thrombosis. [Means for solving the problem]

[0015] The present invention provides iRNA compositions that induce RNA-induced silencing complex (RISC)-mediated cleavage of kallikrein B, the RNA transcript of the plasma (Fletcher factor) 1 (KLKB1) gene, the RNA transcript of the factor XII (F12) gene, or the RNA transcript of the kininogen (KNG1) gene. For simplicity and unless otherwise specified, the term “contact activation pathway gene” as used herein refers to the KLKB1 gene, the F12 gene, or the KNG1 gene. Contact activation pathway genes can be located within cells, for example, within cells in the body of an object such as a human.

[0016] Accordingly, in one embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of factor XII (Hageman factor) (F12), the double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 9 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 10 by three or fewer nucleotides.

[0017] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of factor XII (Hageman factor) (F12), the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a complementary region comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27.

[0018] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of factor XII (Hageman factor) (F12), the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a complementary region comprising at least 15 consecutive nucleotides that differ from nucleotides 2000-2060 of SEQ ID NO: 9 by three or fewer nucleotides. In some embodiments, the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides that differ from nucleotides 2000-2030 of SEQ ID NO: 9 by three or fewer nucleotides. In other embodiments, the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides that differ from nucleotides 2030-2060 of SEQ ID NO: 9 by three or fewer nucleotides. In one embodiment, the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides that differ from nucleotides 2010-2040 of SEQ ID NO: 9 by three or fewer nucleotides. In one embodiment, the antisense strand includes a complementary region containing at least 15 consecutive nucleotides that differ from nucleotides 2010-2035 of SEQ ID NO: 9 by three or fewer nucleotides. In another embodiment, the antisense strand includes a complementary region containing at least 15 consecutive nucleotides that differ from nucleotides 2015-2040 of SEQ ID NO: 9 by three or fewer nucleotides. In yet another embodiment, the antisense strand includes a complementary region containing at least 15 consecutive nucleotides that differ from nucleotides 2015-2045 of SEQ ID NO: 9 by three or fewer nucleotides. In yet another embodiment, the antisense strand includes a complementary region containing at least 15 consecutive nucleotides that differ from nucleotides 2020-2050 of SEQ ID NO: 9 by three or fewer nucleotides. In yet another embodiment, the antisense strand includes a complementary region containing at least 15 consecutive nucleotides that differ from nucleotides 2020-2045 of SEQ ID NO: 9 by three or fewer nucleotides. In yet another embodiment, the antisense strand includes a complementary region containing at least 15 consecutive nucleotides that differ from any one of the ranges of SEQ ID NO: 9 provided in Table 24 by three or fewer nucleotides.In one embodiment, the antisense strand includes a complementary region comprising at least 15 consecutive nucleotides that differ from nucleotides 2018-2040 of SEQ ID NO: 9 by three or fewer nucleotides. In one embodiment, the antisense strand includes a complementary region comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence (5'-UUCAAAGCACUUUAUUGAGUUUC-3') (SEQ ID NO: 25) of the antisense strand of AD-67244 by three or fewer nucleotides. In one embodiment, the sense strand comprises the sense strand nucleotide sequence of AD-67244. In some embodiments, the complementary region comprises nucleotides 15, 16, 17, 18, 19, 20, 21, 22, or 23 that differ from nucleotides 2015-2040 of SEQ ID NO: 9 by three or fewer nucleotides. In some embodiments, the complementary region comprises nucleotides 15, 16, 17, 18, 19, 20, 21, 22, or 23 that differ from nucleotides 2015-2045 of SEQ ID NO: 9 by three or fewer nucleotides. In some embodiments, the complementary region includes nucleotides 15, 16, 17, 18, 19, 20, 21, 22, or 23 that differ from nucleotides 2018-2040 of SEQ ID NO: 9 by three or fewer nucleotides. In some embodiments, the complementary region includes nucleotides 15, 16, 17, 18, 19, 20, 21, 22, or 23 that differ from nucleotides 2018-2045 of SEQ ID NO: 9 by three or fewer nucleotides. In one embodiment, the drug contains at least one modified nucleotide. In another embodiment, all nucleotides of the drug are modified nucleotides. In one embodiment, the drug further includes a ligand, for example, a ligand bound to the 3' end of the sense strand. In one embodiment, the sense strand and the antisense strand are each independently 15-30 nucleotides long. In another embodiment, the sense strand and the antisense strand are each independently 19-25 nucleotides long.

[0019] In one embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of kallikrein B, plasma (Fletcher factor) 1 (KLKB1), the double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by three or fewer nucleotides.

[0020] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of kallikrein B, plasma (Fletcher factor) 1 (KLKB1), the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a complementary region containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in Tables 3, 4, 19A, or 19B.

[0021] In one embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of kininogen 1 (KNG1), the double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 17 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 18 by three or fewer nucleotides.

[0022] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of kininogen 1 (KNG1), the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a complementary region comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in Tables 15, 16, 19E, or 19F.

[0023] In one embodiment, the antisense chain includes a complementary region comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in any one of Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27.

[0024] In one embodiment, the double-stranded RNAi agent provided herein comprises at least one modified nucleotide.

[0025] In one embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of kallikrein B, plasma (Fletcher factor) 1 (KLKB1), the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides differing by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 consecutive nucleotides differing by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 2, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached to its 3' end.

[0026] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of factor XII (Hageman factor) (F12), the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides differing by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 9, and the antisense strand comprises at least 15 consecutive nucleotides differing by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 10, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached to its 3' end.

[0027] In a further embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of kininogen 1 (KNG1), the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides differing by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 17, and the antisense strand comprises at least 15 consecutive nucleotides differing by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 18, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached to its 3' end.

[0028] In certain embodiments, the dsRNA contains at least one modified nucleotide. In certain embodiments, the dsRNA contains four or fewer (i.e., 4, 3, 2, 1, or 0) unmodified nucleotides in the sense strand. In certain embodiments, the dsRNA contains four or fewer (i.e., 4, 3, 2, 1, or 0) unmodified nucleotides in the antisense strand. In certain embodiments, the dsRNA contains four or fewer (i.e., 4, 3, 2, 1, or 0) unmodified nucleotides in both the sense and antisense strands. In certain embodiments, all nucleotides in the sense strand of the dsRNA are modified nucleotides. In certain embodiments, all nucleotides in the antisense strand of the dsRNA are modified nucleotides. In certain embodiments, all nucleotides in both the sense strand and the antisense strand of the dsRNA are modified nucleotides.

[0029] In certain embodiments, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, fixed nucleotides, unfixed nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, non-basic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic, such as vinyl phosphate.

[0030] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of 2'-O-methyl and 2'-fluoro modifications.

[0031] In certain embodiments, the antisense strand of any double-stranded RNAi agent of the present invention contains eight or fewer 2'-fluoro modifications, seven or fewer 2'-fluoro modifications, six or fewer 2'-fluoro modifications, five or fewer 2'-fluoro modifications, four or fewer 2'-fluoro modifications, three or fewer 2'-fluoro modifications, two or fewer 2'-fluoro modifications, one or fewer 2'-fluoro modifications, or one or fewer 2'-fluoro modifications. In other embodiments, the sense strand of any double-stranded RNAi agent of the present invention contains six or fewer 2'-fluoro modifications, five or fewer 2'-fluoro modifications, four or fewer 2'-fluoro modifications, three or fewer 2'-fluoro modifications, two or fewer 2'-fluoro modifications, one or fewer 2'-fluoro modifications, or one or fewer 2'-fluoro modifications.

[0032] In one embodiment, the double-stranded RNAi agent further comprises at least one phosphorothioate nucleotide interbonding. In another embodiment, the double-stranded RNAi agent comprises 6 to 8 phosphorothioate nucleotide interbondings.

[0033] The complementary region may be at least 17 nucleotides long, 18 nucleotides long, 19 nucleotides long, 20 nucleotides long, or 21 nucleotides long.

[0034] In certain embodiments, the complementary region may be 19 to 21 nucleotides long or 21 to 23 nucleotides long.

[0035] In certain embodiments, each strand of the double-stranded RNAi agent is 30 nucleotides or less in length. In certain embodiments, the double-stranded RNAi agent is at least 15 nucleotides in length.

[0036] In certain embodiments, at least one strand of the double-stranded RNAi agent contains a 3' overhang of at least one nucleotide. In certain embodiments, at least one strand contains a 3' overhang of at least two nucleotides.

[0037] In certain embodiments, the double-stranded RNAi agent further comprises a ligand. In certain embodiments, the ligand is conjugated to the 3' end of the sense strand of the dsRNA. In certain embodiments, the ligand is an N-acetylgalactosamine (GalNAc) derivative. In certain embodiments, the ligand is one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker. In certain embodiments, the ligand is [ka] That is the case.

[0038] In a particular embodiment, dsRNA is shown in the following schematic diagram. [ka] It is conjugated to the ligand as shown in the formula, where X is O or S.

[0039] In one embodiment, X is O.

[0040] In one embodiment, the sense array and the antisense array are selected from any one of the arrays listed in any one of Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27.

[0041] In one embodiment, the complementary region consists of one of the antisense sequences listed in any one of Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27.

[0042] In one embodiment, the F12 expression inhibitory dsRNA agent is selected from the group consisting of AD-66170, AD-66173, AD-66176, AD-66125, AD-66172, AD-66167, AD-66165, AD-66168, AD-66163, AD-66116, AD-66126, and AD-67244. In another embodiment, the F12 expression inhibitory dsRNA agent is AD-67244.

[0043] In one embodiment, the dsRNA agent that inhibits KLKB1 expression is selected from the group consisting of AD-65077, AD-65170, AD-65103, AD-65083, AD-65087, AD-65149, AD-64652, AD-65162, AD-65153, AD-65084, AD-65099, and AD-66948. In another embodiment, the dsRNA agent that inhibits KLKB1 expression is AD-66948.

[0044] In one embodiment, the dsRNA agent that inhibits KNG1 expression is selected from the group consisting of AD-66259, AD-66261, AD-66262, AD-66263, AD-6634, and AD-67344. In another embodiment, the dsRNA agent that inhibits KNG1 expression is AD-67344.

[0045] In one embodiment, the present invention provides cells comprising a double-stranded RNAi agent of the present invention that targets KLKB1. In one embodiment, the present invention provides cells comprising a double-stranded RNAi agent of the present invention that targets F12. In a further embodiment, the present invention provides cells comprising a double-stranded RNAi agent of the present invention that targets KNG1.

[0046] In one embodiment, the present invention provides a vector encoding at least one strand of a double-stranded RNAi agent of the present invention that targets KLKB1. In another embodiment, the present invention provides a vector encoding at least one strand of a double-stranded RNAi agent of the present invention that targets F12. In a further embodiment, the present invention provides a vector encoding at least one strand of a double-stranded RNAi agent of the present invention that targets KNG1.

[0047] In one embodiment, the present invention provides a pharmaceutical composition comprising the double-stranded RNAi agent or vector of the present invention for inhibiting the expression of the KLKB1 gene. In another embodiment, the present invention provides a pharmaceutical composition comprising the double-stranded RNAi agent or vector of the present invention for inhibiting the expression of the F12 gene. In a further embodiment, the present invention provides a pharmaceutical composition comprising the double-stranded RNAi agent or vector of the present invention for inhibiting the expression of the KNG1 gene.

[0048] The pharmaceutical compositions provided herein may be administered in a non-buffered solution, such as physiological saline or water, or in a buffer solution, for example, the buffer solution may contain an acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS).

[0049] In one embodiment, the pharmaceutical composition of the present invention comprises a double-stranded RNAi agent and a lipid preparation as described herein.

[0050] In one embodiment, the present invention provides a method for inhibiting KLKB1 expression in cells. The method comprises the steps of: contacting cells with a double-stranded RNAi agent or pharmaceutical composition of the present invention; and maintaining the cells for a time sufficient to obtain degradation of the mRNA transcript of the KLKB1 gene, thereby inhibiting the expression of the KLKB1 gene in the cells.

[0051] In another embodiment, the present invention provides a method for F12 expression in cells. The method comprises the steps of: contacting cells with a double-stranded RNAi agent or pharmaceutical composition of the present invention; and maintaining the cells for a time sufficient to obtain degradation of the mRNA transcript of the F12 gene, thereby inhibiting the expression of the F12 gene in the cells.

[0052] In a further embodiment, the present invention provides a method for KNG1 expression in cells. The method comprises the steps of: contacting cells with a double-stranded RNAi agent or pharmaceutical composition of the present invention; and maintaining the cells for a time sufficient to obtain degradation of the mRNA transcript of the KNG1 gene, thereby inhibiting the expression of the KNG1 gene in the cells.

[0053] In one embodiment, the cells are cells located within the body of an object, such as a human subject.

[0054] In one embodiment, KLKB1 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0055] In one embodiment, F12 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0056] In one embodiment, KNG1 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0057] In one embodiment, the present invention provides a method for treating a subject having a disease or disorder that may benefit from reduced expression of contact activation pathway genes. The method comprises administering a therapeutically effective amount of the double-stranded RNAi agent or pharmaceutical composition of the present invention to the subject, thereby treating the subject.

[0058] In one embodiment, the contact activation pathway gene is KLKB1. In another embodiment, the contact activation pathway gene is F12. In yet another embodiment, the contact activation pathway gene is KNG1.

[0059] In another embodiment, the present invention provides a method for preventing at least one symptom of a subject having a disease or disorder that may benefit from reduced expression of contact-activated pathway genes. The method comprises administering a preventive effective amount of the double-stranded RNAi agent or pharmaceutical composition of the present invention to a subject, thereby preventing at least one symptom of the subject having a disorder that may benefit from reduced expression of contact-activated pathway genes.

[0060] In one embodiment, the contact activation pathway gene is KLKB1. In another embodiment, the contact activation pathway gene is F12. In yet another embodiment, the contact activation pathway gene is KNG1. In one embodiment, the contact activation pathway gene is F12, and the method further comprises the step of administering the double-stranded RNAi agent of the present invention that targets KLKB1. In another embodiment, the contact activation pathway gene is F12, and the method further comprises the step of administering the double-stranded RNAi agent of the present invention that targets KNG1.

[0061] In one embodiment, administration of double-stranded RNAi to a subject results in a decrease in bradykinin levels or a decrease in coagulation factor XII activity.

[0062] In one embodiment, the disorder is a contact-activated pathway-related disorder such as thrombosis, hereditary angioedema (HAE), Flectcher factor deficiency, or essential hypertension.

[0063] In certain embodiments, at least one symptom is an angioedema attack or thrombus formation.

[0064] In one embodiment, the subject is a human.

[0065] In one embodiment, the method further includes the step of administering an anti-KLKB1 antibody or its antigen-binding fragment to a target.

[0066] In one embodiment, the method further includes the step of measuring the bradykinin and / or coagulation factor XII levels of the subject.

[0067] In another embodiment, the present invention provides a method for inhibiting F12 expression in a subject. This method comprises administering a therapeutically effective dose of the double-stranded RNAi agent of the present invention that targets F12 to a subject, thereby inhibiting F12 expression in the subject.

[0068] In one embodiment, the present invention provides a method for inhibiting KLKB1 expression in a subject. The method comprises administering a therapeutically effective dose of the double-stranded RNAi agent of the present invention, which targets KLKB1, to a subject, thereby inhibiting KLKB1 expression in the subject.

[0069] In one embodiment, the present invention provides a method for inhibiting KNG1 expression in a subject. The method comprises administering a therapeutically effective dose of the double-stranded RNAi agent of the present invention, which targets KNG1, to a subject, thereby inhibiting KNG1 expression in the subject.

[0070] In one embodiment, the present invention provides a method for treating a subject with a tendency to form blood clots. The method comprises administering a therapeutically effective amount of the double-stranded RNAi agent of the present invention that targets F12, or a pharmaceutical composition containing the double-stranded RNAi agent of the present invention that targets F12, to the subject, thereby treating the subject.

[0071] In another embodiment, the present invention provides a method for preventing at least one symptom of a subject having a thrombosis-prone condition. The method comprises administering a prophylactic effective amount of the double-stranded RNAi agent of the present invention targeting F12, or a pharmaceutical composition comprising the double-stranded RNAi agent of the present invention targeting F12, to a subject, thereby preventing at least one symptom of the subject.

[0072] In one embodiment, the method further comprises the step of administering the double-stranded RNAi agent of the present invention that targets KLKB1. In another embodiment, the method further comprises the step of administering the double-stranded RNAi agent of the present invention that targets KNG1.

[0073] In one embodiment, the present invention provides a method for treating a subject having hereditary angioedema (HAE). The method comprises administering a therapeutically effective amount of the double-stranded RNAi agent of the present invention that targets F12, or a pharmaceutical composition containing the double-stranded RNAi agent of the present invention that targets F12, to the subject, thereby treating the subject.

[0074] In another embodiment, the present invention provides a method for preventing at least one symptom of a subject having hereditary angioedema (HAE). The method comprises administering a prophylactic effective amount of the double-stranded RNAi agent of the present invention targeting F12, or a pharmaceutical composition comprising the double-stranded RNAi agent of the present invention targeting F12, to a subject, thereby preventing at least one symptom of the subject.

[0075] In one embodiment, the method further comprises the step of administering the double-stranded RNAi agent of the present invention that targets KLKB1. In another embodiment, the method further comprises the step of administering the double-stranded RNAi agent of the present invention that targets KNG1.

[0076] In another embodiment, the present invention provides a method for preventing thrombus formation in subjects at risk of thrombus formation. The method comprises administering a prophylactic effective amount of the double-stranded RNAi agent of the present invention that targets F12, or a pharmaceutical composition containing the double-stranded RNAi agent of the present invention that targets F12, to a subject, thereby inhibiting thrombus formation in the subject at risk of thrombus formation.

[0077] In one embodiment, subjects at risk of thrombus formation have contact activation pathway-related diseases or disorders.

[0078] In one embodiment, the contact-activated pathway-related disorder is a thrombosis-prone condition. In another embodiment, the contact-activated pathway-related disorder is hereditary angioedema (HAE).

[0079] In other embodiments, the contact activation pathway-related disease is Flectcher factor deficiency or essential hypertension.

[0080] In one embodiment, subjects at risk of thrombosis are selected from a group consisting of surgical patients; medical patients; pregnant subjects; postpartum subjects; subjects with a history of thrombosis; subjects receiving hormone replacement therapy; subjects who sit for long periods of time; and obese subjects.

[0081] In one embodiment, the method further comprises the step of administering the double-stranded RNAi agent of the present invention that targets KLKB1. In another embodiment, the method further comprises the step of administering the double-stranded RNAi agent of the present invention that targets KNG1.

[0082] In another embodiment, the present invention provides a method for preventing angioedema attacks in subjects with hereditary angioedema (HAE). The method comprises administering a prophylactic effective amount of the double-stranded RNAi agent of the present invention targeting F12, or a pharmaceutical composition containing the double-stranded RNAi agent of the present invention targeting F12, to the subject, thereby preventing angioedema attacks.

[0083] In one embodiment, the method further comprises the step of administering the double-stranded RNAi agent of the present invention that targets KLKB1. In another embodiment, the method further comprises the step of administering the double-stranded RNAi agent of the present invention that targets KNG1. [Brief explanation of the drawing]

[0084] [Figure 1] This graph shows the suppression of KLKB1 mRNA 7-10 days after a single subcutaneous dose of 1 mg / kg or 3 mg / kg of the indicated drug in wild-type mice. [Figure 2] This graph shows the suppression of F12 mRNA 7 to 10 days after administration of the indicator drug at a single subcutaneous dose of 1 mg / kg or 3 mg / kg, or at a single dose of 1 mg / kg or 10 mg / kg in wild-type mice. [Figure 3] This graph shows the suppression of KNG1 mRNA 7-10 days after administration of a single subcutaneous dose of 1 mg / kg or 3 mg / kg of the indicated drug in wild-type mice. [Figure 4] [Figure 4A] This graph shows the amount of Evans blue pigment in the blood of mice that were administered a single dose of AD-66948 and captopril at doses of 0 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg, on day 7 after administration. [Figure 4B] This graph shows the amount of Evans blue pigment in the intestines of mice that were administered a single dose of AD-66948 and captopril at doses of 0 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg, on day 7 after administration. [Figure 4C] This graph shows the suppression of KLKB1 mRNA in the liver of mice that were administered a single dose of AD-66948 and captopril at doses of 0 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg, on day 7 after administration. [Figure 4D] This graph shows the relative intestinal permeability on day 7 after administration to mice that received single doses of AD-66948 and captopril at doses of 0 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg. [Figure 5][Figure 5A] This graph shows the amount of Evans blue pigment in the blood of mice that were administered a single dose of AD-67244 and captopril at doses of 0 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg, on day 7 after administration. [Figure 5B] This graph shows the amount of Evans blue pigment in the intestines of mice that were administered a single dose of AD-67244 and captopril at doses of 0 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg, on day 7 after administration. [Figure 5C] This graph shows the suppression of F12 mRNA in the liver of mice that were administered a single dose of AD-67244 and captopril at doses of 0 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg, on day 7 after administration. [Figure 5D] This graph shows the relative intestinal permeability on day 7 after administration to mice that received single doses of AD-67244 and captopril at doses of 0 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg. [Figure 6] [Figure 6A] This graph shows the amount of Evans blue pigment in the blood of mice that were administered a single dose of 0 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg of AD-67344 and captopril on day 7 after administration. [Figure 6B] This graph shows the amount of Evans blue pigment in the intestines of mice that were administered a single dose of 0 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg of AD-67344 and captopril on day 7 after administration. [Figure 6C] This graph shows the suppression of KNG1 mRNA in the liver of mice that were administered a single dose of 0 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg of AD-67344 and captopril on day 7 after administration. [Figure 6D] This graph shows the relative intestinal permeability on day 7 after administration to mice that received single doses of AD-67344 and captopril at doses of 0 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg. [Figure 7]The modified nucleotide sequences of indicator double-stranded RNAi agents targeting the KLKB1 gene are shown. F represents a 2'-fluoronucleotide modification; OMe represents a 2'-O-methyl(2'-OMe) nucleotide modification; and s represents a phosphorothioate bond. The figures disclose sequence numbers 2285 to 2302 in order of appearance. [Figure 8] The modified nucleotide sequences of indicator double-stranded RNAi agents targeting the F12 gene are shown. F represents a 2'-fluoronucleotide modification; OMe represents a 2'-O-methyl(2'-OMe) nucleotide modification; and s represents a phosphorothioate bond. The figures disclose sequence numbers 2303 to 2320 in the order they are presented. [Figure 9] The modified nucleotide sequences of indicator double-stranded RNAi agents targeting the KNG1 gene are shown. F represents a 2'-fluoronucleotide modification; OMe represents a 2'-O-methyl(2'-OMe) nucleotide modification; and s represents a phosphorothioate bond. The figures disclose sequence numbers 2321 to 2332 in order of appearance. [Figure 10] [Figure 10A] This graph shows the amount of Evans blue pigment in the ear of mice that were administered a single dose of 0.1 mg / kg, 0.5 mg / kg, or 3 mg / kg of AD-67244 in combination with a single dose of 10 mg / kg of a C1-INH-targeted dsRNA agent on day 7 after administration. Error bars = standard deviation. [Figure 10B] This graph shows dose-dependent F12 mRNA suppression on day 7 after administration of a single dose of 0.1 mg / kg, 0.5 mg / kg, or 3 mg / kg of AD-67244 subcutaneously in combination with a single dose of 10 mg / kg of a C1-INH-targeted dsRNA agent. [Figure 11] This graph shows the suppression of plasma F12 protein in female cynomolgus monkeys after single subcutaneous administration of AD-67244 at doses of 3 mg / kg, 1 mg / kg, 0.3 mg / kg, or 0.1 mg / kg. The plasma F12 levels shown are relative F12 protein levels normalized to the mean baseline F12 protein level before administration. Error bars = standard deviation. [Figure 12]This graph shows the suppression of F12 protein in plasma of wild-type mice administered a single dose of either AD-67244 or AD-74841 at a dose of 0.5 mg / kg. [Figure 13] This graph shows the effect of 5'-terminal modification on the in vivo efficacy of indicator drugs. [Modes for carrying out the invention]

[0085] The present invention provides iRNA compositions that induce RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of contact activation pathway genes (i.e., kallikrein B, plasma (Fletcher factor) 1 (KLKB1) gene, factor XII (Hagemann factor) (F12) gene, or kininogen 1 (KNG1) gene). These genes may be present in cells, for example, in cells within the body of a target such as a human. Using these iRNAs, it becomes possible to target and degrade the mRNA of the corresponding gene (KLKB1 gene, F12 gene, or KNG1 gene) in mammals.

[0086] The RNAi agents of the present invention are designed to target the protein-coding region and the 3'UTR region of the human KLKB1 gene, including the portion of this gene that is conserved in KLKB1 orthologs of other mammalian species. While not intended to be theoretically limited, combinations or partial combinations of the aforementioned properties, as well as specific target sites and / or modifications in these RNAi agents, are thought to confer improved efficacy, stability, potency, persistence, and safety to the RNAi agents of the present invention.

[0087] The iRNAs of the present invention are approximately 30 nucleotides or less in length, for example, 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, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19 The RNA strand (antisense strand) may contain a region of ~21, 19~20, 20~30, 20~29, 20~28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20~21, 21~30, 21~29, 21~28, 21~27, 21~26, 21~25, 21~24, 21~23, or 21~22 nucleotides in length, and this region is substantially complementary to at least a portion of the mRNA transcript of a contact activation pathway gene, i.e., the KLKB1 gene, F12 gene, or KNG1 gene.

[0088] In certain embodiments, the iRNAs of the present invention include an RNA strand (antisense strand) that has a region of at least 19 consecutive nucleotides substantially complementary to at least a portion of the mRNA transcript of a contact activation pathway gene, i.e., the KLKB1 gene, F12 gene, or KNG1 gene, and may include an RNA strand (antisense strand) of a longer length, for example, up to 66 nucleotides, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides. These iRNAs having longer antisense strands preferably include a second RNA strand (sense strand) of 20-60 nucleotides in length, and these sense strands and antisense strands form a double helix of 18-30 consecutive nucleotides.

[0089] The inventors have demonstrated, using in vitro and in vivo assays, that iRNAs targeting contact activation pathway genes potently mediate RNAi, resulting in significant inhibition of the expression of contact activation pathway genes, namely the KLKB1, F12, or KNG1 genes. The inventors have also demonstrated that the RNAi agents of the present invention are highly stable in the cytoplasm and lysosomes. Therefore, methods and compositions containing these iRNAs are useful for treating contact activation pathway-related diseases or disorders, such as thrombosis, HAE, and for preventing at least one symptom of a contact activation pathway-related disease or disorder in subjects with or at risk of developing such a disease or disorder.

[0090] Accordingly, the present invention also provides a method for treating subjects with disorders that may benefit from inhibiting or reducing the expression of contact activation pathway genes, such as contact activation pathway-related diseases, including thrombosis or hereditary angioedema (HAE), using an iRNA composition that causes RNA-induced silencing complex (RISC) mediated cleavage of RNA transcripts of contact activation pathway genes.

[0091] The extremely low dosage of iRNA according to the present invention can, in particular, specifically and efficiently mediate RNA interference (RNAi) to sufficiently inhibit the expression of the corresponding gene (contact activation pathway gene).

[0092] The following detailed description discloses how to prepare and use compositions containing iRNA to inhibit the expression of contact activation pathway genes (i.e., KLKB1 gene, F12 gene, or KNG1 gene), and compositions, uses, and methods for treating subjects with diseases and disorders who may benefit from the inhibition and / or reduction of the expression of contact activation pathway genes (i.e., KLKB1 gene, F12 gene, or KNG1 gene).

[0093] I. Definition To make the present invention easier to understand, several terms are first defined. Furthermore, it should be noted that whenever a variable value or range of values ​​is stated, intermediate values ​​and ranges of the stated values ​​are also intended to be part of the present invention.

[0094] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” means one or more elements, e.g., multiple elements.

[0095] The term "including" is used herein to mean "including but not limited to," and is used synonymously with this phrase.

[0096] The term "or" is used herein to mean the term "and / or" unless the context clearly indicates otherwise, and is used synonymously with this term.

[0097] The term "at least" preceding a number or number sequence is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be included as is evident from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides out of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated characteristic. When "at least" precedes a number sequence or range, it is understood that "at least" may modify each of the numbers in that sequence or range.

[0098] When used herein, the range includes both the upper and lower limits.

[0099] As used herein, the terms “prekallikrein” and “KLKB1” are synonymous with “kallikrein B, plasma (Fletcher factor) 1” and refer to the naturally occurring gene encoding zymogenized kallikrein, prekallikrein. Plasma prekallikrein is converted to plasma kallikrein (also called active kallikrein) by F12a, which then releases bradykinin from high molecular weight kininogen through proteolysis, thereby activating F12. Bradykinin is a peptide that promotes vascular permeability and is present in high levels in HAE patients. For the amino acid and complete coding sequence of the KLKB1 gene reference sequence, see, for example, GenBank accession number GI:78191797 (RefSeq accession number NM_000892.3; SEQ ID NO: 1; SEQ ID NO: 2). For mammalian orthologues of the human KLKB1 gene, see, for example, GenBank accession number GI:544436072 (RefSeq accession number XM_005556482, cynomolgus monkey; SEQ ID NO: 7 and SEQ ID NO: 8); GI:380802470 (RefSeq accession number JU329355, rhesus monkey); GI:236465804 (RefSeq accession number NM_008455, mouse; SEQ ID NO: 3 and SEQ ID NO: 4); GI:162138904 (RefSeq accession number NM_012725, rat; SEQ ID NO: 5 and SEQ ID NO: 6).

[0100] Further examples of KLKB1 mRNA sequences are readily available using publicly available databases, such as GenBank, UniProt, and OMIM.

[0101] As used herein, the terms “coagulation factor XII,” “FXII,” “F12,” “active F12,” and “F12a,” and “Factor XII (Hagemann factor),” used synonymously, refer to the naturally occurring gene encoding the zymogenized form of F12a. F12a is a serine protease (or serine endopeptidase) class enzyme (EC3.4.21.38) that cleaves prekallikrein to form kallikrein, which subsequently releases bradykinin from high molecular weight kininogen to activate F12. For the amino acid and complete coding sequence of the reference sequence of the F12 gene, see, for example, GenBank accession number GI:145275212 (RefSeq accession number NM_000505; SEQ ID NO: 9; SEQ ID NO: 10). For mammalian orthologues of the human F12 gene, see, for example, GenBank accession number GI:544441267 (RefSeq accession number XM_005558647, cynomolgus monkey; SEQ ID NO: 11 and SEQ ID NO: 12); GI:805299477 (RefSeq accession number NM_021489, mouse; SEQ ID NO: 13 and SEQ ID NO: 14); GI:62078740 (RefSeq accession number NM_001014006, rat; SEQ ID NO: 15 and SEQ ID NO: 16).

[0102] Further examples of F12 mRNA sequences are readily available using publicly available databases, such as GenBank, UniProt, and OMIM.

[0103] As used herein, the terms “Fitzgerald factor,” “Williams-Fitzgerald-Flaujeac factor,” “high molecular weight kininogen” (“HMWK” or “HK”), “low molecular weight kininogen” (“LMWK”), and “kininogen 1,” used synonymously with “KNG1,” refer to naturally occurring genes that are alternatively spliced ​​to produce HMWK and LMWK. When HMWK is cleaved by active kallikrein, bradykinin is released. For the amino acid and complete coding sequence of the KNG1 gene reference sequence, see, for example, GenBank accession number GI:262050545 (RefSeq accession number NM_001166451; SEQ ID NO: 17; SEQ ID NO: 18). For mammalian orthologues of the human KNG1 gene, see, for example, GenBank accession number GI:544410550 (RefSeq accession number XM_005545463, cynomolgus monkey; SEQ ID NO: 19 and SEQ ID NO: 20); GI:156231028 (RefSeq accession number NM_001102409, mouse; SEQ ID NO: 21 and SEQ ID NO: 22); GI:80861400 (RefSeq accession number NM_012696, rat; SEQ ID NO: 23 and SEQ ID NO: 23).

[0104] Further examples of KNG1 mRNA sequences are readily available using publicly available databases, such as GenBank, UniProt, and OMIM.

[0105] For simplicity, as used herein, unless otherwise specified, “contact activation pathway gene” refers to the KLKB1 gene, the F12 gene, or the KNG1 gene.

[0106] As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a contact activation pathway gene, including mRNA, which is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence may be long enough to act as a substrate for iRNA-mediated cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during transcription of the contact activation pathway gene. In one embodiment, the target sequence is located within the protein-coding region of the contact activation pathway gene. In another embodiment, the target sequence is located within the 3'UTR of the contact activation pathway gene.

[0107] The target sequence may be approximately 9-36 nucleotides long, for example, approximately 15-30 nucleotides long. For example, the target sequence may be approximately 15-30 nucleotides, 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, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 1 The target sequence may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides long. In some embodiments, the target sequence is about 19-30 nucleotides long. In other embodiments, the target sequence is about 19-25 nucleotides long. In yet another embodiment, the target sequence is about 19-23 nucleotides long. In some embodiments, the target sequence is about 21-23 nucleotides long. Intermediate ranges and lengths between those described above are also considered to be part of the present invention.

[0108] As used herein, the term “sequence-containing chain” refers to an oligonucleotide containing a chain of nucleotides represented by a sequence shown using standard nucleotide nomenclature.

[0109] "G," "C," "A," and "U" generally represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. However, it will be understood that the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides or surrogate replacement moieties, as will be further detailed below (see, for example, Table 2). Those skilled in the art will be well aware that guanine, cytosine, adenine, and uracil may be substituted by other moieties without significantly altering the base-pairing properties of oligonucleotides containing such substitution moieties. For example, but not limited to, nucleotides containing inosine as a base may base-pair with nucleotides containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine may be substituted, for example, with nucleotides containing inosine in the nucleotide sequences of dsRNA characterized in the present invention. In another example, adenine and cytosine at any point in the oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU fluctuation base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods addressed in the present invention.

[0110] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interferant,” as used synonymously herein, refer to agents containing RNA and mediating targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as defined herein. iRNAs lead to sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs regulate (e.g., inhibit) the expression of the KLKB1 gene in cells, for example, in mammalian subjects.

[0111] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as a contact activation pathway gene, i.e., a KLKB1 target mRNA sequence, an F12 target mRNA sequence, or a KNG1 target mRNA sequence, to induce cleavage of the target RNA. Although we do not wish to be constrained by theory, it is thought that long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases in the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Herein, in one embodiment, the present invention relates to single-stranded RNA (siRNA) that is generated in cells and promotes the formation of a RISC complex resulting in the silencing of a target gene, i.e., a contact activation pathway gene. Accordingly, the term "siRNA" is also used herein to refer to the above RNAi.

[0112] In another embodiment, the RNAi agent may be a single-stranded siRNA introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15 to 30 nucleotides and are chemically modified. Designs and tests of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as single-stranded siRNAs chemically modified as described herein or by the methods described in Lima et al., (2012) Cell 150:883-894.

[0113] In another embodiment, the “iRNA” for use in the compositions, uses and methods of the present invention is double-stranded RNA, and is referred herein to as “double-stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, which are shown to have “sense” and “antisense” orientations toward the target RNA, i.e., the contact activation pathway gene, i.e., the KLKB1 gene, the F12 gene, or the KNG1 gene. In one embodiment of the present invention, double-stranded RNA (dsRNA) induces degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0114] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, as used herein, “RNAi agent” may contain ribonucleotides with chemical modifications; an RNAi agent may contain substantial modifications in multiple nucleotides. As used herein, the term “modified nucleotide” independently refers to a nucleotide having a modified sugar moiety, a modified nucleotide-nucleotide bond, and / or a modified nucleic acid base. Therefore, the term “modified nucleotide” encompasses, for example, the substitution, addition, or removal of a functional group or atom to the nucleoside bond, sugar moiety, or nucleic acid base. Suitable modifications for use in the agents of the present invention include all types of modifications disclosed herein or known in the art. Any such modification used in siRNA-type molecules is encompassed by “RNAi agent” for the purposes of this specification and the claims.

[0115] The double-stranded region may be of any length that allows for the specific degradation of the desired target RNA via the RISC pathway, and can range from approximately 9 to 36 base pairs in length, for example, in the range of approximately 15 to 30 base pairs, for example, approximately 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, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27 , 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26 Lengths of approximately 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, or 36 base pairs, such as 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths between those described above are also considered to be part of the present invention.

[0116] The two strands forming the double-stranded structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of one larger molecule and are therefore joined by a contiguous chain of nucleotides between the 3' end of one strand and the 5' end of the other, the joining RNA strands are called a “hairpin loop.” A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twenty, at least 23 or more unpaired nucleotides.

[0117] If the two substantially complementary strands of a dsRNA are composed of separate RNA molecules, these molecules may or may not be covalently linked. If the two strands are covalently linked by means other than a contiguous chain of nucleotides between the 3' end of one strand and the 5' end of the other, the linking structure is called a "linker." RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus the overhangs present in the double helix. In addition to the double helix structure, RNAi agents may contain one or more nucleotide overhangs.

[0118] In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, for example, a contact activation pathway gene, i.e., a KLKB1 target mRNA sequence, an F12 target mRNA sequence, or a KNG1 target mRNA sequence, to lead to the cleavage of the target RNA. Although we do not wish to be constrained by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). When dsRNA is processed by the ribonuclease-III-like enzyme Dicer, it becomes a 19-23 base pair small interfering RNA with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated to form the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0119] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists if the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain or consist of a nucleotide / nucleoside analog containing a deoxynucleotide / nucleoside. The overhang may be the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located at the 5' end, the 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0120] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' and / or 5' ends, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are substituted with nucleoside thiophosphates.

[0121] In certain embodiments, the overhang in the sense strand, the antisense strand, or both may include an extended length longer than 10 nucleotides, for example, 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides. In certain embodiments, the extended overhang is located on the double-stranded sense strand. In certain embodiments, the extended overhang is located at the 3' end of the double-stranded sense strand. In certain embodiments, the extended overhang is located at the 5' end of the double-stranded sense strand. In certain embodiments, the extended overhang is located on the double-stranded antisense strand. In certain embodiments, the extended overhang is located at the 3' end of the double-stranded antisense strand. In certain embodiments, the extended overhang is located at the 5' end of the double-stranded antisense strand. In certain embodiments, one or more nucleotides in the overhang are substituted with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary portion, and therefore the overhang can form a stable hairpin structure under physiological conditions.

[0122] "Smooth" or "blunt-ended" means that there are no unpaired nucleotides at the corresponding end of a double-stranded RNAi agent, i.e., there are no nucleotide overhangs. A "blunt-ended" RNAi agent is a dsRNA that is double-stranded throughout its entire length, i.e., has no nucleotide overhangs at any end of the molecule. The RNAi agents of the present invention include RNAi agents having a nucleotide overhang at one end (i.e., an agent having one overhang and one blunt end) or RNAi agents having nucleotide overhangs at both ends.

[0123] The terms “antisense strand” or “guide strand” refer to, for example, a strand of iRNA, e.g., dsRNA, that contains a region substantially complementary to the target sequence, e.g., KLKB1 mRNA. As used herein, the term “complementary region” refers to a region of the antisense strand that is substantially complementary to the sequence, e.g., the target sequence, e.g., the contact activation pathway gene nucleotide sequence, as defined herein. If the complementary region is not fully complementary to the target sequence, mismatches may exist in the internal or terminal regions of the molecule. Generally, most acceptable mismatches are located in terminal regions, e.g., in 5, 4, 3, 2, or 1 nucleotide at the 5' and / or 3' ends of the iRNA. In one embodiment, the double-stranded RNAi agent of the present invention contains a nucleotide mismatch in the antisense strand. In another embodiment, the double-stranded RNAi agent of the present invention contains a nucleotide mismatch in the sense strand. In one embodiment, the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotide from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, at the 3' terminal nucleotide of the iRNA.

[0124] As used herein, the terms “sense strand” or “passenger strand” refer to a strand of iRNA containing a region substantially complementary to the antisense strand region, as defined herein.

[0125] As used herein, the term “cleavage region” refers to a region located directly adjacent to a cleavage site. A cleavage site is the site in the target where cleavage occurs. In one embodiment, the cleavage region includes three bases directly adjacent to the cleavage site at either end of the cleavage site. In another embodiment, the cleavage region includes two bases directly adjacent to the cleavage site at either end of the cleavage site. In yet another embodiment, the cleavage site occurs specifically at a site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0126] When used herein, unless otherwise specified, the term “complementary” means, as understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under given conditions to form a double-stranded structure, where the first nucleotide sequence is described in relation to a second nucleotide sequence. Such conditions may be, for example, stringent conditions, where stringent conditions may include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, 50°C or 70°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 that may occur within living organisms, may be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of the two sequences, depending on the final use of the hybridized nucleotides.

[0127] In the iRNAs described herein, for example, complementary sequences in dsRNAs include base pairings of one or both nucleotide sequences over the full length of one or both nucleotide sequences of an oligonucleotide or polynucleotide containing a first nucleotide sequence to an oligonucleotide or polynucleotide containing a second nucleotide sequence. Such sequences may be referred to herein as “fully complementary” to each other. However, if the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may be fully complementary, or, when hybridized to double helixes of up to 30 base pairs, while retaining their ability to hybridize under optimal conditions for their final application, e.g., inhibition of gene expression via the RISC pathway, they may form one or more mismatched base pairs, but generally five or fewer, four or fewer, three or fewer, or two or fewer. However, if the two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides and the other oligonucleotide of 23 nucleotides for the purposes described herein may be referred to as “fully complementary” if the longer oligonucleotide contains a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide.

[0128] As used herein, “complementary” sequences may include, or may be entirely formed from, non-Watson-Crick base pairs and / or non-natural and modified nucleotides, insofar as the above requirements related to their hybridizing ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuation or Hoogsteen-type base pairs.

[0129] The terms “complementary,” “fully complementary,” and “substantially complementary” as used herein may be used in relation to matching bases between the sense strand and antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as can be understood from the context in which they are used.

[0130] As used herein, a polynucleotide "substantially complementary to at least a portion of" messenger RNA (mRNA) means a polynucleotide substantially complementary to a contiguous portion of the mRNA of interest (e.g., mRNA encoding a contact activation pathway gene) including the 5'UTR, open reading frame (ORF), or 3'UTR. For example, a polynucleotide is complementary to at least a portion of KLKB1 mRNA if its sequence is substantially complementary to a contiguous portion of the mRNA encoding the KLKB1 gene.

[0131] Accordingly, in some embodiments, the sense strand polynucleotides and antisense polynucleotides disclosed herein are fully complementary to the target contact activation pathway gene sequences.

[0132] In one embodiment, the antisense polynucleotide disclosed herein is fully complementary to the target KLKB1 sequence. In another embodiment, the antisense polynucleotide disclosed herein is substantially complementary to the target KLKB1 sequence and comprises a continuous nucleotide sequence that is at least about 80% complementary over the corresponding region of the nucleotide sequence of either SEQ ID NOs: 1 and 2, or the fragment of either SEQ ID NOs: 1 and 2, and over its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0133] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target KLKB1 sequence and include a continuous nucleotide sequence that is at least about 80% complementary over its entire length to any one sense strand nucleotide sequence in any one of Tables 3, 4, 19A, or 19B, or any fragment of any one antisense strand nucleotide sequence in any one of Tables 3, 4, 19A, or 19B, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0134] In one embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide, wherein the antisense polynucleotide is complementary to a target KLKB1 sequence, and comprises one of the antisense strand nucleotide sequences in any one of Tables 3, 4, 19A, or 19B, or a fragment of one of the antisense strand nucleotide sequences in any one of Tables 3, 4, 19A, or 19B, and a continuous nucleotide sequence that is at least about 80% complementary over its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0135] In one embodiment, the antisense polynucleotide disclosed herein is fully complementary to the target F12 sequence. In another embodiment, the antisense polynucleotide disclosed herein is substantially complementary to the target F12 sequence and comprises a continuous nucleotide sequence that is at least about 80% complementary over the corresponding region of the nucleotide sequence of SEQ ID NO: 9 or 10, or a fragment of SEQ ID NO: 9 or 10, and over its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0136] In other embodiments, the antisense chain polynucleotide is substantially complementary to the target F12 sequence and comprises a continuous nucleotide sequence that is at least about 80% complementary over its entire length to any one of the sense chain nucleotide sequences in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27, or any fragment of any one of the antisense chain nucleotide sequences in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27, and for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0137] In one embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide, wherein the antisense polynucleotide is complementary to a target F12 sequence, and comprises one antisense strand nucleotide sequence in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27, or a fragment of one antisense strand nucleotide sequence in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27, and a continuous nucleotide sequence that is at least about 80% complementary over its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0138] In one embodiment, the sense-strand polynucleotides and antisense polynucleotides disclosed herein are fully complementary to the target KNG1 sequence. In other embodiments, the sense-strand polynucleotides and / or antisense polynucleotides disclosed herein are substantially complementary to the target KNG1 sequence and include a continuous nucleotide sequence that is at least about 80% complementary over the corresponding region of the nucleotide sequence of SEQ ID NO: 17 or 18, or the fragment of SEQ ID NO: 17 or 18, and over its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0139] In other embodiments, the antisense strand polynucleotide is substantially complementary to the target KNG sequence and comprises a sequence of nucleotides that is at least about 80% complementary over its entire length to one of the sense strand nucleotide sequences in any one of 15 or 16, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0140] In one embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide, wherein the antisense polynucleotide is complementary to a target KNG1 sequence, and comprises one of the antisense strand nucleotide sequences in Table 15 or 16, or a fragment of one of the antisense strand nucleotide sequences in Table 15 or 16 and a continuous nucleotide sequence that is at least about 80% complementary over its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0141] Generally, in some embodiments, the majority of the nucleotides in each chain are ribonucleotides, but as described in detail herein, each or both chains may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, “iRNA” may contain ribonucleotides having chemical modifications. Such modifications may include any type of modification disclosed herein or known in the art. Any such modification used in an iRNA molecule is encompassed by “iRNA” for the purposes of this specification and the claims.

[0142] In one aspect of the present invention, the agent for use in the methods and compositions of the present invention is a single-stranded antisense RNA molecule that inhibits target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to the sequence in the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation stoichiometrically by base-pairing with mRNA and physically interfering with the translation mechanism (see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). The single-stranded antisense RNA molecule is about 15 to about 30 nucleotides long and may have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule may contain a sequence of at least about 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any one of the antisense sequences described herein.

[0143] As used herein, “Subject” refers to animals such as primates (humans, non-human primates, e.g., monkeys, and chimpanzees), non-primates (cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, and whales), or mammals, including birds (e.g., ducks or geese). In some embodiments, the subject is a human being, for example, a human being treated or evaluated for a disease, disorder, or condition in which a person may benefit from reduced contact-activated pathway gene expression (i.e., KLKB1 gene expression, F12 gene expression, and / or KNG1 gene expression) and / or replication as described herein; a human being at risk of a disease, disorder, or condition in which a person may benefit from reduced contact-activated pathway gene expression; a human being having a disease, disorder, or condition in which a person may benefit from reduced contact-activated pathway gene expression; and / or a human being treated for a disease, disorder, or condition in which a person may benefit from reduced contact-activated pathway gene expression.

[0144] As used herein, the terms “treat” or “remedy” refer to beneficial or desired outcomes, including, but not limited to, relief or improvement of one or more symptoms associated with contact-activated pathway gene expression (i.e., KLKB1 gene expression, F12 gene expression, and / or KNG1 gene expression) and / or contact-activated pathway protein production (i.e., KLKB1 protein production, F12 protein production, and / or KNG1 protein production), such as thrombogenic tendencies, e.g., thrombus formation, the presence of hyperbradykinin, hereditary angioedema (HAE), e.g., hereditary angioedema type I; hereditary angioedema type II; hereditary angioedema type III; or any other hereditary angioedema caused by hyperbradykinin levels, angioedema attacks, edema and swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals (genetials), prodromal symptoms; laryngeal swelling; non-pruritic rash; nausea; vomiting; and abdominal pain. "Treatment" can also mean an extension of survival compared to the survival expected without treatment.

[0145] In the context of the expression of the target contact-activated pathway gene and / or the production of the contact-activated pathway protein or the level of disease markers or symptoms, the term “lower” refers to a statistically significant decrease in such level. The decrease may be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more, preferably down to a level recognized as within the normal range for individuals without such disorder.

[0146] As used herein, “prevention” or “prevention” means, when used in relation to a disease, disorder or condition in which one may benefit from reduced expression of contact-activated pathway genes and / or production of contact-activated pathway proteins, for example, the formation of venous thrombosis, arterial thrombosis, ventricular thrombosis, thromboembolism, the presence of hyperbradykinin, angioedema attacks, hereditary angioedema type I; hereditary angioedema type II; hereditary angioedema type III; any other hereditary angioedema caused by hyperbradykinin levels; edema and swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals (genetials), prodromal symptoms; laryngeal swelling; non-pruritic rash; nausea; vomiting; abdominal pain, and other symptoms of contact-activated pathway gene expression, or a reduction in the likelihood of developing symptoms associated with such disease, disorder or condition, or a reduction in the frequency and / or duration of symptoms associated with such disease, disorder or condition. The absence of the onset of a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (for example, by at least about 10% on a clinically recognized scale for the disease or disorder), or a delay in symptoms (for example, by several days, weeks, months, or years), is considered effective prevention.

[0147] As used herein, the term “contact-activated pathway-related disorders” refers to diseases or disorders caused by or associated with contact-activated pathway gene expression (i.e., KLKB1 gene expression, F12 gene expression, and / or KNG1 gene expression) or contact-activated pathway protein production (i.e., KLKB1 protein production, F12 protein production, and / or KNG1 protein production). The term “contact-activated pathway-related disorders” includes diseases, disorders, or conditions that may benefit from reduced contact-activated pathway gene expression and / or contact-activated pathway protein activity. Contact-activated pathway-related disorders may be genetic or acquired disorders.

[0148] Non-exclusive examples of contact-activated pathway-related diseases include, for example, thrombosis, hereditary angioedema (HAE) (hereditary angioedema type I; hereditary angioedema type II; hereditary angioedema type III; or any other hereditary angioedema caused by high bradykinin levels), prekallikrein deficiency (hereditary or acquired), also known as Fletcher factor deficiency, malignant essential hypertension, hypertension, and end-stage renal disease.

[0149] In one embodiment, a contact-activated pathway-related disorder is a thrombotic tendency. As used herein, the term “thrombotic tendency” is also referred to as “hypoclomatization” or “pro-thrombosis” and is any disorder or condition associated with blood coagulation abnormalities that increase the risk of thrombosis and thrombosis. As used herein, the term “thrombosis” refers to the process of local coagulation or clot formation (formation of a “thrombus” or “blood clot”) of blood in a part of the circulatory system. A thrombotic tendency may be hereditary, acquired, or a result of environmental conditions. Exemplary hereditary thrombotic tendencies include hereditary antithrombin deficiency, hereditary protein C deficiency, hereditary protein S deficiency, hereditary factor V Leyden thrombosis, and prothrombin (factor II) G20210A. Exemplary acquired thrombotic tendencies include antiphospholipid syndromes. Acquired / environmental thrombosis tendencies can be a result of, for example, trauma, fracture, surgery (e.g., orthopedic surgery, tumor surgery), use of oral contraceptives, hormone replacement therapy, pregnancy, postpartum period, hypercoaguability, history of thrombosis, aging, immobilization (e.g., bed rest for more than 3 days), prolonged travel, metabolic syndrome, and air pollution (see, for example, Previtali, et al. (2011) Blood Transfus 9:120). Therefore, "individuals at risk of thrombosis" include surgical patients (e.g., those undergoing general surgery, oral surgery, orthopedic surgery (e.g., total knee or hip replacement), trauma surgery, and tumor surgery); medical patients (e.g., those with immobilization disorders, those requiring bed rest for more than three days and / or long-term use of intravenous catheters; those with atrial fibrillation; elderly individuals; those with renal impairment; those with artificial heart valves; those with heart failure; those with cancer); pregnant individuals; postpartum individuals; those with a history of thrombosis; those receiving hormone replacement therapy; those sitting for long periods on airplanes or in cars; and obese individuals.

[0150] In one embodiment, the contact-activated pathway-related disorder is hereditary angioedema (HAE). As used herein, “hereditary angioedema” is used synonymously with the term “HAE” and refers to an autosomal dominant disorder caused by mutations in the C1 inhibitor (C1INH), SERPING1) gene, or coagulation factor XII (F12) gene, which cause recurrent edema and swelling in patients. Typical symptoms of HAE include severe swelling of the arms, legs, hands, feet, face, tongue and larynx, abdomen, trunk, and genitals, nausea, vomiting, abdominal pain, and a nonpriuric rash. Bradykinin peptide levels are elevated during HAE attacks or episodes.

[0151] In another embodiment, the contact activation pathway-related disease is prekallikrein deficiency.

[0152] In another embodiment, the contact activation pathway-related disease is malignant essential hypertension.

[0153] In another embodiment, the contact activation pathway-related disease is hypertension.

[0154] In another embodiment, the contact activation pathway-related disease is end-stage renal disease.

[0155] When used herein, “therapeutic dose” is intended to include an amount of RNAi agent sufficient to cause treatment of a disease (e.g., by reducing, improving, or maintaining one or more symptoms of the pre-existing disease or disease) when administered to a patient for the treatment of a subject having HAE and / or contact-activated pathway-related disease. “Therapeutic dose” may vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity, as well as the patient’s medical history, age, weight, family history, genetic structure, stage of the pathological process mediated by contact-activated pathway gene expression, the type of prior or concomitant treatment, if any, and other individual characteristics of the patient being treated.

[0156] When used herein, “Prophylactic effective dose” is intended to contain an amount of RNAi agent sufficient to prevent or improve one or more symptoms of a contact-activated pathway-related disease when administered to a subject who has not yet developed or shown symptoms of the disease but may be predisposed or at risk. Improvement of the disease includes slowing the course of the disease or reducing the severity of the disease if it develops later. The “Prophylactic effective dose” may vary depending on the RNAi agent, the method of administration of the agent, the degree of risk of the disease, and the patient’s medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and other individual characteristics of the patient being treated.

[0157] The “therapeutic effective dose” or “prophylactically effective dose” also includes the amount of RNAi agent that produces some desired local or systemic effect with a reasonable benefit-risk ratio applicable to any treatment. The RNAi agent used in the method of the present invention may be administered in an amount sufficient to produce a reasonable benefit-risk ratio applicable to such treatment.

[0158] The term “sample,” as used herein, includes similar bodily fluids, cells, or tissues taken from a subject, as well as collections of bodily fluids, cells, or tissues present within the body of the subject. Examples of bodily fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples from tissues, organs, or local areas. For example, a sample may originate from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In certain embodiments, a sample may originate from the liver (e.g., the whole liver or a specific part of the liver or a specific type of cell within the liver, such as hepatocytes), the retina or a part of the retina (e.g., retinal pigment epithelium), the central nervous system or a part of the central nervous system (e.g., the ventricles or choroid plexus), or the pancreas or specific cells or parts of the pancreas. In some embodiments, “sample derived from subject” refers to cerebrospinal fluid obtained from the subject. In preferred embodiments, “sample derived from subject” refers to blood or plasma taken from the subject. In a further embodiment, “sample derived from the subject” refers to liver tissue (or a portion thereof) or retinal tissue (or a portion thereof) derived from the subject.

[0159] II. The iRNA of the present invention The present invention provides an iRNA that inhibits the expression of contact activation pathway genes (i.e., the KLKB1 gene, F12 gene, or KNG1 gene). In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of contact activation pathway genes in cells, such as cells in the body of mammals, including humans, that have or are at risk of developing contact activation pathway-related diseases, such as thrombosis or hereditary angioedema. This dsRNA includes an antisense strand having a complementary region complementary to at least a portion of the mRNA formed in the expression of the contact activation pathway gene. This complementary region is approximately 30 nucleotides or less in length (for example, approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). When this iRNA comes into contact with cells expressing contact activation pathway genes, it inhibits the expression of contact activation pathway genes (e.g., human, primate, non-primate, or bird contact activation pathway genes) by at least approximately 10% when assayed by, for example, PCR or branched DNA (bDNA)-based methods, or by protein-based methods such as immunofluorescence analysis using, for example, Western blotting or flow cytometry.

[0160] dsRNA comprises two complementary RNA strands that hybridize to form a double-stranded structure under the conditions in which the dsRNA will be used. One strand of the dsRNA (the antisense strand) contains a complementarity region that is substantially complementary to the target sequence, or generally fully complementary. The target sequence may originate from the mRNA sequence formed during the expression of a contact activation pathway gene (i.e., the KLKB1, F12, or KNG1 gene). The other strand (the sense strand) contains a region complementary to the antisense strand, and the two strands hybridize to form a double-stranded structure when combined under favorable conditions. As described elsewhere in this specification and as known in the art, the complementary sequence of the dsRNA may also be included as a self-complementary region of a single nucleic acid molecule, rather than on a separate oligonucleotide.

[0161] Generally, double-stranded structures are 15-30 base pairs long, for example, 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, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-2 The lengths are 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths between those listed above are also considered to be part of the present invention.

[0162] Similarly, the complementary region of the target sequence is 15-30 nucleotides long, e.g., 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, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 1 The nucleotide lengths are 9-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. Intermediate ranges and lengths between those listed above are also considered to be part of the present invention.

[0163] In some embodiments, the dsRNA is about 15–20 nucleotides long, or about 25–30 nucleotides long. Generally, dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21–23 nucleotides can serve as a substrate for Dicer. As those skilled in the art will also recognize, the RNA region targeted for cleavage is in most cases part of a larger RNA molecule (often an mRNA molecule). Where applicable, the “part” of the mRNA target is a contiguous sequence of mRNA targets long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0164] The double-stranded region is the primary functional part of dsRNA, for example, approximately 9-36 base pairs, for example, approximately 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-3 3, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-2 4, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 1 Those skilled in the art will also recognize that the double-stranded region is 9-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Herein, in one embodiment, a complex of an RNA molecule or an RNA molecule having a double-stranded region of more than 30 base pairs is dsRNA to the extent that it is processed into a functional double-strand of, for example, 15-30 base pairs that targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that in one embodiment, miRNA is dsRNA. In another embodiment, dsRNA is not a natural miRNA. In another embodiment, iRNA agents useful for targeting the expression of contact activation pathway genes are not generated in target cells by cleavage of larger dsRNAs.

[0165] The dsRNAs described herein may further include one or more single-stranded nucleotide overhangs, e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. Nucleotide overhangs may contain or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. Overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0166] dsRNA can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, Inc.), as will be further described later.

[0167] The iRNA compounds of the present invention can be prepared using a two-step procedure. First, the individual strands of a double-stranded RNA molecule are prepared separately. Next, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase, solid-phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotide strands containing non-natural or modified nucleotides can be readily prepared. The single-stranded oligonucleotides of the present invention can be prepared using solution-phase, solid-phase organic synthesis, or both.

[0168] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand is selected from the group of sequences provided in any one of Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27, and the corresponding antisense strand of the sense strand is selected from the group of sequences provided in any one of Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27.

[0169] In one embodiment, the sense strand is selected from the group of sequences provided in any one of Tables 3, 4, 19A, and 19B, and the corresponding antisense strand of the sense strand is selected from the group of sequences provided in any one of Tables 3, 4, 19A, and 19B. In this embodiment, one of these two sequences is complementary to the other of these two sequences, and one of these sequences is substantially complementary to the sequence of mRNA produced in the expression of the KLKB1 gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide described as the sense strand in any one of Tables 3, 4, 19A, and 19B, and the second oligonucleotide described as the corresponding antisense strand of the sense strand in any one of Tables 3, 4, 19A, and 19B. In one embodiment, substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0170] In one embodiment, the sense strand is selected from the group of sequences provided in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27, and the corresponding antisense strand of the sense strand is selected from the group of sequences provided in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27. In this embodiment, one of these two sequences is complementary to the other of these two sequences, and one of these sequences is substantially complementary to the mRNA sequence produced in the expression of the F12 gene. Accordingly, in this embodiment, the dsRNA comprises two oligonucleotides, one of which is described as the sense strand in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27, and the second oligonucleotide is described as the corresponding antisense strand in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27. In one embodiment, substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0171] In one embodiment, the sense strand is selected from the group of sequences provided in any one of Tables 15, 16, 19E, and 19F, and the corresponding antisense strand of the sense strand is selected from the group of sequences provided in any one of Tables 15, 16, 19E, and 19F. In this embodiment, one of these two sequences is complementary to the other of these two sequences, and one of these sequences is substantially complementary to the mRNA sequence produced in the expression of the KNG1 gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide described as the sense strand in any one of Tables 15, 16, 19E, and 19F, and the second oligonucleotide described as the corresponding antisense strand of the sense strand in any one of Tables 15, 16, 19E, and 19F. In one embodiment, substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0172] While some of the sequences in Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27 are described as modified and / or conjugated sequences, it will be understood that the RNA of the iRNA of the present invention, for example, the dsRNA of the present invention, may include any one of the sequences shown in Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27, which are unmodified, unconjugated, and / or modified and / or conjugated in a manner different from those described in these tables.

[0173] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of about 20 to about 23 base pairs, for example, 21 base pairs, have been found to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA double-stranded structures may also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, thanks to the properties of the oligonucleotide sequences provided in any one of Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27, the dsRNAs described herein may include at least one strand of a minimum length of 21 nucleotides. It is naturally expected that shorter double-stranded sequences obtained by subtracting just a few nucleotides from one or both ends of any one of the sequences in Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27 may be equally effective compared to the dsRNAs described above.Therefore, dsRNA having at least 15, 16, 17, 18, 19, 20 consecutive nucleotides or more derived from one of the sequences in Tables 3, 4, 19A, and 19B, and whose inhibitory ability on KLKB1 gene expression differs from that of dsRNA containing the complete sequence by only about 5, 10, 15, 20, 25, or 30% or less, and having at least 15, 16, 17, 18, 19, 20 consecutive nucleotides or more derived from one of the sequences in Tables 9, 10, 19C, 19D, 20, and 21, and It is intended that the present invention includes dsRNAs whose inhibitory ability on F12 gene expression differs from that of a dsRNA containing the complete sequence by only about 5, 10, 15, 20, 25, or 30% or less, and dsRNAs having at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides or more derived from one of the sequences in Tables 15, 16, 19E, and 19F, and whose inhibitory ability on KNG1 gene expression differs from that of a dsRNA containing the complete sequence by only about 5, 10, 15, 20, 25, or 30% or less.

[0174] In addition, the RNAs provided in any one of Tables 3, 4, 19A, and 19B identify RISC-mediated cleavage-prone sites in the KLKB1 transcript, the RNAs provided in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27 identify RISC-mediated cleavage-prone sites in the F12 transcript, and the RNAs provided in any one of Tables 15, 16, 19E, and 19F identify RISC-mediated cleavage-prone sites in the KNG1 transcript. Accordingly, the present invention further features iRNAs that target within the range of one of these sites. As used herein, an iRNA is said to target within a specific range of sites in an RNA transcript if the iRNA promotes cleavage of the transcript within any of the ranges of those specific sites. Such iRNAs generally contain at least about 15 consecutive nucleotides derived from one of the sequences provided to any one of Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27, coupled with an additional nucleotide sequence taken from a region adjacent to the selection sequence in the contact activation pathway gene.

[0175] Target sequences are generally about 15–30 nucleotides long, but the suitability of specific sequences within this range to guide the cleavage of any given target RNA varies. While the various software packages and guidelines described herein provide guidance for identifying the optimal target sequence for any given gene target, empirical methods can also be employed, where a “window” or “mask” of a given size (21 nucleotides as an example) is placed literally or figuratively (including in silico) on the target RNA sequence to identify sequences within a size range in which they can act as target sequences. The next potential target sequence can be identified by gradually shifting the sequence “window” one nucleotide upstream or downstream of the initial target sequence position until a complete set of possible sequences is identified for any given target size of choice. This process, along with the systematic synthesis and testing of identified sequences (using assays described herein or known in the art) to identify the optimally functioning sequence, can identify the RNA sequence that best mediates the inhibition of target gene expression when targeted with an iRNA agent. Therefore, for example, a sequence identified in any one of Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27 represents an effective target sequence, but further optimization of inhibition efficiency can be achieved by gradually "shifting the window" one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory properties.

[0176] Furthermore, for example, further optimization may be achieved by systematically adding or removing nucleotides to any sequence identified in any one of Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27 to generate longer or shorter sequences, and by testing the resulting sequences by shifting the longer or shorter size window upward or downward from that point towards the target RNA. In addition, the efficiency of inhibition may be further improved by combining this method for generating novel candidate targets in inhibition assays known in the art and / or described herein with testing the efficacy of iRNAs based on those target sequences. Furthermore, such optimized sequences may be modified, for example, by introducing modified nucleotides described herein or known in the art, adding or altering overhangs, or by other modifications known in the art and / or described herein to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermal stability, improving membrane permeable delivery, targeting to specific sites or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes).

[0177] The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatched region is not located in the center of the complementary region. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, for a 23-nucleotide iRNA agent, the strand complementary to the region of the contact activation pathway gene generally contains no mismatches in the central 13 nucleotides. Using the methods described herein or methods known in the art, it is possible to determine whether an iRNA containing a mismatch with the target sequence is effective in inhibiting the expression of the contact activation pathway gene. Considering the effectiveness of iRNAs with mismatches in inhibiting the expression of the contact activation pathway gene is particularly important when it is known that a particular complementary region in the contact activation pathway gene has polymorphic sequence variation within the population.

[0178] III. Modified iRNA of the present invention In one embodiment, the RNA of the iRNA of the present invention, e.g., dsRNA, is unmodified and does not contain, for example, any chemical modifications and / or conjugates known in the art and described herein. In another embodiment, the RNA of the iRNA of the present invention, e.g., dsRNA, is chemically modified to improve stability or other beneficial properties. In a particular embodiment of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In another embodiment of the present invention, all of the nucleotides of the iRNA of the present invention are modified. The iRNA of the present invention that "substantially all of its nucleotides are modified" is mostly modified but not completely modified and may contain five or fewer, four or fewer, three or fewer, two or fewer, or one or fewer unmodified nucleotides. In some embodiments, substantially all of the nucleotides of the iRNA of the present invention are modified, and the iRNA includes eight or fewer 2'-fluoro modifications on the sense strand (e.g., seven or fewer 2'-fluoro modifications, six or fewer 2'-fluoro modifications, five or fewer 2'-fluoro modifications, four or fewer 2'-fluoro modifications, three or fewer 2'-fluoro modifications, or two or fewer 2'-fluoro modifications) and six or fewer 2'-fluoro modifications on the antisense strand (e.g., five or fewer 2'-fluoro modifications, four or fewer 2'-fluoro modifications, three or fewer 2'-fluoro modifications, or two or fewer 2'-fluoro modifications). In other embodiments, all nucleotides of the iRNA of the present invention are modified, and the iRNA includes eight or fewer 2'-fluoro modifications on the sense strand (e.g., seven or fewer 2'-fluoro modifications, six or fewer 2'-fluoro modifications, five or fewer 2'-fluoro modifications, four or fewer 2'-fluoro modifications, three or fewer 2'-fluoro modifications, or two or fewer 2'-fluoro modifications) and six or fewer 2'-fluoro modifications on the antisense strand (e.g., five or fewer 2'-fluoro modifications, four or fewer 2'-fluoro modifications, three or fewer 2'-fluoro modifications, or two or fewer 2'-fluoro modifications).

[0179] Nucleic acids addressed in the present invention may be synthesized and / or modified by methods well established in the art, such as those described herein by reference in “Current protocols in nucleic acid chemistry,” Beaucage, S. Let al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Modifications include, for example, terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugate, inverted linkage) or 3'-terminal modifications (conjugate, DNA nucleotide, inverted linkage, etc.); base modifications, e.g., substitution with stable bases, unstable bases, or bases that base-pair with a wide range of partners, base removal (non-basic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and / or skeletal modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing a modified skeleton or lacking natural nucleoside-to-nucleoside bonds. RNAs having a modified skeleton include, in particular, those that do not have a phosphorus atom in their skeleton. For the purposes of this specification and as is sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside skeleton can also be considered as oligonucleosides. In some embodiments, the modified iRNA has a phosphorus atom in its internucleoside skeleton.

[0180] Examples of modified RNA backbones include phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methylphosphonate, and other alkylphosphonates including 3'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-aminophosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and boranophosphate having a normal 3'-5' linkage, their 2'-5' linkage analogs, and those having an inverted polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms.

[0181] Representative U.S. patents teaching the preparation of the phosphorus-containing bond described above include, but are not limited to, U.S. Patent No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,195; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; and No. 5,32 Specification No. 1,131; Specification No. 5,399,676; Specification No. 5,405,939; Specification No. 5,453,496; Specification No. 5,455,233; Specification No. 5,466,677; Specification No. 5,476,925 Specification No. 5,519,126; Specification No. 5,536,821; Specification No. 5,541,316; Specification No. 5,550,111; Specification No. 5,563,253; Specification No. 5,571,799; Specification No. 5,587 ,361 Specification; 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, Examples include U.S. Patent No. 639; No. 6,608,035; No. 6,683,167; No. 6,858,715; No. 6,867,294; No. 6,878,805; No. 7,015,315; No. 7,041,816; No. 7,273,933; No. 7,321,029; and U.S. Reissue Patent No. RE39464, the entire contents of each of these are incorporated herein by reference.

[0182] Modified RNA backbones that do not contain phosphorus atoms within have a backbone formed by short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatom and alkyl or cycloalkyl nucleoside linkages, or one or more short-chain heteroatom or heterocyclic nucleoside linkages. These include those having a morpholino linkage (partially formed from the sugar moiety of a nucleoside); a siloxane backbone; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl 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 CH2 component parts.

[0183] Representative U.S. patents teaching the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 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 by reference.

[0184] In other embodiments, suitable RNA mimetic compounds are considered for use in iRNA, where both the sugar and nucleoside bonds, i.e., the nucleotide unit backbone, are replaced with novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimetic compound, that has been shown to have excellent hybridization properties is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bonded directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262, the entire contents of which are incorporated herein by reference. Further PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0185] Some embodiments of the present invention include RNA having a phosphorothioate skeleton and oligonucleosides having a heteroatom skeleton, in particular including the -CH2-NH-CH2-, --CH2-N(CH3)-O-CH2- [known as the methylene(methylimino) or MMI skeleton], --CH2-ON(CH3)-CH2--, --CH2-N(CH3)-N(CH3)-CH2- and -N(CH3)-CH2-CH2- [wherein the natural phosphodiester skeleton is represented as -OPO-CH2--], and the amide skeleton of the aforementioned U.S. Patent No. 5,602,240. In some embodiments, the RNA described herein has the morpholino skeleton structure of the aforementioned U.S. Patent No. 5,034,506.

[0186] Modified RNA can also contain one or more substituted sugar moieties. The iRNAs taken up herein, for example, dsRNA, can contain at the 2'-position one of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 alkyl or C2-C 10 can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2).nOCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA is at the 2'-position C1-C 10The modifications include lower alkyl groups, substituted lower alkyl groups, alkali groups, aralkyl groups, O-alkaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalator groups, groups that improve the pharmacodynamic properties of iRNA, or groups that improve the pharmacokinetic properties of iRNA, and one of other substituents having similar properties. In some embodiments, the modifications include 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O--CH2CH2OCH3) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications are 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., the O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2, as described in the following examples herein.

[0187] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions in the iRNA's RNA, particularly on the 3' terminal nucleotide or at the 3' and 5' positions of the sugar in the 2'-5' linked dsRNA. The iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; Nos. 5,118,800; Nos. 5,319,080; Nos. 5,359,044; Nos. 5,393,878; Nos. 5,446,137; Nos. 5,466,786; Nos. 5,514,785; Nos. 5,519,134; and Nos. 5,567,81 Examples include Specification No. 1; Specification No. 5,576,427; Specification No. 5,591,722; Specification No. 5,597,909; Specification No. 5,610,300; Specification No. 5,627,053; Specification No. 5,639,873; Specification No. 5,646,265; Specification No. 5,658,873; Specification No. 5,670,633; and Specification No. 5,700,920, some of which are owned by the same person as the present application. The entire contents of each of the above are incorporated herein by reference.

[0188] The RNA of iRNA may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include deoxythymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, 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-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, and 5-uracil (pseudo This includes uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 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, as well as other synthetic and natural nucleic acid bases such as 3-deazaguanine and 3-deazaadenine.Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in Concise Encyclopedia Of Polymer Science and Engineering, pp. 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed in the present 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. The 5-methylcytosine substituent has been shown to increase nucleic acid double-strand stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp.276–278), and is an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0189] Representative U.S. patents teaching the above-mentioned modified nucleic acid bases and some other modified nucleic acid base preparations include, but are not limited to, U.S. Patent 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; and 5,594,1 Examples include Specification No. 21, No. 5,596,091; No. 5,614,617; No. 5,681,941; No. 5,750,692; No. 6,015,886; No. 6,147,200; No. 6,166,197; No. 6,222,025; No. 6,235,887; No. 6,380,368; No. 6,528,640; No. 6,639,062; No. 6,617,438; No. 7,045,610; No. 7,427,672; and No. 7,495,088, the entire contents of each of these are incorporated herein by reference.

[0190] The RNA of iRNA can also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a bridge of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a bridge that connects two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in certain embodiments, the agent of the present invention may include one or more fixed nucleic acids (LNAs). A fixed nucleic acid is a nucleotide having a modified ribose moiety, the ribose moiety including an additional bridge that connects the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively “fixes” the ribose in its 3'-endo structural configuration. The addition of immobilized nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention may be one or more bicyclic nucleosides containing a 4'-2' bridge.Examples of such 4'-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 known as "restricted ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and its analogues; see, for example, U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and its analogues; see, for example, U.S. Patent No. 8,278,2 Examples include: (see Patent No. 83); 4'-CH2-N(OCH3)-2' (and its analogues; see, e.g., U.S. Patent No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (where R is H, C1-C12 alkyl), or protecting groups (see, e.g., U.S. Patent 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(=CH2)-2' (and its analogues; see, e.g., U.S. Patent No. 8,278,426). The entire contents of each of these are incorporated herein by reference.

[0191] Further representative U.S. patents and publications teaching the preparation of fixed nucleic acid nucleotides include, but are not limited to, U.S. Patent 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; and 7 Examples include U.S. Patent Publication No. 427,672; No. 7,569,686; No. 7,741,457; No. 8,022,193; No. 8,030,467; No. 8,278,425; No. 8,278,426; No. 8,278,283; U.S. Patent Publication No. 2008 / 0039618; and U.S. Patent Publication No. 2009 / 0012281, the entire contents of each of these are incorporated herein by reference.

[0192] For example, any of the above bicyclic nucleosides having one or more stereochemical sugar configurations, including α-L-ribofuranose and β-D-ribofuranose, can be prepared (see International Publication No. 99 / 14226).

[0193] The RNA of the iRNA may also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” is a fixed nucleic acid containing a bicyclic sugar moiety including a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is in an S configuration referred to herein as “S-cEt”.

[0194] The iRNA of the present invention may also include one or more “contourally restricted nucleotides” (“CRNs”). A CRN is a nucleotide analog having a linker that connects the C2' and C4' carbons or the C3 and -C5' carbons of ribose. CRNs fix the ribose ring to a stable configuration and increase its hybridization affinity to mRNA. The linker is long enough to position oxygen in an optimal location for stability and affinity, and reduces puckering of the ribose ring.

[0195] Representative publications teaching some of the above preparations of CRN include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and PCT Publication No. 2013 / 036868, the entire contents of which are incorporated herein by reference.

[0196] One or more nucleotides of the iRNA of the present invention may also include hydroxymethyl-substituted nucleotides. A "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also known as an "unlocked nucleic acid" ("UNA") modification.

[0197] Representative U.S. patent publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Application Publications 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of which are incorporated herein by reference.

[0198] Potentially stable modifications to the ends of RNA molecules may include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl 4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3”-phosphate, inverted base dT (idT), etc. Disclosure of these modifications can be found in PCT Publication No. International Publication No. 2011 / 005861.

[0199] Other nucleotide modifications of the iRNA of the present invention include 5' phosphates or 5' phosphate mimetic models, such as 5' terminal phosphates or phosphate mimetic models for the antisense strand of an RNAi agent. Preferred phosphate mimetic models are described, for example, in U.S. Patent Application Publication No. 2012 / 0157511 (all of which are incorporated herein by reference).

[0200] A. Modified iRNA containing the motif of the present invention In certain embodiments of the present invention, examples of double-stranded RNAi agents include agents having chemical modifications disclosed in U.S. Provisional Patent Application No. 61 / 561,710 filed November 18, 2011, or PCT / US2012 / 065691 filed November 16, 2012, respectively, the entire contents of which are incorporated herein by reference. As shown herein and in U.S. Provisional Patent Application No. 61 / 561,710 or PCT Application No. PCT / US2012 / 065691, better results are obtained by introducing one or more motifs of three identical modifications on a triple nucleotide into the sense and / or antisense strands of the RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent may be modified or completely modified. The introduction of these motifs disrupts the modification pattern of the sense and / or antisense strands, if present. RNAi agents can be optionally conjugated with a GalNAc derivative ligand, for example, on the sense strand. The resulting RNAi agents exhibit superior gene silencing activity.

[0201] More specifically, it was surprisingly discovered that the gene silencing activity of the RNAi agent was significantly improved when the sense and antisense strands of the double-stranded RNAi agent were completely modified to have one or more motifs of three identical modifications on a triple nucleotide sequence at or near the cleavage site of at least one strand of the RNAi agent.

[0202] Therefore, the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., a contact activation pathway gene, i.e., the KLKB1 gene, the F12 gene, or the KNG1 gene) in vivo. The RNAi agent includes a sense strand and an antisense strand. Each strand of the RNAi agent can be in the range of 12 to 30 nucleotides in length. For example, each strand can be 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

[0203] The sense strand and the antisense strand typically form a double-stranded RNA (''dsRNA''), also referred to herein as an ''iRNA agent''. The double-stranded region of the iRNA agent can be 12 to 30 nucleotide pairs in length. For example, the double-stranded region can be 14 to 30 nucleotide pairs in length, 17 to 30 nucleotide pairs in length, 27 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 21 nucleotide pairs in length, 17 to 19 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, 21 to 25 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0204] In one embodiment, the RNAi agent may contain one or more overhang regions and / or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhang may be 1 to 6 nucleotides long, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. The overhang may result from one strand being longer than the other, or from two strands of equal length being staggered. The overhang may form a mismatch with the target mRNA, or it may be complementary to or different from the target gene sequence. The first and second strands may also be joined by additional bases or other non-base linkers, for example, to form a hairpin.

[0205] In one embodiment, each nucleotide in the overhang region of the RNAi agent may independently be a modified or unmodified nucleotide, including, but not limited to, 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof, including 2'-sugar modifications. For example, TT may be an overhang sequence for any end on any strand. The overhang may form a mismatch with the target mRNA, or it may be complementary to the targeted gene sequence, or it may be a different sequence.

[0206] 5'- or 3'-overhangs in the sense strand, antisense strand, or both strands of an RNAi agent can be phosphorylated. In one embodiment, the overhang region comprises two nucleotides having a phosphorothioate between them, where the two nucleotides may be the same or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is located in the antisense strand. In one embodiment, this 3'-overhang is located in the sense strand.

[0207] RNAi agents may contain only one overhang that can enhance RNAi interference activity without affecting their overall stability. For example, a single-stranded overhang may be located at the 3' end of the sense strand or the 3' end of the antisense strand. RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa. Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end and a blunt end at the 5' end. While we do not wish to be constrained by theory, asymmetric blunt ends at the 5' end and 3' end overhangs of the antisense strand are favorable for introducing guide strands into RISC processes.

[0208] In one embodiment, the RNAi agent is a 19-nucleotide long double-ended bluntmer, and the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides from the 5' end at positions 7, 8, and 9. The antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides from the 5' end at positions 11, 12, and 13.

[0209] In another embodiment, the RNAi agent is a 20-nucleotide blunt-ended double strand, the sense strand containing at least one motif of three 2'-F modifications in three consecutive nucleotides from the 5' end at positions 8, 9, and 10. The antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides from the 5' end at positions 11, 12, and 13.

[0210] In yet another embodiment, the RNAi agent is a 21-nucleotide blunt-ended double strand, the sense strand containing at least one motif of three 2'-F modifications in three consecutive nucleotides from the 5' end at positions 9, 10, and 11. The antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides from the 5' end at positions 11, 12, and 13.

[0211] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, with one end of the RNAi agent being blunt and the other end comprising two nucleotide overhangs. Preferably, the two nucleotide overhangs are located at the 3' end of the antisense strand.

[0212] If two nucleotide overhangs are located at the 3' end of the antisense strand, there may be two phosphorothioate nucleotide interbonds between the three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotides. In one embodiment, the RNAi agent further has two phosphorothioate nucleotide interbonds between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, all nucleotides in the sense and antisense strands of the RNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In one embodiment, each residue is independently modified, for example, with 2'-O-methyl or 3'-fluoro in alternating motifs. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0213] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the sense strand being 25-30 nucleotides long, with positions 1-23 of the first strand containing at least 8 ribonucleotides starting from the 5' terminal nucleotide (position 1); the antisense strand being 36-66 nucleotides long, with at least 8 ribonucleotides starting from the 3' terminal nucleotide, forming a double helix with positions 1-23 of the sense strand paired with the sense strand; at least 3' terminal nucleotides of the antisense strand not paired with the sense strand, with up to 6 consecutive 3' terminal nucleotides not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; and 10-30 consecutive nucleotides of the 5' end of the antisense strand not paired with the sense strand. It contains an oside, thereby forming a single-stranded 5' overhang of 10–30 nucleotides; at least the 5' and 3' terminal nucleotides of the sense strand are bases that pair with the nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length so as to reduce the expression of the target gene when the double-stranded nucleic acid is introduced into mammalian cells; the sense strand contains at least one motif of three 2'-F modifications in a triple nucleotide, where at least one of the motifs is located at or near the cleavage site; the antisense strand contains at least one motif of three 2'-O-methyl modifications in a triple nucleotide at or near the cleavage site.

[0214] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand having a nucleotide length of at least 25 and no more than 29, and a second strand having a length of no more than 30 nucleotides, comprising at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, and the double-stranded region is at least 25 nucleotides long, the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the length of the second strand so that when the RNAi agent is introduced into mammalian cells, the expression of the target gene is reduced, and dicer cleavage of the RNAi agent preferentially yields the siRNA including the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. Optionally, RNAi agents may further contain ligands.

[0215] In one embodiment, the sense strand of the iRNA agent comprises at least one motif of three identical modifications in a triple nucleotide sequence, one of which is located at a cleavage site of the sense strand.

[0216] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif of three identical modifications in a triple nucleotide sequence, one of which is located at or near the cleavage site of the antisense strand.

[0217] In RNAi agents with a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically located near positions 10, 11, and 12 from the 5' end. Therefore, the three identical modification motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide in the double-stranded region from the 5' end of the antisense strand. The cleavage sites in the antisense strand can also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0218] The sense strand of an RNAi agent may contain at least one motif of three identical modifications in a triple nucleotide sequence at the cleavage site; the antisense strand may have at least one motif of three identical modifications in a triple nucleotide sequence at or near the cleavage site. When the sense and antisense strands form a dsRNA double helix, the sense and antisense strands may be aligned such that one motif of three nucleotides in the sense strand and one motif of three nucleotides in the antisense strand have at least one nucleotide duplication, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may be duplicated, or all three nucleotides may be duplicated.

[0219] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications in a triple nucleotide sequence. The first motif may be located at or near a cleavage site on the strand, and the other motifs may be wing modifications. The term “wing modification” as used herein refers to a motif located on a different part of the strand, away from the motif at or near the cleavage site on the same strand. The wing modifications are adjacent to the first motif or separated by at least one or more nucleotides. If the motifs are directly adjacent to each other, their chemical structures are different; if the motifs are separated by one or more nucleotides, their chemical structures may be the same or different. Two or more wing modifications may be present. For example, if two wing modifications are present, each wing modification may be located at one end relative to the first motif at or near the cleavage site, or on either side of the lead motif.

[0220] Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications in a triple nucleotide sequence, with at least one of the motifs located at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in a sequence similar to those present in the sense strand.

[0221] In one embodiment, the wing modification in the sense or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0222] In another embodiment, the wing modification in the sense or antisense strand of the RNAi agent typically does not contain the first one or two paired nucleotides within the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0223] If the sense strand and antisense strand of the RNAi agent each contain at least one wing modification, the wing modification may be located at the same end of the double-stranded region and may have one, two, or three nucleotide duplicates.

[0224] If the sense strand and antisense strand of an RNAi agent each contain at least two wing modifications, the sense strand and antisense strand may be aligned such that two modifications from one strand are each located at one end of the double-stranded region and have one, two, or three nucleotide duplicates; two modifications from one strand are each located at the other end of the double-stranded region and have one, two, or three nucleotide duplicates; and two modifications from one strand are located on each side of the read motif and have one, two, or three nucleotide duplicates in the double-stranded region.

[0225] In one embodiment, all nucleotides in the sense and antisense strands of an RNAi agent, including nucleotides that are part of a motif, may be modified. Each nucleotide may be modified with the same or different modifications, which may include changes to one or more of the unbound phosphate oxygens and / or bound phosphate oxygens; changes to components of the ribose sugar, e.g., the 2' hydroxyl of the ribose sugar; large-scale substitution of the phosphate moiety by a "dephospho" linker; modification or substitution of native bases; and substitution or modification of the ribose-phosphate backbone.

[0226] Because nucleic acids are polymers of subunits, many modifications, such as modifications to bases, phosphate moieties, or unbound oxygen atoms of phosphate moieties, are located at repeating positions within the nucleic acid. In some cases, modifications may be present at all desired positions in the nucleic acid, but often this is not the case. For example, modifications may be present only at the 3' or 5' end, or only in the terminal region, e.g., at a position on the terminal nucleotide or at the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications may be present in the double-stranded region, the single-stranded region, or both. Modifications may be present only in the double-stranded region of RNA, or only in the single-stranded region of RNA. For example, phosphorothioate modifications at unbound oxygen positions may be present at only one or both ends, or only in the terminal region, e.g., at a position on the terminal nucleotide or at the last 2, 3, 4, 5, or 10 nucleotides of the strand, or in both the double-stranded and single-stranded regions, particularly at the ends. The 5' end or both ends may be phosphorylated.

[0227] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes (surrogates) in single-stranded overhangs, e.g., 5' or 3' overhangs, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of ribose sugars by modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'-F), or 2'-O-methyl modifications instead of ribosaccharides in nucleic acid bases, and modifications of phosphate groups, e.g., phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0228] In one embodiment, each residue in the sense and antisense chains is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. The chains may contain two or more modifications. In one embodiment, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro.

[0229] At least two distinct modifications are typically present in the sense and antisense chains. These two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others.

[0230] In one embodiment, N a and / or N b This includes alternating pattern modifications. As used herein, the term “alternating motif” refers to a motif having one or more modifications, where each modification is present in alternating nucleotides on a single chain. Alternating nucleotides may refer to one modification on every other nucleotide, one modification on every three nucleotides, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motifs may be “ABABABABABAB···”, “AABBAABBAABB···”, “AABAABAABAAB···”, “AAABAAABAAAB···”, “AAABBBAAABBB···”, or “ABCABCABCABC···”.

[0231] The types of modifications included in alternating motifs can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand may be selected from several possibilities of modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0232] In one embodiment, the RNAi agent of the present invention includes a modification pattern of alternating motifs in the sense strand that is shifted relative to the modification pattern of alternating motifs in the antisense strand. This shift may be such that the modification groups of the nucleotides in the sense strand correspond to different modification groups of the nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with an antisense strand in a dsRNA double strand, the alternating motifs in the sense strand may begin with "ABABAB" from 5' to 3' of the strand, and the alternating motifs in the antisense strand may begin with "BABABA" from 5' to 3' of the strand within the double-stranded region. As another example, the alternating motifs in the sense strand may begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motifs in the antisense strand may begin with "BBAABBAA" from 5' to 3' of the strand within the double-stranded region, thereby resulting in a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

[0233] In one embodiment, the RNAi agent comprises a pattern of alternating 2'-O-methyl and 2'-F modifications in the sense strand, which initially has a shift relative to the pattern of alternating 2'-O-methyl and 2'-F modifications in the antisense strand, i.e., a 2'-O-methyl modified nucleotide in the sense strand base-pairs with a 2'-F modified nucleotide in the antisense strand, and vice versa. The sense strand may begin at position 1 with a 2'-F modification, and the antisense strand may begin at position 1 with a 2'-O-methyl modification.

[0234] The introduction of one or more motifs of three identical modifications on a triple nucleotide sequence into the sense strand and / or antisense strand disrupts the initial modification pattern present in the sense strand and / or antisense strand. This disruption of the modification pattern in the sense strand and / or antisense strand, by introducing one or more motifs of three identical modifications on a triple nucleotide sequence into the sense strand and / or antisense strand, unexpectedly enhances the gene silencing activity against the target gene.

[0235] In one embodiment, when three identical modification motifs on three consecutive nucleotides are introduced into any of the chains, the modifications of nucleotides adjacent to the motif are different from the modifications of the motif. For example, a portion of the sequence containing the motif is "···N a YYYN b ..." where "Y" represents the modification of three identical modification motifs in three consecutive nucleotides, and "N a " and "N b " represents a modification of a nucleotide adjacent to the motif "YYY", which is different from the modification of Y, and N a and N b These can be the same or different modifications. Or, N a and / or N b This may or may not exist if a wing modifier is present.

[0236] The RNAi agent may further contain at least one phosphorothioate or methylphosphonate internucleotide bond. The modification of the phosphorothioate or methylphosphonate internucleotide bond may be present on any nucleotide in the sense strand, antisense strand, or both strands, at any position on the strand. For example, the modification of the internucleotide bond may be present on all nucleotides in the sense strand and / or antisense strand; each modification of the internucleotide bond may be present in an alternating pattern in the sense strand and / or antisense strand; or the sense strand or antisense strand may contain modifications of both internucleotide bonds in an alternating pattern. The alternating pattern of the internucleotide bond modifications in the sense strand may be the same as or different from that in the antisense strand, and the alternating pattern of the internucleotide bond modifications in the sense strand may have a shift relative to the alternating pattern of the internucleotide bond modifications in the antisense strand. In one embodiment, a double-standed RNAi agent contains 6 to 8 phosphorothioate internucleotide bonds. In one embodiment, the antisense strand includes two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end, and the sense strand includes at least two phosphorothioate nucleotide interlinks at either its 5' or 3' end.

[0237] In one embodiment, RNAi includes a modification of phosphorothioate or methylphosphonate internucleotide bonds in the overhang region. For example, the overhang region may include two nucleotides having a phosphorothioate or methylphosphonate internucleotide bond between them. The internucleotide bond modification may also be formed to bind the overhang nucleotide to a terminal paired nucleotide in the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides may be bound by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, there may be further phosphorothioate or methylphosphonate internucleotide bonds that bind the overhang nucleotide to a paired nucleotide adjacent to the overhang nucleotide. For example, there may be at least two phosphorothioate internucleotide bonds between three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. These three terminal nucleotides may be located at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand.

[0238] In one embodiment, two nucleotide overhangs are located at the 3' end of the antisense strand, with two phosphorothioate nucleotide interbonds between the three terminal nucleotides, two of which are overhang nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotides. Optionally, the RNAi agent may further have two phosphorothioate nucleotide interbonds between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

[0239] In one embodiment, the RNAi agent includes mismatches with the target, intra-double-strand mismatches, or combinations thereof. Mismatches may occur in overhang regions or double-strand regions. Base pairs may be evaluated based on their tendency to promote dissociation or melting (e.g., with respect to the free energy of binding or dissociation of a particular pair, the simplest method being to examine each pair individually, although similar or equivalent analyses may also be used). With respect to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical or non-canonical pairings (as described elsewhere in this specification), are preferred over canonical (A:T, A:U, G:C) pairings; pairings containing universal bases are preferred over canonical pairings.

[0240] In one embodiment, the RNAi agent comprises at least one of the first one, two, three, four, or five base pairs in the double-stranded region from the 5' end of the antisense strand, independently selected from the group A:U, G:U, and I:C, and a mismatch pair to facilitate the dissociation of the antisense strand at the 5' end of the double-stranded region, such as a non-canonical or non-canonical pair or a pair containing a universal base.

[0241] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0242] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, the 3' ends of the sense strand and / or antisense strand have a short sequence of deoxythymine nucleotides, for example, two dT nucleotides.

[0243] In one embodiment, the sense strand sequence is given by formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I) (In the formula: i and j are independently either 0 or 1; p and q are independently between 0 and 6; each N a However, each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b However, independently, it represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p and n q However, independently, it represents an overhanging nucleotide; Here, Nb and Y do not have the same modifications; (XXX, YYY, and ZZZ each independently represent one motif of three identical modifications in a triple nucleotide sequence.) This can be represented by [formula]. Preferably, all YYY are 2'-F modified nucleotides.

[0244] In one embodiment, N a and / or N b This includes alternating pattern modifications.

[0245] In one embodiment, the YYY motif is located at or near the sense strand cleavage site. For example, if the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif may be located at or near the sense strand cleavage site, starting from the first nucleotide from the 5' end; or optionally, starting from the 5' end and counting from the first paired nucleotide in the double-stranded region (e.g., it may be located at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13).

[0246] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense chain can be represented by the following formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib); 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id).

[0247] If the sense chain is represented by formula (Ib), then N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0248] If the sense chain is expressed as equation (Ic), then N bThis represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0249] If the sense chain is represented as expression (Id), then each N b This independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6. Each N a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0250] X, Y, and Z can each be the same or different from one another.

[0251] In another embodiment, i is 0, j is 0, and the sense chain can be represented by the following equation: 5'n p -N a -YYY-N a -n q 3'(Ia).

[0252] If the sense chain is represented by equation (Ia), then each N a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0253] In one embodiment, the antisense strand sequence of RNAi is given by formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3'(II) (In the formula: k and l are independently either 0 or 1; p' and q' are each independently between 0 and 6; each N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p 'and n q ' independently represents an overhanging nucleotide; Here, N b 'and Y' do not have the same modifier; X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications in a triple nucleotide sequence. It can be represented by:

[0254] In one embodiment, N a 'and / or N b ' includes alternating pattern modifications.

[0255] The Y'Y'Y' motif is located at or near the cleavage site of the antisense strand. For example, if the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif may be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, starting from the first nucleotide from the 5' end; or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif is located at positions 11, 12, and 13.

[0256] In one embodiment, all Y'Y'Y' motifs are 2'-OMe modified nucleotides.

[0257] In one embodiment, k is 1, l is 0, or k is 0, l is 1, or both k and l are 1.

[0258] Therefore, the antisense strand can be represented by the following formula: 5’n q’ -N a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N a ’-n p’ 3’(IIb); 5’n q’ -N a ’-Y’Y’Y’-N b ’-X’X’X’-n p’ 3’(IIc); or 5’n q’ -N a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N b ’-X’X’X’-N a ’-n p’ 3’(IId).

[0259] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15, or 2 to 10.

[0260] When the antisense strand is represented by formula (IIc), N b ’ represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15, or 2 to 10.

[0261] When the antisense strand is represented by formula (IId), each N b' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6.

[0262] In another embodiment, when k is 0 and l is 0, the antisense chain can be represented by the following formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia).

[0263] If the antisense chain is represented by equation (IIa), then each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0264] X', Y', and Z' can each be the same or different from one another.

[0265] Each nucleotide in the sense and antisense strands can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' can, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0266] In one embodiment, the sense strand of the RNAi agent may, if the double-stranded region has 21 nucleotides, include YYY motifs located at positions 9, 10, and 11 of the strand, starting from the first nucleotide from the 5' end; or optionally, starting from the 5' end, starting from the first paired nucleotide in the double-stranded region; where Y represents a 2'-F modification. The sense strand may further include XXX or ZZZ motifs as wing modifications at the opposite end of the double-stranded region; where XXX and ZZZ independently represent a 2'-OMe modification or a 2'-F modification, respectively.

[0267] In one embodiment, the antisense strand may contain a Y'Y'Y' motif located at positions 11, 12, and 13 of the strand, starting from the first nucleotide from the 5' end; or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end; where Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region; where X'X'X' and Z'Z'Z' independently represent a 2'-OMe modification or a 2'-F modification.

[0268] Each sense strand represented by any one of the above equations (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense strand represented by any one of the above equations (IIa), (IIb), (IIc), and (IId).

[0269] Therefore, the RNAi agent for use in the method of the present invention may include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi double strand is given by formula (III): Sense: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) (In the formula: i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; each N a and N a ’ However, each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b and N b ’ However, independently, it represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; Here, Each n may or may not exist. p ',n p , n q ', and n q However, independently, it represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides. It is represented by [this].

[0270] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0271] An exemplary combination of sense and antisense strands that form an RNAi double helix includes the following formula: 5'n p -N a -YYY-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a’ -n q ’ 5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId)

[0272] When an RNAi agent is represented by formula (IIIb), each N b Each N independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. a Each independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0273] When an RNAi agent is represented by formula (IIIc), each N b , N b ' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a Each independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0274] When an RNAi agent is represented by formula (IIId), each N b , N b ' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a , N a' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. a , N a ', N b and N b Each of these independently includes alternating pattern modifications.

[0275] When an iRNA agent is represented by formula (IIId), each N b , N b ' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a , N a ’ This independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. a , N a ', N b and N b ’ Each of these independently includes alternating pattern modifications.

[0276] In equations (III), (IIIa), (IIIb), (IIIc), and (IIId), X, Y, and Z may be the same as or different from each other.

[0277] If an RNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), then at least one Y nucleotide may form a base pair with one of the Y' nucleotides; or at least two Y nucleotides may form base pairs with the corresponding Y' nucleotides; or all three Y nucleotides may form base pairs with the corresponding Y' nucleotides.

[0278] If the RNAi agent is represented by formula (IIIb) or (IIId), then at least one Z nucleotide may form a base pair with one of the Z' nucleotides; or at least two Z nucleotides may form base pairs with the corresponding Z' nucleotides; or all three Z nucleotides may form base pairs with the corresponding Z' nucleotides.

[0279] If an RNAi agent is represented by formula (IIIc) or (IIId), then at least one X nucleotide may form a base pair with one of the X' nucleotides; or at least two X nucleotides may form a base pair with the corresponding X' nucleotide; or all three X nucleotides may form a base pair with the corresponding X' nucleotide.

[0280] In one embodiment, modifications on the Y nucleotide differ from modifications on the Y' nucleotide, modifications on the Z nucleotide differ from modifications on the Z' nucleotide, and / or modifications on the X nucleotide differ from modifications on the X' nucleotide.

[0281] In one embodiment, if the RNAi agent is represented by formula (IIId), then N a The modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, if the RNAi agent is represented by formula (IIId), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is bound to an adjacent nucleotide via a phosphorothioate bond. In yet another embodiment, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p' is bound to an adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker (described below). In another embodiment, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The sense strand is bound to an adjacent nucleotide via a phosphorothioate bond, and the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0282] In one embodiment, if the RNAi agent is represented by formula (IIIa), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The sense strand is bound to an adjacent nucleotide via a phosphorothioate bond, and the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0283] In one embodiment, the RNAi agent is a multimer comprising at least two double strands represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the double strands being joined by a linker. The linker may be cleavable or incleavable. Optionally, the multimer further comprises a ligand. Each double strand may target the same gene or two different genes; or each double strand may target the same gene at two different target sites.

[0284] In one embodiment, the RNAi agent is a multimer comprising three, four, five, six or more double strands represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), where the double strands are linked by linkers. The linkers may be cleavable or incleavable. Optionally, the multimer further comprises ligands. Each double strand may target the same gene or two different genes; or each double strand may target the same gene at two different target sites.

[0285] In one embodiment, two RNAi agents represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are bound to each other at one or both of their 5' and 3' ends and optionally conjugated to a ligand. Each RNAi agent may target the same gene or two different genes; or each RNAi agent may target the same gene at two different target sites.

[0286] Various publications describe multimeric RNAi agents that can be used in the methods of the present invention. Such publications include International Publication No. 2007 / 091269, U.S. Patent No. 7858769, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887, and International Publication No. 2011 / 031520, the entire contents of which are incorporated herein by reference.

[0287] As will be described in more detail below, RNAi agents containing conjugations of one or more carbohydrate moieties can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety is conjugated to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent may be replaced by another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is conjugated. A ribonucleotide subunit in which the ribose sugar of the subunit is thus substituted is referred herein to a ribose-substituted modified subunit (RRMS). The cyclic carrier may be a carbocyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms are heteroatoms, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic system, or may contain two or more rings, e.g., a fused ring. The cyclic carrier may be a fully saturated ring system, or may contain one or more double bonds.

[0288] Ligands can be bound to polynucleotides via a carrier. The carrier comprises (i) at least one “skeletal attachment point,” preferably two “skeletal attachment points,” and (ii) at least one “tethering attachment point.” As used herein, “skeletal attachment point” refers to a bond that is available for and suitable for incorporating the carrier into the ribonucleic acid skeleton, which may contain a functional group, e.g., a hydroxyl group, or generally a skeleton, e.g., a phosphate, or a modified phosphate, e.g., sulfur. A “tethering attachment point” (TAP) refers, in some embodiments, to a constituent ring atom of a cyclic carrier connecting a selected moiety, e.g., a carbon atom or heteroatom (different from the atoms providing the skeletal attachment points). This moiety may be, for example, a carbohydrate, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Therefore, cyclic carriers often contain functional groups, such as amino groups, or generally provide bonds suitable for the incorporation or tethering of other chemical components, such as ligands, into the constituent ring.

[0289] The iRNA agent may be conjugated to a ligand via a carrier, which may be a cyclic group or a cyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the cyclic group is selected from a selinol skeleton or a diethanolamine skeleton.

[0290] In certain specific embodiments, the RNAi agent used in the method of the present invention is an agent selected from the group of agents listed in any one of Tables 3, 4, 9, 10, 15, 16, 19A, 19B, 19C, 19D, 19E, 19F, 20, 21, 23, 24, 26, and 27. In one embodiment, this agent is any one of the agents listed in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27. These agents may further include ligands.

[0291] IV. iRNA conjugated to a ligand Another modification of the iRNA of the present invention involves chemically attaching one or more ligands, portions, or conjugates to the RNA to improve the activity, cell distribution, or cell uptake of the iRNA.These parts are not limited to, but include the cholesterol portion (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), thioethers, for example, beryl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), and thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992,20:533-538), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al.,EMBO J,1991,10:1111-1118; Kabanov et al.,FEBS Lett.,1990,259:327-330; Svinarchuk et al.,Biochimie,1993,75:49-54), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett.,1995,36:3651-3654; Shea et al.,Nucl.Acids Examples of lipid moieties include Res., 1990, 18:3777-3783, polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0292] In one embodiment, the ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand provides, for example, improved affinity to selected targets (e.g., molecules, cells, or cell types), compartments (e.g., compartments of cells or organs), body tissues, organs, or regions compared to species without such ligands. Preferred ligands do not participate in double-strand pairing in double-stranded nucleic acids.

[0293] Ligands may include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands may also be synthetic polymers, such as recombinant or synthetic molecules like synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-coglycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudo-peptide polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helix peptides.

[0294] The ligand may also include a target group, e.g., a cell or tissue targeting agent, e.g., lectins, glycoproteins, lipids or proteins, or antibodies that bind to a specific cell type, such as kidney cells. The target group may be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or RGD peptide, or RGD peptide mimetic or aptamer.

[0295] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetate, 1-pyrenebutyric 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 (cholenic acid Examples include acids, dimethoxytrityl, or phenoxazine, and peptide complexes (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole complex, Eu3+ tetraaza macrocyclic complex), dinitrophenyl, HRP, or AP.

[0296] Ligands can be proteins, such as glycoproteins, or peptides, such as coligands, or antibodies, such as molecules that have specific affinity for antibodies that bind to specific cell types, such as hepatocytes. Ligands may also include hormones and hormone receptors. Ligands may also include lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, or non-peptide species such as polyvalent mannose, polyvalent fucose, or aptamers. Ligands may be, for example, lipopolysaccharides, activators of 38MAP kinase, or activators of NF-κB.

[0297] A ligand can be a substance, such as a drug, that can improve the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton, such as by disrupting the cellular microtubules, microfilaments, and / or intermediate filaments. The drug may be, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoserbine.

[0298] In some embodiments, the ligands bound to the iRNAs described herein act as pharmacokinetic modulators (PK modulators). Examples of PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, and vitamins. Exemplary PK modulators, but not limited to, include cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing several phosphorothioate bonds are also known to bind to serum proteins; therefore, short oligonucleotides, such as oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate bonds in their backbone, are also suitable as ligands (e.g., PK modulators) in the present invention. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK modulators in the embodiments described herein.

[0299] The ligand-conjugated oligonucleotides of the present invention can be synthesized using oligonucleotides having reactive pendant functional groups, such as those derived from the binding of a binding molecule to the oligonucleotide (as described below). These reactive oligonucleotides may react directly with commercially available ligands, synthetic ligands having any of the various protecting groups, or ligands to which the binding moiety is bound.

[0300] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). Any other means known in the art for such synthesis may be used in addition to or instead of these. Similar techniques for preparing other oligonucleotides, such as phosphorothioates and alkylated derivatives, are also known.

[0301] In the ligand-conjugate oligonucleotides and ligand molecules having sequence-specific binding nucleosides of the present invention, the oligonucleotides and oligonucleosides can be assembled in a suitable DNA synthesis apparatus using standard nucleotide or nucleoside precursors, nucleotide or nucleoside conjugate precursors already having a binding site, ligand-nucleotide or nucleoside conjugate precursors already having a ligand molecule, or building blocks containing non-nucleoside ligands.

[0302] When a nucleotide conjugate precursor already having a binding site is used, the synthesis of a sequence-specific binding nucleoside is typically completed first, after which the ligand molecule reacts with the binding site to form a ligand-conjugate oligonucleotide. In one embodiment, the oligonucleotide or binding nucleoside of the present invention is synthesized by an automated synthesizer using commercially available standard and non-standard phosphoramidites commonly used in oligonucleotide synthesis, as well as phosphoramidites derived from ligand-nucleoside conjugates.

[0303] A. Lipid conjugates In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues of the body, such as non-renal target tissues. For example, the target tissue may be the liver, including the parenchymal cells of the liver. Other molecules capable of binding to HSA may also be used as ligands. For example, naproxen or aspirin may be used. Lipid or lipid-based ligands may be used to (a) increase the resistance of the conjugate to degradation, (b) increase the targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, such as HSA.

[0304] The binding of conjugates to target tissues can be inhibited, for example, controlled, using lipid-based ligands. For instance, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidneys and therefore less likely to be removed from the body. Conjugates can be targeted to the kidneys using lipids or lipid-based ligands that bind more weakly to HSAs.

[0305] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, the lipid-based ligand binds to HSA with sufficient affinity such that the conjugate is distributed to non-renal tissue. However, the affinity is preferably not so strong that the HSA-ligand binding cannot be reversed.

[0306] In another preferred embodiment, the lipid-based ligand binds weakly to or does not bind at all to the HSA so that the conjugate is preferably distributed to the kidney. Other portions targeting renal cells may also be used instead of or in addition to the lipid-based ligand.

[0307] In another embodiment, the ligand is a portion taken up by target cells, e.g., proliferating cells, e.g., a vitamin. These are particularly useful for treating, for example, undesirable cell proliferation, whether malignant or non-malignant, disorders characterized by, for example, cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by target cells such as hepatocytes. HAS and low-density lipoprotein (LDL) are also included.

[0308] B. Cell permeability agents In another embodiment, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, this agent is amphiphilic. Exemplary agents are peptides such as tat or antennopedia. If this agent is a peptide, it may be modified by including the use of peptidyl mimes, inverted isomers, non-peptide or pseudopeptide bonds, and D-amino acids. The helical agent is preferably an α-helix agent, which preferably has lipophilic and oleophobic phases.

[0309] The ligand may be a peptide or a peptide mimetic. Peptide mimes (also referred to herein as oligopeptide mimes) are molecules capable of folding into a distinct three-dimensional structure similar to that of natural peptides. Binding of peptides and peptide mimes to iRNA agents can affect the pharmacokinetic distribution of the iRNA, for example, by enhancing cell recognition and absorption. The peptide or peptide mimetic portion may be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0310] The peptide or peptide mimetic may be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., mainly consisting of Tyr, Trp, or Phe). The peptide moiety may be a dendrimer peptide, a constrained peptide, or a cross-linked peptide. In another alternative example, the peptide moiety may contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 26). RFGF analogues containing hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 27)) can also be target moieties. The peptide moiety can be a "delivery" peptide capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 28)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 29)) have been shown to function as delivery peptides. Peptides or peptide mimetic compounds can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al.). (al., Nature, 354:82-84, 1991). Examples of peptides or peptide mimetic compounds linked to dsRNA agents via monomer units incorporated for cell targeting purposes include peptides such as arginine-glycine-aspartate (RGD)-peptides or RGD mimetic compounds. The peptide portion can range in length from approximately 5 to approximately 40 amino acids. The peptide portion may have structural modifications, such as to enhance stability or direct conformational properties. Any of the structural modifications described below may be used.

[0311] The RGD peptide moiety for use in the compositions and methods of the present invention may be linear or cyclic and may be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissues. RGD-containing peptides and peptide mimetic forms may include D-amino acids and synthetic RGD mimetic forms. In addition to RGD, other moieties targeting integrin ligands may be used. Preferred conjugates of these ligands target PECAM-1 or VEGF.

[0312] "Cell-permeable peptides" are capable of permeating cells, such as microbial cells like bacteria or fungal cells, or mammalian cells like human cells. Peptides that permeate microbial cells may be, for example, α-helix linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenecin), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides may also contain nuclear localization signals (NLS). For example, cell-permeable peptides may be bifidopphimotic peptides such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0313] C. Carbohydrate Conjugate In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids as described herein, and the compositions are suitable for in vivo therapeutic use. As used herein, “carbohydrate” means a compound that is a carbohydrate itself, consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with each carbon atom bonded to an oxygen, nitrogen, or sulfur atom; or a compound having a carbohydrate moiety as part thereof, consisting of one or more monosaccharide units, each monosaccharide unit having at least six carbon atoms (which may be linear, branched, or cyclic), with each carbon atom bonded to an oxygen, nitrogen, or sulfur atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gum. Examples of specific monosaccharides include sugars with a HBV of 100% or higher (e.g., HBV, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., HBV, C6, C7, or C8).

[0314] In one embodiment, the carbohydrate conjugate used in the composition and method of the present invention is a monosaccharide. In another embodiment, the carbohydrate conjugate used in the composition and method of the present invention is [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0315] In one embodiment, the monosaccharide is [ka] These include N-acetylgalactosamine.

[0316] Other representative carbohydrate conjugates used in the embodiments described herein include, but are not limited to, [ka] (Formula XXIII) [where one of X or Y is an oligonucleotide, the other is hydrogen] is included.

[0317] In certain embodiments of the present invention, GalNAc or a GalNAc derivative is bound to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is bound to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is bound to the iRNA agent of the present invention via a trivalent linker.

[0318] In one embodiment, the double-stranded RNAi agent of the present invention comprises one GalNAc or GalNAc derivative bound to an iRNA agent. In another embodiment, the double-stranded RNAi agent of the present invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently bound to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0319] In some embodiments, for example, when the two strands of the iRNA agent of the present invention are part of a larger molecule forming a hairpin loop containing multiple unpaired nucleotides, with the 3' end of one strand connected to the 5' end of the other strand by an unbroken nucleotide chain, each unpaired nucleotide within the hairpin loop may independently contain GalNAc or a GalNAc derivative linked via a monovalent linker.

[0320] In one embodiment, the carbohydrate conjugate further comprises one or more of the above-described ligands, such as PK modifiers and / or cell-permeable peptides, but is not limited to these.

[0321] Further carbohydrate conjugates suitable for use in the present invention include those described in International Publication No. 2014 / 179620 and International Publication No. 2014 / 179627 of the PCT, the contents of which are incorporated herein by reference in their entirety.

[0322] D. Linker In one embodiment, the conjugates or ligands described herein may be bound to iRNA oligonucleotides using a variety of linkers that may be cleavable or incleavable.

[0323] The term "linker" or "bonding group" refers to an organic part that connects two parts of a compound, for example, a covalent bond between two parts of a compound. Linkers are typically directly bonded or atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or, but are not limited to, substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, substituted or unsubstituted alkynyls, arylalkyls, arylalkenyls, arylalkynyls, heteroarylalkyls, heteroarylalkenyls, heteroarylalkynyls, heterocyclylalkyls, heterocyclylalkenyls, heterocyclylalkynyls, aryls, heteroaryls, heterocyclyl, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroarylalkyls, alkylheteroarylalkenyls, alkylheteroarylalkynyls, alkenylheteroaryl Alkyl alkyl, alkenyl heteroaryl alkenyl, alkenyl heteroaryl alkynyl, alkynyl heteroaryl alkyl, alkynyl heteroaryl alkenyl, alkynyl heteroaryl alkynyl, alkyl heterocyclyl alkyl, alkyl heterocyclyl alkenyl, alkyl heterocyclyl alkynyl, alkenyl heterocyclyl alkyl, alkenyl heterocyclyl alkenyl, alkenyl heterocyclyl alkynyl, alkynyl heterocyclyl alkyl, alkynyl heterocyclyl a This comprises chains of atoms such as lukenyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, and alkynylheteroaryl (where one or more methylene atoms may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic structures); where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker has about 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 16, or 8 to 16 atoms.

[0324] The cleavable binding group is sufficiently stable outside the cell, but after entering the target cell, it is cleaved, releasing the two parts held together by the linker. In a preferred embodiment, the cleavable binding group is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or more, or at least about 100 times faster in the target blood or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum) within the target cell or under a first reference condition (which may be selected to mimic or represent conditions found in blood or serum).

[0325] Cleavable binding groups are sensitive to the presence of cleavage agents, such as pH, redox potential, or degradable molecules. Generally, cleavage agents are more widely present in cells than in serum or blood, or are found at higher levels or activity. Examples of such degrading agents include redox agents selected for specific substrates or lacking substrate specificity, such as mercaptans that can degrade redox-cleavable binding groups by oxidizing or reducing enzymes present in cells; esterases; agents capable of creating endosomes or acidic environments, such as agents that result in a pH of 5 or less; enzymes, peptidases (which may be substrate-specific), and phosphatases that can hydrolyze or degrade acid-cleavable binding groups by acting as general acids.

[0326] Cleavable binding groups, such as disulfide bonds, can be pH sensitive. While human serum has a pH of 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers, by having cleavable binding groups that are cleaved at a favorable pH, will release cationic lipids from ligands within the cell or into desired compartments of the cell.

[0327] A linker may contain cleavable binding groups that can be cleaved by specific enzymes. The type of cleavable binding group incorporated into the linker may depend on the target cell. For example, a ligand targeting the liver may bind to cationic lipids via a linker containing an ester group. Because hepatocytes are rich in esterases, this linker will be cleaved more efficiently within hepatocytes than in esterase-deficient cell types. Other esterase-rich cell types include lung, renal cortex, and testicular cells.

[0328] Linkers containing peptide bonds can be used to target peptidase-rich cell types such as hepatocytes and synovial cells.

[0329] Generally, the suitability of a candidate cleavable binding group can be evaluated by testing the ability of a degradation agent (or degradation condition) to cleave the candidate binding group. It would also be desirable to test the ability of the candidate cleavable binding group to resist cleavage in the blood or in contact with other non-target tissues. Therefore, the relative sensitivity to cleavage between the first and second conditions can be determined, with the first condition selected to demonstrate cleavage within target cells and the second condition selected to demonstrate cleavage in other tissues or biological fluids, such as blood or serum. This evaluation can be performed in a cell-free system, intracellularly, in cell cultures, in organs or tissue cultures, or in whole animals. It may be useful to perform the initial evaluation in cell-free or culture conditions and confirm it with further evaluation in whole animals. In a preferred embodiment, a useful candidate compound is cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0330] i. Redox-cleavable bond groups In one embodiment, the cleavable binding group is a redox-cleavable binding group that is cleaved after reduction or oxidation. An example of a reductively cleavable binding group is a disulfide binding group (-SS-). To determine whether a candidate cleavable binding group is a suitable “reductively cleavable binding group” or suitable for use with, for example, a specific iRNA moiety and a specific targeting agent, one may look to the methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art, which mimics the rate of cleavage that may be observed in cells, for example, target cells. A candidate can also be evaluated under conditions selected to mimic blood or serum conditions, in one of which the candidate compound is cleaved by about 10% or less in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of candidate compounds can be determined using standard enzyme kinetic assays under conditions selected to mimic an intracellular medium and compared to conditions selected to mimic an extracellular medium.

[0331] ii. Phosphate-based cleavable binding groups In another embodiment, the cleavable linker includes a phosphate-based cleavable binding group. The phosphate-based cleavable binding group is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves the phosphate group in a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based binding groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0332] iii. Acid-cleavable bonding groups In another embodiment, the cleavable linker includes an acid-cleavable binding group. An acid-cleavable binding group is a binding group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable binding group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or less), or by a drug such as an enzyme that can act as a general acid. Within cells, certain low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable binding groups. Examples of acid-cleavable binding groups include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups may be represented by the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0333] iv. Ester-based bonding groups In another embodiment, the cleavable linker comprises an ester-based cleavable binding group. The ester-based cleavable binding group is cleaved by enzymes such as esterases and amylases in cells. Examples of ester-based cleavable binding groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester-cleavable binding group is represented by the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0334] v. Peptide-based cleavage groups In yet another embodiment, the cleavable linker includes a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. The peptide-based cleavable group is a peptide bond formed between amino acids to give oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not contain an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to give peptides and proteins. The peptide-based cleavable group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give peptides and proteins, and does not include all amide functional groups. The peptide-based cleavable linking group is represented by the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0335] In one embodiment, the iRNA of the present invention is conjugated with a carbohydrate via a linker. Non-limiting examples of iRNA-carbohydrate conjugation with a linker of the compositions and methods of the present invention include, but are not limited to, [ka] [ka] [ka] For example, if one of X or Y is an oligonucleotide, the other is hydrogen.

[0336] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives linked via a divalent or trivalent branched linker.

[0337] In one embodiment, the dsRNA of the present invention is given by formulas (XXXII) to (XXXV): [ka] Conjugated to a divalent or trivalent branched linker selected from the group of structures shown in any of the following: During the ceremony: Each instance of q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represents a number between 0 and 20, and the repeating units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C Each of these is either absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O, each appearing independently; Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C However, each instance is independent, either non-existent, alkylene, or substituted alkylene, where one or more methylene groups are O, S, S(O), SO2, N(R) N It may be interrupted or terminated by one or more of the following: C(R')=C(R"), C≡C, or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B, R 5A , R 5B , R 5C Each of these terms appears independently, either as nonexistent, or as NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocycline; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C However, each represents a ligand; that is, each instance is independently a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a However, it is either H or an amino acid side chain. Trivalent conjugate GalNAc derivatives are used with RNAi agents, and formula (XXXV): [ka] It is particularly useful for inhibiting the expression of target genes such as those mentioned above. In the formula, L 5A , L 5B and L 5C However, this represents monosaccharides such as GalNAc derivatives.

[0338] Suitable divalent and trivalent branched linkage groups for conjugation to GalNAc derivatives include, but are not limited to, the structures listed above as formulas II, VII, XI, X, and XIII.

[0339] Representative patents teaching the preparation of RNA conjugates include, but are not limited to, U.S. Patent 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; and 5,414,077. No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779 4,789,737; 4,824,941; 4,835,263; 4,876,335; Specification No. 4,904,582; Specification No. 4,958,013; Specification No. 5,082,830; Specification No. 5,112,963; Specification 5 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,27 Specification No. 2,250; Specification No. 5,292,873; Specification No. 5,317,098; Specification No. 5,371,241, Specification No. 5,391, Specification No. 723; Specification No. 5,416,203, Specification No. 5,451,463; Specification No. 5,510,475; Specification No. 5,512,66 Specification No. 7; Specification Nos. 5,514,785; Specification Nos. 5,565,552; Specification Nos. 5,567,810; Specification Nos. 5,574,142; Specification Nos. 5,585,481; Specification Nos. 5,587,371; Specification Nos. 5,595,726; Specification Nos. 5,597,696; Specification Nos. 5,599,923; Specification Nos. 5,599,928 and 5,688,941; Specification Nos. 6,294,664; Specification Nos. 6,320,017; Specification Nos. 6,576,752; Specification Nos. 6,783,931; Specification Nos. 6,900,297;References include Patent No. 7,037,646 and Patent No. 8,106,022, the entire contents of which are incorporated herein by reference.

[0340] It is not necessary for all positions of a given compound to be uniformly modified; in fact, two or more of the above modifications can be incorporated into a single compound or even into a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0341] In relation to the present invention, a “chimeric” iRNA compound or “chimeric” is an iRNA compound, preferably a dsRNA, that comprises two or more chemically distinct regions, each composed of at least one monomeric unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically comprise at least one region, where the RNA is modified to give the iRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to a target nucleic acid. Further regions of the iRNA can serve as substrates for enzymes capable of cleaving RNa:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNa strand of an RNA:DNA double-stranded DNA. Thus, activation of RNase H results in cleavage of the RNA target, thereby significantly increasing the efficiency of iRNA inhibition of gene expression. Consequently, when chimeric dsRNAs are used, equivalent results are often obtained with shorter iRNAs compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of RNA targets can typically be detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0342] In some cases, the RNA of an iRNA can be modified with a non-ligand group. Several non-ligand molecules have been conjugated to iRNAs to improve their activity, cell distribution, or cell uptake, and procedures for such conjugations are available in the scientific literature.Such non-ligand portions include 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), thioethers, for example, hexyl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), and thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992,20:533), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al.,EMBO J.,1991,10:111; Kabanov et al.,FEBS Lett.,1990,259:327; Svinarchuk et al.,Biochimie,1993,75:49), phospholipids, 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), polyamine or polyethylene glycol chains (Manoharan et al. It contained lipid moieties such as al., Nucleosides & Nucleotides, 1995, 14:969, adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugate protocol involves the synthesis of RNA having aminolinkers at one or more positions in its sequence. The amino group is then reacted with the conjugated molecule using a suitable coupling agent or activating reagent. The conjugate reaction can be carried out using RNA still bound to a solid support or after cleavage of RNA in the solution phase. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0343] V. Delivery of iRNA according to the present invention The iRNA of the present invention can be delivered to cells in a target, such as a human target (e.g., a target requiring an iRNA agent, such as a target with a disease, disorder, or pathological condition related to contact activation pathway gene expression), in several different ways. For example, delivery may be carried out by contacting the cells with the iRNA of the present invention either in vitro or in vivo. In vivo delivery may also be carried out directly by administering a composition containing the iRNA, such as dsRNA, to the target. Alternatively, in vivo delivery may be carried out indirectly by administering one or more vectors that encode and lead to the expression of the iRNA. These alternatives are further described below.

[0344] In general, any method for delivering nucleic acid molecules (in vitro or in vivo) may be adapted for use with the iRNA of the present invention (see, for example, Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. 94 / 02595, which are incorporated herein by reference in their entirety). In the case of in vivo delivery, factors to be considered for delivering the iRNA molecule include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, for example, by direct injection or transplantation into tissue, or by local administration of the formulation. Local administration to the treatment site can maximize the local concentration of the agent, limit exposure of the agent to systemic tissues that may be adversely affected or degraded by the agent, and reduce the total dose of the iRNA molecule administered. Several studies have demonstrated successful knockdown of gene products when iRNA is administered locally. For example, intravitreal injection of VEGF dsRNA into cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injection into mice (Reich, SJ. et al., (2003) Mol.Vis.9:210-216) both demonstrated prevention of neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral administration of dsRNA in mice reduced tumor volume (Pille, J. et al., (2005) Mol.Ther.11:267-274) and extended the survival of tumor-bearing mice (Kim, WJ. et al., (2006) Mol.Ther.14:343-350; Li, S. et al., (2007) Mol.Ther.15:515-523).RNA interference can be delivered locally to the central nervous system by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and delivered locally to the lungs by intranasal administration (Howard, KA. et al. Success has also been demonstrated by al., (2006) Mol.Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For systemic administration of iRNA for the treatment of disease, the RNA can be modified or delivered using a drug delivery system; both methods play a role in preventing the rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of RNA or drug carriers can also enable targeting of the iRNA composition to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemical binding to lipophilic groups such as cholesterol, which improves cellular uptake and prevents degradation. For example, when iRNA conjugated to the lipophilic cholesterol portion of ApoB was systemically administered to mice, knockdown of apoB mRNA was achieved in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of iRNA to aptamers has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO. et al., (2006) Nat. Biotechnol. 24:1005-1015).In alternative embodiments, iRNA may be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of negatively charged iRNA molecules and enhance interactions with negatively charged cell membranes, enabling efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers may bind to iRNA or be induced to form vesicles or micelles that enclose the iRNA (see, e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of iRNA when administered systemically. Methods for preparing and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, for example, Sorensen, DR., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN. et al. (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS 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 iRNA include DOTAP (Sorensen, DR., et al (2003), see above; Verma, UN. et al., (2003), see above), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al., (2006) Nature 441:111-114), cardiolipin (Chien, PY. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of Examples include (print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In one embodiment, the iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrin can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in whole.

[0345] A. The iRNA of the present invention encoded by a vector iRNAs targeting contact activation pathway genes can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A. et al., International PCT Publication No. 00 / 22113, Conrad, International PCT Publication No. 00 / 22114, and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (approximately several hours to several weeks) or persistent (several weeks to several months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which may be embedded or non-embedded vectors. Transgenes can also be constructed to allow inheritance as extrachromosomal plasmids (see Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0346] Individual strands of iRNA can be transcribed from a promoter in an expression vector. If two separate strands are expressed to produce, for example, dsRNA, two separate expression vectors can be co-introduced into target cells (for example, by transfection or infection). Alternatively, each individual strand of dsRNA can be transcribed by a promoter located on the same expression plasmid. In one embodiment, dsRNA is expressed as a reverse repeat polynucleotide joined by a linker polynucleotide sequence to have a stem-loop structure.

[0347] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for iRNA expression described herein can be produced using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from many commercial sources. Such vectors are usually provided that contain restriction sites convenient for inserting the desired nucleic acid segment. Delivery of iRNA expression vectors may be systemic delivery, for example, by intravenous or intramuscular administration, by administration to target cells transplanted from a patient and then reintroduction into the patient, or by any other means that enable introduction into the desired target cells.

[0348] iRNA expression plasmids can be transfected into target cells as a complex with a cationic lipid carrier (e.g., oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO®). Multiple lipid transfections for iRNA-mediated knockdown targeting different regions of the target RNA over a period of one week or more are also envisioned in this invention. The success of vector introduction into host cells can be monitored using various known methods. For example, transient transfection can be indicated using a reporter such as a fluorescent marker such as green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured by using markers that confer resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance, to transfected cells.

[0349] Viral vector systems that may be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentivirus vectors and Moloney's mouse leukemia virus; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) orthopox, e.g., vaccinia virus vectors or avian pox, e.g., poxvirus vectors such as canarypox or fowlpox; and (j) helper-dependent or attenuated adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not be incorporated into the cell genome. The construct may optionally include a viral sequence for transfection. Alternatively, the construct may be incorporated into an episomal replication-capable vector, e.g., EPV and EBV vectors. Constructs for the recombinant expression of iRNA generally require regulatory elements, such as promoters and enhancers, to ensure iRNA expression within the target cell. Other embodiments of vectors and constructs are considered and will be discussed further later.

[0350] A vector useful for iRNA delivery will contain sufficient regulatory elements (promoter, enhancer, etc.) to express the iRNA in the desired target cells or tissues. These regulatory elements may be selected to provide either constitutive or regulatory / inducible expression.

[0351] iRNA expression can be precisely regulated, for example, by using inducible regulatory sequences sensitive to specific physiological regulators, such as blood glucose levels or hormones (Docherty et al., 1994, FASEB J.8:20-24). Suitable inducible expression systems for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art will be able to select an appropriate regulatory / promoter sequence based on the intended use of the iRNA transgene.

[0352] Viral vectors containing nucleic acid sequences encoding iRNAs may be used. For example, retroviral vectors may be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for proper packaging of the viral genome and integration into host cell DNA. The nucleic acid sequence encoding the iRNA is cloned into one or more vectors that facilitate the delivery of the nucleic acid to the patient. Further details on retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references demonstrating the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Possible lentiviral vectors for use include, for example, HIV-based vectors described in U.S. Patent No. 6,143,520; No. 5,665,557; and No. 5,981,276, which are incorporated herein by reference.

[0353] Adenoviruses are also considered for use in iRNA delivery according to the present invention. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelium. Adenoviruses naturally infect respiratory epithelium and cause mild disease. Other targets of adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) provide an overview of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrate the use of adenovirus vectors to deliver genes to rhesus monkey respiratory epithelium. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155; Mastrangeli et al. (1992), J. Clin. Invest. 91:225-234 (1993); PCT Publication International Pamphlet No. 94 / 12649; and Wang et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing the iRNAs addressed in this invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0354] Adeno-associated virus (AAV) vectors can also be used to deliver the iRNA of the present invention (Walsh et al., Proc.Soc.Exp.Biol.Med.204:289-300(1993); U.S. Patent No. 5,436,146). In one embodiment, the iRNA may be expressed as two distinct, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNAs addressed in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher KJ et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Patent Application Publication No. 94 / 13788; and International Patent Application Publication No. 93 / 24641, which are fully disclosed herein by reference.

[0355] Other viral vectors suitable for delivering the iRNA of the present invention include vaccinia viruses, such as attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, and poxviruses such as avian poxes like fowlpox or canarypox.

[0356] The directivity of a viral vector can be modified by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins as needed. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mocola, etc. AAV vectors can be constructed to target different cells by manipulating the vector to express different capsid protein serotypes. See, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the full disclosure of which is incorporated herein by reference.

[0357] A vector-based pharmaceutical formulation may contain the vector in an acceptable diluent, or it may contain a sustained-release matrix into which the gene delivery vehicle is embedded. Alternatively, if a complete gene delivery vector, such as a retroviral vector, can be produced intact from recombinant cells, the pharmaceutical formulation may contain one or more cells that produce the gene delivery system.

[0358] VI. Pharmaceutical composition of the present invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNA of the present invention. In one embodiment, the present invention provides a pharmaceutical composition comprising the iRNA as described herein and a pharmaceutically acceptable carrier. The iRNA-containing pharmaceutical composition is useful for the treatment of diseases or disorders related to the expression or activity of contact activation pathway genes (i.e., the KLKB1 gene, the F12 gene, and / or the KNG1 gene). Such pharmaceutical compositions are formulated based on a delivery method. One example is a composition formulated for systemic administration via parenteral delivery, for example, by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma by intracerebral infusion, for example, by continuous pump infusion. The pharmaceutical composition of the present invention may be administered in a dose sufficient to inhibit the expression of contact activation pathway genes.

[0359] Such pharmaceutical compositions are formulated based on the delivery method. One example is a composition formulated for systemic administration by parenteral delivery, such as intravenous (IV) delivery, or for subcutaneous delivery. Another example is a composition formulated for direct delivery to the liver by intrahepatic infusion, such as by continuous pump infusion.

[0360] The pharmaceutical composition of the present invention may be administered in a dose sufficient to inhibit the expression of contact activation pathway genes. Generally, preferred doses of the iRNA of the present invention may range from about 0.001 to about 200.0 milligrams per kilogram of body weight per day of the recipient, generally ranging from about 1 to 50 mg per kilogram of body weight per day. Typically, preferred doses of the iRNA of the present invention may range from about 0.1 mg / kg to about 5.0 mg / kg, preferably from about 0.3 mg / kg to about 3.0 mg / kg. Repeated-dose regimens may include periodic administration of therapeutic doses of iRNA, such as every other day or once a year. In certain embodiments, iRNA is administered about once a month to about four times a year (i.e., every three months).

[0361] After the initial treatment regimen, the treatment can be administered at a reduced frequency.

[0362] Those skilled in the art will recognize that certain factors, including but not limited to the severity of the disease or illness, previous treatments, the overall health and / or age of the subject, and other pre-existing diseases, may influence the dose and time required to effectively treat the subject. Furthermore, treatment of the subject with a therapeutically effective amount of the composition may consist of a single treatment or a series of treatments. The effective dose and in vivo half-life for each iRNA encompassed by the present invention can be estimated using conventional methodologies or based on in vivo studies using appropriate animal models as described elsewhere herein.

[0363] Advances in mouse genetics have led to the creation of numerous mouse models for studying various human diseases, including disorders that may benefit from reduced expression of contact activation pathway genes.

[0364] The pharmaceutical composition of the present invention may be administered in several ways, depending on whether local or systemic treatment is required and the site to be treated. Administration may be local (e.g., by a transdermal patch), pulmonary administration by inhalation or blowing of powder or aerosol, for example, using a sprayer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral administration. Parenteral administration may be intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; for example, subcutaneous administration using an implantable device; or, for example, intracranial administration by intraparenchymal, intrathecal or intraventricular administration.

[0365] iRNAs can be delivered to target specific tissues, such as the liver (e.g., hepatocytes in the liver).

[0366] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, droplets, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc., are required or may be desired. Covered condoms and gloves may also be useful. Suitable topical formulations include those in which the iRNA characterizing the present invention is a mixture with a topically delivered agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleyltetramethylaminopropyl DOTAP and dioleylphosphatidylethanolamine DOTMA). The iRNAs characterizing the present invention can be encapsulated in liposomes or can form complexes with liposomes, particularly cationic liposomes. Alternatively, the iRNAs may be complexed with lipids, particularly 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, dicaplate, tricaplate, monoolein, dilaurin, glyceryl 1-monocaplate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 Examples include alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, or diglycerides; or pharmaceutically acceptable salts thereof). Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0367] A. iRNA preparations containing membrane molecular assemblies iRNAs for use in the compositions and methods of the present invention can be formulated for delivery in membrane molecular assemblies, such as liposomes or micelles. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one or more bilayers. Liposomes include monolayer and multilayer vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior and usually does not contain the iRNA composition, but may in some cases. Liposomes are useful for the transport and delivery of active ingredients to the site of action. Because the liposome membrane is structurally similar to that of biological membranes, when a liposome adheres to a tissue, the bilayer of the liposome fuses with the bilayer of the cell membrane. As the fusion of the liposome and the cell progresses, the aqueous contents of the interior containing the iRNA are delivered to the cell, where the iRNA can specifically bind to target RNA and mediate the delivery. In some cases, liposomes can also be specifically targeted, for example, to direct iRNAs towards a particular cell type.

[0368] Liposomes containing iRNA agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component may be an amphiphilic cationic lipid or a lipid conjugate. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholates, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. Next, the iRNA agent preparation is added to the micelles containing the lipid component. The cationic groups in the lipids interact with the iRNA agent and condense around the iRNA agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposomal formulation of the iRNA agent.

[0369] If necessary, a support compound to aid condensation may be added during the condensation reaction, for example, by controlled addition. For example, the support compound may be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH may also be adjusted to aid condensation.

[0370] Methods for generating stable polynucleotide delivery vehicles incorporating polynucleotide / cationic lipid complexes as components of the delivery vehicle are further described, for example, in International Publication No. 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation is described by Felgner, PLet al., Proc. Natl. Acad. Sci. USA 8:7413-7417, 1987; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; Bangham et al., M. Mol. 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. This may also include one or more embodiments of the exemplary methods described in al., Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid assemblies of a suitable size for use as a delivery vehicle include sonication, freeze-thaw, and extrusion (see, e.g., Mayer et al., Biochim. Biophys. Acta 858:161, 1986). If consistently small (50–200 nm) and relatively uniform assemblies are desired, microfluidization may be used (Mayhew et al., Biochim. Biophys. Acta 775:169, 1984). These methods are readily adaptable for packaging iRNA preparations into liposomes.

[0371] Liposomes are divided into two major classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are transported into endosomes. The acidic pH within the endosomes causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0372] pH-sensitive and negatively charged liposomes capture nucleic acids rather than complex them. Since both nucleic acids and lipids are similarly charged, repulsion occurs rather than complex formation. Nevertheless, some nucleic acids are captured within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding thymidine kinase genes to a cell monolayer in a culture medium. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269~274).

[0373] One major type of liposome composition contains phospholipids other than naturally derived phosphatidylcholine. For example, neutral liposome compositions may be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic membrane-fused liposomes are primarily formed from dioleylphosphatidylethanolamine (DOPE). Other types of liposome compositions are formed from phosphatidylcholine (PC), such as soy PC and egg PC. Other types are formed from mixtures of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0374] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent Nos. 5,283,185; U.S. Patent Nos. 5,171,678; International Publication No. 94 / 00569; International Publication No. 93 / 24640; International Publication No. 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.

[0375] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been tested, and their usefulness in drug delivery to the skin has been determined. Cyclosporine-A was delivered to the dermis of mouse skin using nonionic liposome formulations containing Novasome® I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome® II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). The results showed that such nonionic liposome systems are effective in promoting the deposition of cyclosporine-A in different layers of the skin (Hu et al. STPPharma.Sci., 1994, 4(6)466).

[0376] Liposomes also include “stereostabilized” liposomes, and as used herein, this term refers to liposomes containing one or more specific lipids, which, when incorporated into the liposome, result in an enhanced cyclic lifespan compared to liposomes lacking such specific lipids. An example of a stereostabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome contains (A) monosialoganglioside G M1These include (B) those containing one or more glycolipids, or those derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. While not bound by any particular theory, in the art, with regard to sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivativeized lipids, the enhanced circulating half-life of these sterically stabilized liposomes is thought to be due to reduced uptake into cells by the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0377] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NYAcad. Sci., 1987, 507, 64) developed monosialoganglioside G to improve the blood half-life of liposomes. M1 The capabilities of galactocerebroside sulfate and phosphatidylinositol have been reported. These findings are detailed by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). Both U.S. Patent No. 4,837,028 and International Publication No. 88 / 04924, both granted to Allen et al., describe (1) sphingomyelin and (2) ganglioside G M1 Alternatively, liposomes containing galactocerebroside sulfate are disclosed. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimiristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 (Lim et al.).

[0378] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes cannot fuse with the cell membrane as efficiently, but they can be taken up by macrophages in vivo and used to deliver iRNA agents to macrophages.

[0379] Further advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can encapsulate a wide range of water-soluble and lipid-soluble drugs; and liposomes can protect iRNA agents encapsulated within 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 liposomal formulations are lipid surface charge, vesicle size, and the aqueous volume of the liposome.

[0380] Using N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), a positively charged synthetic cationic lipid, small liposomes can be formed that spontaneously interact with nucleic acids and fuse with negatively charged lipids in the cell membrane of tissue culture cells to form lipid-nucleic acid complexes capable of delivering iRNA agents (see, for example, Felgner, Plet al., Proc. Natl. Acad. Sci. USA 8:7413-7417, 1987, and U.S. Patent No. 4,897,355 for a description of its use with DOTMA and DNA).

[0381] 1,2-bis(oleyloxy)-3-(trimethylammonia)propane (DOTAP), a DOTMA analog, can be used in combination with phospholipids to form DNA complex vesicles. Lipofectin® (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids into tissue culture cells containing positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. When sufficiently positively charged liposomes are used, the net charge of the resulting complex is also positive. The positively charged complex thus prepared spontaneously attaches to negatively charged cell surfaces, fuses with the cell membrane, and efficiently delivers functional nucleic acids, for example, into tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl portion is linked by an ester rather than an ether linkage.

[0382] Other reported cationic lipid compounds include those conjugated to one of two types of lipids, such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam®, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide ("DPPES"), and those conjugated to various moieties including carboxyspermine (see, for example, U.S. Patent No. 5,171,678).

[0383] Another cationic lipid conjugate involves the derivatization of lipids by cholesterol formulated into liposomes in combination with DOPE ("DC-Chol") (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, produced 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). In certain cell lines, these liposomes containing conjugated cationic lipids have been shown to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for oligonucleotide delivery are described in International Publication No. 98 / 39359 and International Publication No. 96 / 37194.

[0384] Liposome formulations are particularly well-suited for topical administration, and they offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver iRNA agents to the skin. In some implementations, liposomes are used to deliver iRNA agents to epidermal cells and to facilitate their penetration into dermal tissue, such as the skin. For example, liposomes can be applied topically. Local delivery of drugs formulated as liposomes to the skin has been reported (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. Jand Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al., Gene 56:267-276, 1987; Nicolau, C. et al. (1987) Meth. Enz. 149:157-176, 1987; Straubinger, R. Rand Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Rand See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987.

[0385] Furthermore, nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, were investigated to determine their usefulness in drug delivery to the skin. Nonionic liposome formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver drugs to the dermis of mouse skin. Such formulations containing iRNA agents are useful for treating skin diseases.

[0386] iRNA-containing liposomes can be made highly deformable. Such deformability can allow liposomes to permeate pores smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposome. Transfersomes can be made by adding surface edge activators, usually surfactants, to a standard liposome composition. Transfersomes containing iRNA agents can be delivered to keratinocytes in the skin, for example, by subcutaneous infection. To traverse intact mammalian skin, the lipid vesicles must permeate a series of micropores, each having a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. Furthermore, due to their lipid properties, these transfersomes can be self-optimizing (e.g., adaptable to the shape of hair follicles), self-repairing, often reaching their targets without rupture, and often self-loading.

[0387] Other formulations suitable for the present invention are described in U.S. Provisional Patent Application No. 61 / 018,616, filed on 2 January 2008; No. 61 / 018,611, filed on 2 January 2008; No. 61 / 039,748, filed on 26 March 2008; No. 61 / 047,087, filed on 22 April 2008; and No. 61 / 051,528, filed on 8 May 2008. PCT Application PCT / US2007 / 080331, filed on 3 October 2007, also describes formulations suitable for the present invention.

[0388] Transfersomes are another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can also be described as lipid droplets, which, due to their high deformability, can easily permeate smaller pores than droplets. Transfersomes are adaptable to the environment in which they are used, for example, self-optimal (adapting to the shape of pores in the skin), self-repairing, often reaching their targets without fragmentation, and frequently self-loading. To construct transfersomes, it is possible to add surface edge activators, usually surfactants, to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a serum albumin-containing solution.

[0389] Surfactants have found broad applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and ranking the numerous different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The properties of the hydrophilic group (also known as the "head") provide the most useful means of classifying different surfactants used in formulations (Rieger, "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0390] When a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of applications in pharmaceutical and cosmetic products and can be used across a wide range of pH values. Generally, their HLB values ​​range from 2 to about 18, depending on their structure. Examples of 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. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0391] When a surfactant molecule retains a negative charge when dissolved or dispersed in water, it is classified as anionic. Anionic surfactants include carboxylates such as soap, acyl lactylates, acylamides of amino acids, sulfate esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0392] When a surfactant molecule retains a positive charge when dissolved or dispersed in water, it is classified as a cationic surfactant. Examples of cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0393] A surfactant is classified as amphoteric if its molecule has the ability to possess either a positive or negative charge. Examples of amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines, and phosphatides.

[0394] The use of surfactants in drug products, formulations, and emulsions is outlined (Rieger, “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0395] iRNA for use in the methods of the present invention may also be provided as micelle formulations. A “micelle” is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic parts of the molecules face inward and the hydrophilic parts remain in contact with the surrounding aqueous phase. The opposite arrangement exists when the environment is hydrophobic.

[0396] Mixed micelle formulations suitable for transdermal delivery include aqueous solutions of siRNA compositions and alkali metals C8-C8. 22 It can be prepared by mixing alkyl sulfates and micelle-forming compounds. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocolanyglycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogues, polydocanol alkyl ethers and their analogues, chenodeoxycholates, deoxycholates, and mixtures thereof. The micelle-forming compounds may be added simultaneously with or after the addition of alkali metal alkyl sulfates. Mixed micelles are formed with substantially any type of mixture of components, but vigorous mixing is preferred to provide smaller sized micelles.

[0397] In one method, a first micelle composition containing an siRNA composition and at least an alkali metal alkyl sulfate is prepared. The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, a micelle composition is prepared by mixing an siRNA composition, an alkali metal alkyl sulfate, and at least one micelle-forming compound, followed by adding the remaining micelle-forming compounds while vigorously mixing.

[0398] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect it from bacterial growth. Alternatively, phenol and / or m-cresol may be added together with the micelle-forming components. An isotonic agent such as glycerin may also be added after the formation of the mixed micelle composition.

[0399] In the delivery of micelle formulations as a spray, the formulation can be placed in an aerosol dispenser, and the dispenser is filled with a propellant. Under pressure, the propellant is in liquid form in the dispenser. The ratio of components is adjusted so that there is one aqueous phase and one propellant phase, i.e., one phase exists. If two phases exist, for example, by a metering valve, the dispenser needs to be shaken before dispensing a portion of the contents. The dosage of the drug is dispensed in the form of a fine spray from the metering valve.

[0400] The propellant may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ethers, and diethyl ethers. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

[0401] The specific concentration of essential components can be determined by relatively simple experiments. For oral absorption, it is often desirable to increase the dose to, for example, at least two or three times the dose used for injection or administration via the gastrointestinal tract.

[0402] B. Lipid particles The iRNA, i.e., dsRNA, of the present invention may be completely encapsulated in a lipid preparation, for example, in LNP, or may form other nucleic acid-lipid particles.

[0403] As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNPs typically include cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs are extremely useful for systemic application because they have an extended circulating lifetime after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include "pSPLPs," which contain encapsulated condensant-nucleic acid complexes, as shown in PCT Publication International Publication No. 00 / 03683. The particles of the present invention typically have an average particle size of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. In addition, when nucleic acids are present in the nucleic acid-lipid particles of the present invention, they are resistant to degradation by nucleases in aqueous solutions. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent No. 5,976,567; U.S. Patent No. 5,981,501; U.S. Patent No. 6,534,484; U.S. Patent No. 6,586,410; U.S. Patent No. 6,815,432; U.S. Patent Publication No. 2010 / 0324120; and PCT International Publication No. 96 / 40964.

[0404] In one embodiment, the lipid-to-drug ratio (mass / mass ratio) (e.g., lipid-to-dsRNA ratio) may be within the range of approximately 1:1 to approximately 50:1, approximately 1:1 to approximately 25:1, approximately 3:1 to approximately 15:1, approximately 4:1 to approximately 10:1, approximately 5:1 to approximately 9:1, or approximately 6:1 to approximately 9:1. Ranges between the above ranges are also considered to be part of the present invention.

[0405] Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleyloxy)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-dilinolelenyloxy-N,N-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcarbamoyloxy-3-di Methylaminopropane (DLin-C-DAP), 1,2-Dilinoley oxy-3-(dimethylamino)acetoxypropane (Dlin-DAC), 1,2-Dilinoley oxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoley-3-dimethylaminopropane (DLinDaP), 1,2-Dilinoley thio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleyl-2-Linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoley oxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoley-3-trimethylaminopropane chloride (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-propanediol (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 analogues thereof The following may be used: (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-yl4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylazanegiyl)didodecane-2-ol (Tech G1), or mixtures thereof. Cationic lipids can constitute approximately 20 mol% to 50 mol%, or even 40 mol%, of the total lipids present in the particles.

[0406] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane may be used to prepare lipid-siRNA nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0407] In one embodiment, the lipid-siRNA particles contain 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 10% DSPC, 40% cholesterol, and 10% PEG-C-DOMG (mol percent), with a particle size of 63.0 ± 20 nm and a siRNA / lipid ratio of 0.027.

[0408] Ionic / non-cationic lipids include, without limitation, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleyl-phosphatidylethanolamine 4-(N The anionic or neutral lipids may include -maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidy(phosphatidy)ethanolamine (SOPE), cholesterol, or mixtures thereof. If cholesterol is included, the non-cationic lipids may constitute about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles.

[0409] The conjugate lipids that inhibit particle aggregation can be, for example, polyethylene glycol (PEG) lipids containing, non-limitingly, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C8). The amount of conjugate lipids that prevent particle aggregation can be 0 mol% to about 20 mol%, or 2 mol%, of the total lipids present in the particles.

[0410] In some embodiments, the nucleic acid-lipid particles further contain, for example, about 10 mol% to about 60 mol% or about 48 mol% of the total lipids present in the particles, which is cholesterol.

[0411] In one embodiment, lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) may be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Each stock solution in ethanol may be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-Ceramide C16, 100 mg / ml. The stock solutions of ND98, cholesterol, and PEG-Ceramide C16 may then be combined, for example, in a molar ratio of 42:48:10. The combined lipid solution can be mixed with an aqueous dsRNA solution (e.g., in sodium acetate (pH 5)) such that the final ethanol concentration is approximately 35–45% and the final sodium acetate concentration is approximately 100–300 mM. Lipid-dsRNA nanoparticles are usually formed spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., with a 100 nm cutoff) using a thermobarrel extruder, such as a Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step may be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged with phosphate-buffered saline (PBS) of approximately pH 7, e.g., approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4. [ka]

[0412] The LNP01 formulation is described, for example, in the brochure International Publication No. 2008 / 042973, which is incorporated herein by reference.

[0413] Further exemplary lipid-dsRNA formulations are listed in Table 1.

[0414] [Table 1]

[0415] [Table 2]

[0416] DSPC: Distearoylphosphatidylcholine DPPC: Dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with an average molecular weight of 2000) PEG-DSG: PEG-distylylglycerol (C18-PEG, or PEG-C18) (PEG with an average molecular weight of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with an average molecular weight of 2000) Formulations comprising SNALP(l,2-dilinolenyloxy-N,N-dimethylaminepropane (DLinDMA)) are described in International Publication No. 2009 / 127060, filed on April 15, 2009, which is incorporated herein by reference.

[0417] Formulations containing XTC are described, for example, in U.S. Provisional Patent Application No. 61 / 148,366 filed on 29 January 2009; U.S. Provisional Patent Application No. 61 / 156,851 filed on 2 March 2009; U.S. Provisional Patent Application No. 61 / 228,373 filed on 24 July 2009; U.S. Provisional Patent Application No. 61 / 239,686 filed on 3 September 2009; and International Application PCT / US2010 / 022614 filed on 29 January 2010 (these are incorporated herein by reference).

[0418] Formulations containing MC3 are described, for example, in U.S. Patent Application Publication No. 2010 / 0324120, filed on June 10, 2010 (all of which are incorporated herein by reference).

[0419] Formulations containing ALNY-100 are described, for example, in International Patent Application PCT / US09 / 63933, filed on November 10, 2009 (as incorporated herein by reference).

[0420] Formulations containing C12-200 are described in U.S. Provisional Patent Application No. 61 / 175,770, filed on May 5, 2009, and International Application No. PCT / US10 / 33777, filed on May 5, 2010 (both of which are incorporated herein by reference).

[0421] Compositions and formulations for oral administration include powders or granules, fine particles, nanoparticles, turbidiants, or liquids in water or a non-aqueous medium, capsules, gel capsules, medicine bags, tablets, or small tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing agents, or binders are desired. In some embodiments, the oral formulation is administered with one or more permeabilizing surfactants and chelating agents, containing the dsRNA characteristic of the present invention. Suitable surfactants include fatty acids and / or esters or salts thereof, and bile acids and / or salts thereof. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydrofusidate, 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-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, combinations of osmotic enhancers, such as fatty acid / salt combinations with bile acids / salts, are used. One exemplary combination is lauric acid, capric acid, and the sodium salt of UDCA. Further osmotic enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNAs characterizing the present invention can be delivered orally in granular form, including spray-dried particles or those complexed to form micro or nanoparticles.Examples of dsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxetanes, polyalkylcyanoacrylates; cationic gelatin, albumin, starch, acrylates, polyethylene glycol (PEG), and starch; polyalkylcyanoacrylates; DEAE-derivativeized polyimines, pullulan, cellulose, and starch. Preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P (TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcyano(cyano)acrylate), DEAE-methacrylate, DEAE-hexylacrylate Examples include benzoate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid-coglycolic acid (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations for dsRNA, and such formulations, are described in U.S. Patent No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Patent No. 6,747,014, each of which is incorporated herein by reference.

[0422] Compositions and formulations for parenteral, intraparenchymal (into the brain), subarachnoid, intraventricular, or intrahepatic administration include sterile aqueous solutions, which may also include buffers, diluents, and other suitable additives such as, but not limited to, osmotic enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0423] The pharmaceutical compositions of the present invention include, but are not limited to, liquid formulations, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components, including, but are not limited to, pre-formed liquid formulations, self-emulsifying solids, and self-emulsifying semi-solids. When treating liver diseases such as hepatocarcinoma, formulations targeting the liver are particularly preferred.

[0424] The pharmaceutical formulations of the present invention, which can conveniently exist in unit dosage forms, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of associating the active ingredient with a pharmaceutical carrier or excipient. Generally, the formulations are prepared by homogeneously and intimately associating the active ingredient with a liquid carrier or a micronized solid carrier or both, and then, if necessary, shaping the product.

[0425] The compositions of the present invention can be formulated in any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, softgels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. The aqueous suspension may further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0426] C. Further Formulations i. Emulsion The composition of the present invention can be prepared and formulated as an emulsion. An emulsion is typically a heterogeneous system in which one liquid is dispersed in another liquid, usually in the form of droplets with a diameter greater than 0.1 μm (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and (See Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p.335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). An emulsion is often a two-layer system containing two immiscible liquid phases that are closely mixed and dispersed with each other. Generally, emulsions can be either water in oil (w / o) or oil in water (o / w). When the aqueous phase is finely divided and dispersed as microdroplets in the oil phase of the mass, the resulting composition is called a water in oil (w / o) emulsion. Alternatively, when the oil phase is finely divided and dispersed as microdroplets in the aqueous phase of the mass, the resulting composition is called an oil in water (o / w) emulsion.Emulsions may contain additional components in addition to the dispersed phase and active drug, which may exist as solutions in the aqueous phase, the oil phase, or as separate phases themselves. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as needed. Pharmaceutical emulsions can be polyemulsions consisting of three or more phases, such as oil-in-water (o / w / o) and water-in-oil (w / o / w) emulsions. Such complex formulations often offer certain advantages not provided by simple two-component emulsions. A polyemulsion in which individual oil droplets of an o / w emulsion surround smaller water droplets constitutes a w / o / w emulsion. Similarly, a system of oil droplets surrounded by water droplets stabilized in a continuous phase of oil provides an o / w / o emulsion.

[0427] Emulsions are characterized by having little to no thermodynamic stability. Often, the dispersion or discontinuous phase of an emulsion is well dispersed externally or within a continuous phase and maintained in this form through emulsifiers or the viscosity of the formulation. Any of the phases of the emulsion may be semi-solid or solid, as in the case of emulsion-type ointment bases and creams. Other means of stabilizing an emulsion include the use of emulsifiers that may be incorporated into any of the phases of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorbent bases, and finely dispersed solids (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0428] Synthetic surfactants, also known as surfactants, have found broad applicability in emulsion formulation and are outlined in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p.199). Surfactants are typically amphiphilic and contain both hydrophilic and hydrophobic parts. The ratio of hydrophilicity to hydrophobicity in a surfactant is called the hydrophilic / lipophilic balance (HLB), and it is a valuable tool for classifying and selecting surfactants during the preparation of pharmaceutical formulations. Surfactants can be classified into different types based on the properties of their hydrophilic groups: nonionic, anionic, cationic, and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285).

[0429] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorbent bases, such as anhydrous lanolin and hydrophilic petrolatum, incorporate water to form w / o emulsions while retaining their hydrophilic properties to maintain their semi-solid consistency. Micronized solids are used as good emulsifiers, particularly in surfactant combinations and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate, and colloidal aluminum magnesium silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

[0430] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion. 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, NY, volume 1, p.335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.199).

[0431] Hydrophilic colloids include naturally occurring rubbers such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), synthetic polymers, cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomer, cellulose ether, and carboxyvinyl polymer). These disperse in water or swell in water to form a colloidal solution that stabilizes the emulsion by forming a strong interfacial film around droplets of the dispersed phase and by increasing the viscosity of the outer phase.

[0432] Because emulsions often contain numerous components such as carbohydrates, proteins, sterols, and phosphatides that readily support microbial growth, these formulations frequently incorporate preservatives. Commonly used preservatives in formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, p-hydroxybenzoic acid esters, and boric acid. Antioxidants are also typically added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may include free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, as well as antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0433] The application of emulsion formulations via cutaneous, oral, and parenteral routes, as well as their manufacturing methods, are outlined in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are widely used due to their ease of formulation and their effectiveness in terms of absorption and bioavailability (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutritional preparations are commonly found in materials administered orally as o / w emulsions.

[0434] ii. Microemulsion In one embodiment of the present invention, the iRNA and nucleic acid composition is formulated as a microemulsion. A microemulsion can be defined as a system of water, oil, and an amphiphilic substance that is a single optically isotropic and thermodynamically stable liquid solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, a microemulsion is prepared by first dispersing the oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, generally an alcohol of an intermediate chain length, to form a clear system. Therefore, microemulsions are described as thermodynamically stable, isotropically transparent dispersions consisting of two immiscible liquids stabilized by an interfacial film of surfactant molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are typically prepared using a combination of 3 to 5 components, including oil, water, surfactant, co-surfactant, and electrolyte. Whether a microemulsion is water in oil (w / o) or oil in water (o / w) depends on the properties of the oil and surfactant used, as well as the structure of the polar head and hydrocarbon tail of the surfactant molecule and its geometric packing (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0435] Phenomenological methods using phase diagrams have been extensively studied, providing those skilled in the art with broad knowledge about methods for formulating microemulsions (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a thermodynamically stable droplet formulation that forms spontaneously.

[0436] Surfactants used in the preparation of microemulsions include, without limitation, ionic surfactants, nonionic 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), and decaglycerol decaoleate (DAO750), either alone or in combination with auxiliary surfactants. Auxiliary surfactants, typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, penetrate the surfactant film and, as a result, increase interfacial fluidity by forming an irregular film through the resulting void spaces between surfactant molecules. However, microemulsions can be prepared without the use of auxiliary surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but not limited to, water, an aqueous solution of a drug, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase can be, but not limited to, Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C) 12 ) Mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolated glycerides, saturated polyglycolated C8-C 10 It may contain materials such as glycerides, vegetable oils, and silicone oils.

[0437] Microemulsions are of particular interest from the standpoint of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed for improving the oral bioavailability of peptide-containing drugs (see, for example, U.S. Patents 6,191,105, 7,063,860, 7,070,802, and 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth.Find.Exp.Clin.Pharmacol., 1993, 13, 205). Microemulsions offer advantages such as improved drug solubilization, protection of drugs from enzymatic hydrolysis, potentially improved drug absorption due to altered membrane fluidity and permeability by surfactant induction, ease of preparation, ease of oral administration beyond solid dosage forms, improved clinical efficacy, and reduced toxicity (see, for example, U.S. Patent Nos. 6,191,105, 7,063,860, 7,070,802, and 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). In many cases, microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating heat-sensitive drugs, peptides, or iRNAs. Microemulsions are also effective for the efficient delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to promote increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improved local cellular uptake of iRNAs and nucleic acids.

[0438] The microemulsion of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and permeation enhancers to improve the properties of the formulation and enhance the absorption of iRNA and nucleic acids of the present invention. Permeation enhancers used in the microemulsion 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 is discussed above.

[0439] iii. Particulates The iRNA agent of the present invention may be incorporated into particles, such as microparticles. Microparticles may be produced by spray drying, but may also be produced by other methods including freeze-drying, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

[0440] iv. Penetration enhancers In one embodiment, the present invention utilizes various penetration enhancers to efficiently deliver nucleic acids, particularly iRNA, to animal skin. Most drugs exist in solution in both ionized and non-ionized forms. However, typically only lipid-soluble or lipophilic drugs readily cross cell membranes. It has been found that non-lipophilic drugs can also cross cell membranes if the membrane being crossed is treated with a penetration enhancer. In addition to assisting the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also improve the penetration of lipophilic drugs.

[0441] Penetration enhancers can be classified as belonging to one of five major categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactant agents (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above types of penetration enhancers is described in more detail below.

[0442] Surfactants (or "surface-activating agents") are chemical substances that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved absorption of iRNA through mucous membranes. In addition to bile salts and fatty acids, examples of 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, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluoro compound emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0443] Various fatty acids and their derivatives that 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-monoleyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, and their C 1~20 Examples include alkyl esters (e.g., methyl, isopropyl, and t-butyl), as well as their monoglycerides and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, for example, Touitou, E., et al., Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews i...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of factor XII (Hageman factor) (F12), wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 9 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 10 by three or fewer nucleotides.

2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of factor XII (Hageman factor) (F12), wherein the dsRNA agent comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in any one of Tables 9, 10, 19C, 19D, 20, 21, 23, 24, 26, and 27.

3. A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of the factor XII (Hageman factor) (F12) gene, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region containing at least 15 consecutive nucleotides that differ from nucleotides 2000 to 2060 of SEQ ID NO: 9 by three or fewer nucleotides.

4. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of kallikrein B, plasma (Fletcher factor) 1 (KLKB1), wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by three or fewer nucleotides.

5. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of kallikrein B, plasma (Fletcher factor) 1 (KLKB1), wherein the dsRNA agent comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in Table 3, 4, 19A, or 19B.

6. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of kininogen 1 (KNG1), wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 17 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 18 by three or fewer nucleotides.

7. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of kininogen 1 (KNG1), wherein the dsRNA agent comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in Tables 15, 16, 19E, or 19F.

8. The dsRNA agent according to any one of claims 1 to 7, wherein the dsRNA agent comprises at least one modified nucleotide.

9. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of factor XII (Hageman factor) (F12), wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region. The sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 9 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 10 by three or fewer nucleotides. Substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and A dsRNA agent in which the sense strand is conjugated to a ligand bound to its 3' end.

10. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of kallikrein B, plasma (Fletcher factor) 1 (KLKB1), wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region. The sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by three or fewer nucleotides. Substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and A dsRNA agent in which the sense strand is conjugated to a ligand bound to its 3' end.

11. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of kininogen 1 (KNG1), wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region. The sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 17 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 18 by three or fewer nucleotides. Substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and A dsRNA agent in which the sense strand is conjugated to a ligand bound to its 3' end.

12. The dsRNA agent according to any one of claims 9 to 11, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified.

13. The dsRNA agent according to any one of claims 8 to 12, wherein at least one of the modified nucleotides is selected from the group consisting of deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, fixed nucleotides, unfixed nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramides, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic.

14. The dsRNA agent according to any one of claims 8 to 12, wherein at least one of the modified nucleotides is selected from the group consisting of 2'-O-methyl and 2'-fluoro modifications.

15. The dsRNA agent according to claim 13 or 14, further comprising at least one phosphorothioate nucleotide interbonding.

16. The dsRNA agent according to claim 15, comprising 6 to 8 phosphorothioate nucleotide interbonds.

17. The dsRNA agent according to any one of claims 1 to 7, wherein the complementary region is at least 17 nucleotides long.

18. The dsRNA agent according to any one of claims 1 to 7, wherein the complementary region is 19 to 30 nucleotides long.

19. The dsRNA agent according to claim 18, wherein the complementary region is 21 nucleotides long.

20. The dsRNA agent according to claim 18, wherein the complementary region is 21 to 23 nucleotides long.

21. The dsRNA agent according to claim 18, wherein the complementary region is 19 nucleotides long.

22. A dsRNA agent according to any one of claims 1 to 21, wherein each chain is 30 nucleotides or less in length.

23. A dsRNA agent according to any one of claims 1 to 21, wherein each chain is independently 19 to 30 nucleotides long.

24. A dsRNA agent according to any one of claims 1 to 20, wherein each strand is independently 19 to 25 nucleotides long.

25. A dsRNA agent according to any one of claims 1 to 23, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

26. A dsRNA agent according to any one of claims 1 to 23, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

27. A dsRNA agent according to any one of claims 1 to 7, further comprising a ligand.

28. The dsRNA agent according to claim 27, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

29. The dsRNA agent according to any one of claims 9 to 11 and 28, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

30. The ligand is 【Chemistry 1】 The dsRNA agent according to claim 29.

31. The dsRNA agent is shown in the following schematic diagram. 【Chemistry 2】 The dsRNA agent according to claim 30, conjugated to a ligand as shown in the formula, wherein X is O or S.

32. The dsRNA agent according to claim 31, wherein X is O.

33. The dsRNA agent according to claim 1, wherein the agent is selected from the group consisting of AD-66170, AD-66173, AD-66176, AD-66125, AD-66172, AD-66167, AD-66165, AD-66168, AD-66163, AD-66116, AD-66126, and AD-67244.

34. The dsRNA agent according to claim 4, wherein the agent is selected from the group consisting of AD-65077, AD-65170, AD-65103, AD-65083, AD-65087, AD-65149, AD-64652, AD-65162, AD-65153, AD-65084, AD-65099, and AD-66948.

35. The dsRNA agent according to claim 6, wherein the agent is selected from the group consisting of AD-66259, AD-66261, AD-66262, AD-66263, AD-6634, and AD-67344.

36. Cells containing the dsRNA agent according to any one of claims 1 to 35.

37. A vector encoding at least one strand of a dsRNA agent according to any one of claims 1 to 3.

38. A vector encoding at least one strand of the dsRNA agent according to claim 4 or 5.

39. A vector encoding at least one strand of the dsRNA agent according to claim 6 or 7.

40. A pharmaceutical composition for inhibiting the expression of the KLKB1 gene, comprising a dsRNA agent according to any one of claims 4, 5, and 10, or a vector according to claim 38.

41. A pharmaceutical composition for inhibiting the expression of the F12 gene, comprising a dsRNA agent according to any one of claims 1 to 3 and 9, or a vector according to claim 37.

42. A pharmaceutical composition for inhibiting the expression of the KNG1 gene, comprising a dsRNA agent according to any one of claims 6, 7, and 11, or a vector according to claim 39.

43. The pharmaceutical composition according to any one of claims 40 to 42, wherein the dsRNA agent is administered in a non-buffered environment.

44. The pharmaceutical composition according to claim 43, wherein the non-buffer solution is physiological saline or water.

45. The pharmaceutical composition according to any one of claims 40 to 42, wherein the dsRNA agent is administered with a buffer solution.

46. The pharmaceutical composition according to claim 45, wherein the buffer solution comprises an acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.

47. The pharmaceutical composition according to claim 45, wherein the buffer solution is phosphate-buffered saline (PBS).

48. A pharmaceutical composition comprising a dsRNA agent and a lipid preparation according to any one of claims 1 to 35.

49. A method for inhibiting KLKB1 expression in cells, (a) a step of contacting the cells with a dsRNA agent according to any one of claims 4, 5, and 10, or a pharmaceutical composition according to any one of claims 40 and 43 to 48; (b) A method comprising the step of maintaining the cells produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the KLKB1 gene, thereby inhibiting the expression of the KLKB1 gene in the cells.

50. A method for inhibiting F12 expression in cells, (a) a step of contacting the cells with a dsRNA agent according to any one of claims 1 to 3 and 9, or a pharmaceutical composition according to any one of claims 41 and 43 to 48; (b) A method comprising the step of maintaining the cells produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the F12 gene, thereby inhibiting the expression of the F12 gene in the cells.

51. A method for inhibiting KNG1 expression in cells, (a) a step of contacting the cells with a dsRNA agent according to any one of claims 6, 7, and 11, or a pharmaceutical composition according to any one of claims 42 to 48; (b) A method comprising the step of maintaining the cells produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the KNG1 gene, thereby inhibiting the expression of the KNG1 gene in the cells.

52. The method according to any one of claims 49 to 51, wherein the cells are located in the body of the subject.

53. The method according to claim 50, wherein the subject is a human.

54. The method according to any one of claims 49, 52, and 53, wherein the KLKB1 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

55. The method according to any one of claims 50, 52, and 53, wherein the F12 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

56. The method according to any one of claims 51 to 53, wherein the KNG1 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

57. A method for treating a subject having a disease or disorder that may benefit from reduced expression of a contact activation pathway gene, comprising the step of administering a therapeutically effective amount of a dsRNA agent according to any one of claims 1 to 11, or a pharmaceutical composition according to any one of claims 40 to 48, to the subject, thereby treating the subject.

58. A method for preventing at least one symptom of a disease or disorder in which a person may benefit from reduced expression of a contact activation pathway gene, comprising the step of administering to the person a preventive amount of a dsRNA agent according to any one of claims 1 to 11 or a pharmaceutical composition according to any one of claims 40 to 48, thereby preventing at least one symptom of the person having a disorder in which a person may benefit from reduced expression of a contact activation pathway gene.

59. The method according to claim 57 or 58, wherein administration of the dsRNA to the subject results in a decrease in bradykinin levels or a decrease in coagulation factor XII activity.

60. The method according to claim 57 or 58, wherein the disorder is a contact activation pathway-related disorder.

61. The method according to claim 59, wherein the contact activation pathway-related disease is characterized by a tendency to form thrombi.

62. The method according to claim 60, wherein the contact activation pathway-related disease is hereditary angioedema (HAE).

63. The method according to claim 60, wherein the contact activation pathway-related disease is Fletcher factor deficiency.

64. The method according to claim 60, wherein the contact activation pathway-related disease is essential hypertension.

65. The method according to claim 58, wherein at least one of the symptoms is an angioedema attack.

66. The method according to claim 58, wherein at least one of the symptoms is thrombus formation.

67. The method according to claim 57 or 58, wherein the subject is a human.

68. The method according to any one of claims 57 to 67, further comprising the step of administering an anti-KLKB1 antibody or an antigen-binding fragment thereof to the subject.

69. The method according to claim 57, wherein the method comprises the step of administering a therapeutically effective amount of the dsRNA agent according to any one of claims 1 to 3 or 9 to the subject, and the method further comprises the step of administering the dsRNA agent according to any one of claims 4, 5, and 10 to the subject.

70. The method according to claim 57, wherein the method comprises the step of administering a therapeutically effective amount of the dsRNA agent according to any one of claims 1 to 3 or 9 to the subject, and the method further comprises the step of administering the dsRNA agent according to any one of claims 6, 7, and 11 to the subject.

71. The method according to claim 58, wherein the method comprises the step of administering a prophylactic effective amount of a dsRNA agent according to any one of claims 1 to 3 or 9 to the subject, and the method further comprises the step of administering a dsRNA agent according to any one of claims 4, 5, and 10 to the subject.

72. The method according to claim 58, wherein the method comprises the step of administering a prophylactic effective amount of a dsRNA agent according to any one of claims 1 to 3 or 9 to the subject, and the method further comprises the step of administering a dsRNA agent according to any one of claims 6, 7, and 11 to the subject.

73. The method according to claim 57 or 58, further comprising the step of measuring the bradykinin and / or coagulation factor XII levels of the subject.

74. A method for treating a subject having a tendency to form blood clots, the method comprising the step of administering a therapeutically effective amount of a dsRNA agent according to any one of claims 1 to 3 or 9, or a pharmaceutical composition according to any one of claims 41 and 43 to 48, to the subject, thereby treating the subject.

75. A method for preventing at least one symptom of a subject having a tendency to form blood clots, comprising the step of administering to the subject a preventively effective amount of a dsRNA agent according to any one of claims 1 to 3 or 9, or a pharmaceutical composition according to any one of claims 41 and 43 to 48, thereby preventing at least one symptom of the subject.

76. The method according to claim 72 or 73, further comprising the step of administering the dsRNA agent according to any one of claims 4, 5, or 10 to the subject.

77. The method according to claim 72 or 73, further comprising the step of administering the dsRNA agent according to any one of claims 6, 7, or 11 to the subject.

78. A method for treating a subject having hereditary angioedema (HAE), comprising the step of administering a therapeutically effective amount of a dsRNA agent according to any one of claims 1 to 3 or 9, or a pharmaceutical composition according to any one of claims 41 and 43 to 48, to the subject, thereby treating the subject.

79. A method for preventing at least one symptom of a subject having hereditary angioedema (HAE), comprising the step of administering to the subject a preventive amount of a dsRNA agent according to any one of claims 1 to 3 or 9, or a pharmaceutical composition according to any one of claims 41 and 43 to 48, thereby preventing at least one symptom of the subject.

80. The method according to claim 76 or 77, further comprising the step of administering the dsRNA agent according to any one of claims 4, 5, or 10 to the subject.

81. The method according to claim 76 or 77, further comprising the step of administering the dsRNA agent according to any one of claims 6, 7, or 11 to the subject.

82. A method for inhibiting the expression of KLKB1 in a target, A method comprising the step of administering a therapeutically effective amount of the dsRNA agent according to any one of claims 4, 5, or 10 to the subject, thereby inhibiting the expression of KLKB1 in the subject.

83. A method for inhibiting the expression of F12 in a target, A method comprising the step of administering a therapeutically effective amount of the dsRNA agent according to any one of claims 1 to 3 or 9 to the subject, thereby inhibiting the expression of F12 in the subject.

84. A method for inhibiting the expression of KNG1 in a target, A method comprising the step of administering a therapeutically effective amount of the dsRNA agent according to any one of claims 6, 7, or 11 to the subject, thereby inhibiting the expression of KNG1 in the subject.

85. A method for preventing thrombus formation in a subject at risk of thrombus formation, comprising the step of administering a preventively effective amount of a dsRNA agent according to any one of claims 1 to 3 or 9, or a pharmaceutical composition according to any one of claims 41 and 43 to 48, to the subject, thereby inhibiting thrombus formation in the subject at risk of thrombus formation.

86. The method according to claim 85, wherein the subject at risk of thrombus formation has a contact activation pathway-related disease or disorder.

87. The method according to claim 86, wherein the contact activation pathway-related disease is characterized by a tendency to form thrombi.

88. The method according to claim 86, wherein the contact activation pathway-related disease is hereditary angioedema (HAE).

89. The method according to claim 86, wherein the contact activation pathway-related disease is Fletcher factor deficiency.

90. The method according to claim 86, wherein the contact activation pathway-related disease is essential hypertension.

91. The method according to claim 85, wherein the subject at risk of thrombus formation is selected from the group consisting of surgical patients; internal medicine patients; pregnant subjects; postpartum subjects; subjects with a history of thrombosis; subjects receiving hormone replacement therapy; subjects sitting for long periods of time; and obese subjects.

92. The method according to claim 83, further comprising the step of administering the dsRNA agent according to any one of claims 4, 5, or 10 to the subject.

93. The method according to claim 83, further comprising the step of administering the dsRNA agent according to any one of claims 6, 7, or 11 to the subject.

94. A method for preventing an angioedema attack in a subject having hereditary angioedema (HAE), comprising the step of administering a preventive amount of a dsRNA agent according to any one of claims 1 to 3 or 9, or a pharmaceutical composition according to any one of claims 41 and 43 to 48, to the subject, thereby preventing an angioedema attack in the subject.

95. The method according to claim 94, further comprising the step of administering the dsRNA agent according to any one of claims 4, 5, or 10 to the subject.

96. The method according to claim 94, further comprising the step of administering the dsRNA agent according to any one of claims 6, 7, or 11 to the subject.

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