Factor xi rnai agent compositions and methods of use thereof

Abstract

RNA interference (RNAi) agents for inhibiting the expression of Factor XI (FXI) are provided, as are pharmaceutical compositions comprising one or more FXI RNAi agents together with one or more excipients capable of delivering the RNAi agent(s) to a liver cell in vivo. Delivery of the FXI RNAi agent(s) to liver cells in vivo provides for inhibition of FXI gene expression and can provide for treatment of myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, pulmonary embolism, ischemic stroke, transient ischemic attack, retinal artery occlusion, mesenteric ischemia, renal vein thrombosis, cerebral venous sinus thrombosis, venous thromboembolism due to deep vein thrombosis, and peripheral artery disease, as well as diseases and disorders associated with angioedema, thrombosis, and / or coagulation.

Description

Attorney Docket No. 809030.000060FACTOR XI RNAi AGENT COMPOSITIONS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U. S. Provisional Patent Application No. 63 / 725,232, filed November 26, 2024, entitled “FACTOR XI RNAi AGENT COMPOSITIONS AND METHODS OF USE THEREOF,” the disclosure of which is incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The disclosure relates to double stranded ribonucleic acid interference agents (RNAi agents as used herein) and compositions targeting the Factor XI (FXI) gene, as well as methods of inhibiting expression of a FXI gene and methods of treating subjects having an FXI-associated disease or disorder, such as venous thromboembolism, using such RNAi agents and compositions.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been filed electronically in extensible Markup Language format and is hereby incorporated by reference in its entirety. Said XML file, created on November 24, 2025, is named 80903000060_CTI0001PCT_ST26.v2.xml and is 1,049,634 Bytes in size.BACKGROUND OF THE DISCLOSURE

[0004] Factor XI (also known as plasma thromboplastin antecedent (PTA), FXI, Factor 11, or Fll) is the zymogen form of Factor Xia, which functions in the blood coagulation process. The FXI protein plays a key role in the intrinsic pathway of clot formation, where FXI is activated by Factor XII to FXIa, which subsequently activates Factor IX, leading to thrombin generation and fibrin clot formation. Unlike other coagulation factors that are primarily activated through external trauma, FXI functions as part of an internal “amplification loop” that augments thrombin generation and stabilizes blood clot formation.

[0005] FXI is a plasma protein and serine protease. Structurally, FXI is synthesized in the liver as a glycoprotein and circulates as a homodimer with each monomer possessing a molecular weight of approximately 80 kDa.Attorney Docket No. 809030.000060

[0006] In contrast to well-characterized hemophilias, spontaneous hemorrhaging is rarely observed in Factor XI deficiency (sometimes referred to as Hemophilia C), and patients having FXI deficiency typically only exhibit bleeding generally related to surgery or trauma. Given its role in coagulation, FXI is also an anti -thrombotic target. A need exists for agents capable of specific knockdown of FXI.SUMMARY OF THE DISCLOSURE

[0007] For purposes of summarizing the invention and the advantages achieved over the prior art, certain objects and advantages of the invention have been described herein. Of course, it is to be understood that not necessarily all such objects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example, those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objects or advantages as may be taught or suggested herein.

[0008] As described below, the present disclosure features RNAi agents capable of inhibiting FXI levels in a cell, tissue or subject, as well as uses thereof, e.g., for experimental, commercial development, and / or therapeutic applications.

[0009] In an aspect, the disclosure provides a double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises a sense strand and an antisense strand, and wherein said antisense strand comprises a region of complementarity which comprises at least 12 contiguous nucleotides differing by no more than 3 nucleotides from any one of 88-172. Optionally, the antisense strand includes at least 13 contiguous nucleotides different by no more than 3 nucleotides form any one of SEQ ID NOs: 88-172. Optionally, the antisense strand includes at least 14 contiguous nucleotides different by no more than 3 nucleotides form any one of SEQ ID NOs: 88-172. Optionally, the antisense strand includes at least 15 contiguous nucleotides different by no more than 3 nucleotides form any one of SEQ ID NOs: 88-172. Optionally, the stretch of contiguous nucleotides of the antisense strand differs by no more than two nucleotides, optionally by no more than one nucleotide, or optionally has no mismatches with any one of the respective sense strands of such duplexes.

[0010] In an aspect, the disclosure provides a double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agentAttorney Docket No. 809030.000060comprises a sense strand and an antisense strand, wherein said sense strand comprises at least 12 contiguous nucleotides differing by no more than 3 nucleotides from any one of SEQ ID NOs: 3-87; and wherein said antisense strand comprises at least 12 contiguous nucleotides differing by no more than 3 nucleotides from any one of SEQ ID NOs: 88-172. Optionally, the sense strand includes at least 13 contiguous nucleotides differing by no more than 3 nucleotides from any one of SEQ ID NOs: 3-87. Optionally, the sense strand includes at least 14 contiguous nucleotides differing by no more than 3 nucleotides from any one of SEQ ID NOs: 3-87. Optionally, the sense strand includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of SEQ ID NOs: 3-87. Optionally, the stretch of contiguous nucleotides of the sense strand differs by no more than two nucleotides, optionally by no more than one nucleotide, or optionally has no mismatches with any one of the respective antisense strands of such duplexes. Optionally, the sense strand comprises a region having 80% sequence identity with any one or more of SEQ ID NOs: 25, 26, 50, 62, and / or 68. Optionally, the antisense strand comprises a region of complementarity having 80% sequence identity with any one or more of SEQ ID NOs: 110, 111, 135, 147, and / or 153.

[0011] In an aspect, the disclosure provides a double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FX1), wherein said RNAi agent comprises a sense strand and an antisense strand, wherein said antisense strand comprises a region of complementarity which comprises at least 14 contiguous nucleotides differing by no more than 3 nucleotides from the complement of any one of SEQ ID NOs: 343-427. Optionally, the antisense strand comprises a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the complement of any one of SEQ ID NOs: 343-427. Optionally, the antisense strand comprises a region of complementarity which comprises at least 16 contiguous nucleotides differing by no more than 3 nucleotides from the complement of any one of SEQ ID NOs: 343-427. Optionally, the antisense strand comprises a region of complementarity which comprises at least 17 contiguous nucleotides differing by no more than 3 nucleotides from the complement of any one of SEQ ID NOs: 343-427.

[0012] In some embodiments, at least one of the sense strand and the antisense strand comprises a targeting ligand.

[0013] In some embodiments, the targeting ligand targets a receptor which mediates delivery to a liver tissue.Attorney Docket No. 809030.000060

[0014] In some embodiments, the targeting ligand enhances hepatocyte cell delivery of the RNAi agent.

[0015] In some embodiments, the targeting ligand binds the Asialoglycoprotein receptor (ASGPR) on hepatocyte cells.

[0016] In some embodiments, the targeting ligand is one or more GalNAc moieties attached through a bivalent or trivalent branched linker.

[0017] In some embodiments, the ligand is attached to the 3' end, the 5' end, or the 3' and 5' end of the sense strand.

[0018] In some embodiments, the targeting ligand is a triantennary GalNAc ligand.

[0019] In some embodiments, the targeting ligand is attached to the RNAi agent at the 3'-terminal nucleotide residue of the sense strand.

[0020] In some embodiments, the targeting ligand includes a structure selected from among the following:Attorney Docket No. 809030.000060AcHNAttorney Docket No. 809030.000060. NOPfH -CNEtAttorney Docket No. 809030.000060QAc / 0^'■•■■ CPGHO. OH AcHN £HO HO, OH 'A _ <4 H H HO NAcHN A HO, OH / AoH H I N -AAcHM A

[0021] In some embodiments, the targeting ligand is conjugated via a bio-cleavable linker selected from among DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0022] In an aspect, the disclosure provides a double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agentAttorney Docket No. 809030.000060comprises a sense strand and an antisense strand, wherein said sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 3-87, wherein a substitution of a uracil for any thymine in SEQ ID NOs: 3-87 does not count as a difference that contributes to said differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 3-87; and wherein said antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 88-172, wherein a substitution of a uracil for any thymine in SEQ ID NOs: 88-172 does not count as a difference that contributes to said differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 88-172, wherein at least one of said sense strand and said antisense strand comprises one or more tris(GalNAc) moieties conjugated to one or more terminal nucleotide position, optionally via a linker or carrier.

[0023] In some embodiments, the double stranded RNAi agent comprises at least one modified nucleotide.

[0024] In some embodiments, all of the nucleotides of the sense strand are modified nucleotides.

[0025] In some embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides.

[0026] In some embodiments, all of the nucleotides of the sense strand are modified nucleotides.

[0027] In some embodiments, all of the nucleotides of the antisense strand are modified nucleotides.

[0028] In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0029] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3 '-terminal deoxy -thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a T-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2'-hydroxly-modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, aAttorney Docket No. 809030.000060phosphorami date, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a 2'-deoxy -2 '-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, 3 '-terminal deoxy -thymine nucleotides (dT), a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a nucleotide comprising a 5 '-methylphosphonate group, a nucleotide comprising a 5' phosphate or 5' phosphate mimic, a nucleotide comprising vinyl phosphonate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA) S-Isomer, a terminal nucleotide linked to a cholesteryl derivative, a deoxyribonucleotide, and a dodecanoic acid bisdecylamide group.

[0030] In some embodiments, the modifications on the nucleotides are selected from 2'-O-methyl and 2'-fluoro modifications.

[0031] In some embodiments, the RNAi agent further includes at least one phosphorothioate intemucleotide linkage.

[0032] In some embodiments, the double stranded RNAi agent comprises 6-8 phosphorothioate intemucleotide linkages.

[0033] In some embodiments, the region of complementarity is at least 17 nucleotides in length.

[0034] In some embodiments, the region of complementarity is 19-23 nucleotides in length.

[0035] In some embodiments, the region of complementarity is 19 nucleotides in length.

[0036] In some embodiments, each strand is no more than 30 nucleotides in length.

[0037] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide.

[0038] In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0039] In some embodiments, the region of complementarity comprises any one of the antisense sequences in any one of SEQ ID NOs: 88-172.

[0040] In some embodiments, the region of complementarity consists of any one of the antisense sequences in any one of SEQ ID NOs: 88-172.Attorney Docket No. 809030.000060

[0041] In some embodiments, the RNAi agent further includes a phosphate or phosphate mimic at the 5 '-end of the antisense strand.

[0042] In some embodiments, the phosphate mimic is a vinyl-phosphonate 2'-OMe-nucleotide. Optionally, the phosphate mimic is a vinyl-phosphonate 2'-OMe-uracil (vinu).

[0043] In some embodiments, the RNAi agent comprises at least one modified nucleotide selected from the group consisting of a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, and a nucleotide comprising vinyl phosphonate, optionally wherein the RNAi agent comprises at least one of each of the following modifications: 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, and a nucleotide comprising vinyl phosphonate.

[0044] In some embodiments, the RNAi agent comprises a pattern of modified nucleotides as shown in any one of SEQ ID NOs: 173-342.

[0045] In some embodiments, the double stranded region is 12-30 nucleotide pairs in length.

[0046] some embodiments, the double stranded region is 17-23 nucleotide pairs in length.

[0047] some embodiments, the double stranded region is 17-25 nucleotide pairs in length.

[0048] some embodiments, the double stranded region is 23-27 nucleotide pairs in length.

[0049] In some embodiments, the double stranded region is 19-21 nucleotide pairs in length.

[0050] some embodiments, the double stranded region is 21-23 nucleotide pairs in length.

[0051] In some embodiments, each strand has 12-30 nucleotides.

[0052] In some embodiments, each strand has 19-30 nucleotides.

[0053] In some embodiments, the modifications on the nucleotides are selected from the group consisting of LNA, glycol nucleic acid (GNA), HNA, CeNA, 2'-methoxy ethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof, preferably wherein the modifications on nucleotides are selected from the group consisting of 2'-O-methyl, 2'-fluoro, and combinations thereof.Attorney Docket No. 809030.000060

[0054] In some embodiments, the modifications on the nucleotides are 2'-O-methyl or 2'-fluoro modifications.

[0055] In some embodiments, the base pair at the 1 position of the 5 '-end of the antisense strand of the duplex is an AU base pair.

[0056] In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0057] In some embodiments, the sense strand has a total of 19 nucleotides and the antisense strand has a total of 21 nucleotides.

[0058] In some embodiments, said RNAi agent is selected from the group of RNAi agents listed in any one of SEQ ID NOs: 173-342.

[0059] In some embodiments, all of the nucleotides of said sense strand and all of the nucleotides of said antisense strand comprise a modification.

[0060] In an aspect, the disclosure provides a cell containing any of the aforementioned RNAi agents.

[0061] In an aspect, the disclosure provides a pharmaceutical composition for inhibiting expression of FXI including any of the aforementioned RNAi agents.

[0062] In some embodiments, the RNAi agent is administered in an unbuffered solution.

[0063] In some embodiments, said unbuffered solution is saline or water.

[0064] In some embodiments, said RNAi agent is administered with a buffer solution.

[0065] In some embodiments, said buffer solution comprises acetate, citrate, prolamine, carbonate, phosphate, or any combination thereof.

[0066] In some embodiments, said buffer solution is phosphate buffered saline (PBS).

[0067] In an aspect, the disclosure provides a method of inhibiting expression of Factor XI (FXI) in a cell, the method including the steps of: (a) contacting the cell with any of the aforementioned RNAi agents or any of the aforementioned pharmaceutical compositions; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an FXI gene, thereby inhibiting expression of FXI in the cell.

[0068] In some embodiments, the cell is within a subject.

[0069] In some embodiments, the subject is a human.

[0070] In some embodiments, the subject is selected from the group consisting of a rhesus monkey, a cynomolgus monkey, a mouse, and a rat.Attorney Docket No. 809030.000060

[0071] In some embodiments, the human subject suffers from a disease or disorder selected from the group consisting of myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, pulmonary embolism, ischemic stroke, transient ischemic attack, retinal artery occlusion, mesenteric ischemia, renal vein thrombosis, cerebral venous sinus thrombosis, arterial thrombosis, venous thrombosis, venous thromboembolism due to deep vein thrombosis, peripheral artery disease, and thrombosis due to artificial surfaces (e.g., catheter associated thrombosis, medical device thrombosis).

[0072] In some embodiments, the disease or disorder is myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, pulmonary embolism, or ischemic stroke.

[0073] In some embodiments, the FXI expression is inhibited by at least about 30%. Optionally, the FXI expression is inhibited in human hepatocytes by from about 30% to about 90%. Optionally, the FXI expression is inhibited in human hepatocytes by from about 40% to about 95% or more.

[0074] In an aspect, the disclosure provides a method of treating a subject having a disorder that would benefit from a reduction in FXI expression, including the step of administering to the subject a therapeutically effective amount of any of the aforementioned RNAi agents or any of the aforementioned pharmaceutical compositions, thereby treating said subject.

[0075] In some embodiments, the subject suffers from an FXI-associated disorder.

[0076] In some embodiments, the subject is a human.

[0077] In some embodiments, the FXI-associated disorder is myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, pulmonary embolism, or ischemic stroke.

[0078] In some embodiments, the method further includes the step of administering an additional therapeutic agent to the subject.

[0079] In some embodiments, the method further includes the step of administering a pharmaceutically acceptable excipient to the subject. Optionally, the pharmaceutically acceptable excipient is an additive (e.g., a buffer, a pH modifier, or the like).

[0080] In some embodiments, the RNAi agent is administered at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0081] In some embodiments, the RNAi agent is administered to the subject intravenously or subcutaneously.Attorney Docket No. 809030.000060

[0082] In some embodiments, the method reduces the expression of a target gene in a liver tissue. Optionally, the target gene is expressed by a hepatocyte, optionally wherein the hepatocyte is a primary hepatocyte.

[0083] In an aspect, the disclosure provides a method of inhibiting the expression of FXI in a subject, the method including the step of: administering to said subject a therapeutically effective amount of any of the aforementioned RNAi agents or any of the aforementioned pharmaceutical compositions, thereby inhibiting the expression of FXI in said subject.

[0084] In an aspect, the disclosure provides a double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises a sense strand and an antisense strand, and wherein said antisense strand comprises a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense strand nucleobase sequences of a duplex selected from the group consisting of SEQ ID NOs: 88-172.

[0085] In some embodiments, the RNAi agent comprises one or more modifications selected from the group consisting of a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a nucleotide comprising a glycol nucleic acid (GNA), a phosphorothioate (PS), a vinyl-phosphonate 2'-OMe-nucleotide (e.g., a vinyl-phosphonate 2'-OMe-uracil (vinu)), optionally wherein said RNAi agent comprises at least one of each modification selected from the group consisting of a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a phosphorothioate and a vinyl-phosphonate 2'-OMe-uracil (vinu). In some embodiments, the RNAi agent comprises one or more deoxyribonucleotides.

[0086] In some embodiments, the RNAi agent comprises four or more PS modifications, optionally six to ten PS modifications, optionally six PS modifications.

[0087] In some embodiments, each of the sense strand and the antisense strand of the RNAi agent comprises a 5 '-terminus and a 3 '-terminus, and wherein the RNAi agent comprises six PS modifications positioned at the penultimate and ultimate intemucleotide linkages from the 5'-terminus of the sense and the respective 3'- and 5 '-termini of the antisense strands of the RNAi agent.

[0088] In some embodiments, each of the sense strand and the antisense strand of the RNAi agent comprises a 5 '-terminus and a 3 ’-terminus, and wherein the RNAi agent comprises two or more 2'-fluoro modified nucleotides, optionally wherein each of the sense strand and the antisenseAttorney Docket No. 809030.000060strand of the RNAi agent comprises two or more 2'-fluoro modified nucleotides, optionally wherein the 2'-fluoro modified nucleotides are located on the sense strand at nucleobase positions 5, 7, 8, and 9 from the 5 '-terminus of the sense strand and on the antisense strand at nucleobase positions 2, 6, 8, 9, 14 and 16 from the 5'-terminus of the antisense strand.

[0089] In some embodiments, each of the sense strand and the antisense strand of the RNAi agent comprises a 5 '-terminus and a 3 ’-terminus, and wherein the RNAi agent comprises two or more 2'-fluoro modified nucleotides, optionally wherein each of the sense strand and the antisense strand of the RNAi agent comprises two or more 2'-fluoro modified nucleotides, optionally wherein the 2'-fluoro modified nucleotides are located on the sense strand at nucleobase positions 7, 9, 10, and 11 from the 5 '-terminus of the sense strand and on the antisense strand at nucleobase positions 2, 6, 8, 9, 14 and 16 from the 5'-terminus of the antisense strand.

[0090] In some embodiments, each of the sense strand and the antisense strand of the RNAi agent comprises a 5 '-terminus and a 3 ’-terminus, and wherein the RNAi agent comprises two or more 2'-O-methyl modified nucleotides, optionally wherein each of the sense strand and the antisense strand of the RNAi agent comprises two or more 2'-O-methyl modified nucleotide nucleotides, optionally wherein the 2'-O-methyl modified nucleotide nucleotides are located on the sense strand at nucleobase positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5'-terminus of the sense strand and on the antisense strand at nucleobase positions 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 from the 5'-terminus of the antisense strand.

[0091] In some embodiments, each of the sense strand and the antisense strand of the RNAi agent comprises a 5 '-terminus and a 3 ’-terminus, and wherein the RNAi agent comprises two or more 2'-O-methyl modified nucleotides, optionally wherein each of the sense strand and the antisense strand of the RNAi agent comprises two or more 2'-O-methyl modified nucleotides, optionally wherein the 2'-O-methyl modified nucleotides are located on the sense strand at nucleobase positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 from the 5'-terminus of the sense strand and on the antisense strand at nucleobase positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 from the 5'-terminus of the antisense strand.

[0092] In some embodiments, each of the sense strand and the antisense strand of the RNAi agent comprises a 5 '-terminus and a 3 '-terminus, and wherein the RNAi agent comprises one or more vinu modifications, optionally wherein the RNAi agent comprises a single vinu modification at the 5 '-terminus of the antisense strand.Attorney Docket No. 809030.000060

[0093] In some embodiments, each of the sense strand and the antisense strand of the RNAi agent comprises a 5'-terminus and a 3'-terminus, wherein each of the sense strand and the antisense strand of the RNAi agent comprises 2'-fluoro modified nucleotides located on the sense strand at nucleobase positions 5, 7, 8, and 9 from the 5 '-terminus of the sense strand and on the antisense strand at nucleobase positions 2, 6, 8, 9, 14 and 16 from the 5'-terminus of the antisense strand, wherein each of the sense strand and the antisense strand of the RNAi agent comprises 2'-O-methyl modified nucleotide nucleotides located on the sense strand at nucleobase positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5'-terminus of the sense strand and on the antisense strand at nucleobase positions 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 from the 5 '-terminus of the antisense strand, and wherein the RNAi agent comprises a single vinu modification at the 5 '-terminus of the antisense strand.

[0094] In some embodiments, each of the sense strand and the antisense strand of the RNAi agent comprises a 5'-terminus and a 3'-terminus, wherein each of the sense strand and the antisense strand of the RNAi agent comprises 2'-fluoro modified nucleotides located on the sense strand at nucleobase positions 7, 9, 10, and 11 from the 5 '-terminus of the sense strand and on the antisense strand at nucleobase positions 2, 6, 8, 9, 14 and 16 from the 5'-terminus of the antisense strand, wherein each of the sense strand and the antisense strand of the RNAi agent comprises 2'-O-methyl modified nucleotide nucleotides located on the sense strand at nucleobase positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 from the 5 '-terminus of the sense strand and on the antisense strand at nucleobase positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 from the 5 '-terminus of the antisense strand, and wherein the RNAi agent comprises a single vinu modification at the 5'-terminus of the antisense strand.

[0095] In an aspect, the disclosure provides a method for treating or preventing a FXI-associated disease or disorder in a subject, the method including the step of administering to said subject a therapeutically effective amount of any of the aforementioned RNAi agents or any of the aforementioned pharmaceutical compositions, thereby treating or preventing a FXI-associated disease or disorder in the subject.

[0096] In some embodiments, the FXI-associated disease or disorder is selected from the group consisting of myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, pulmonary embolism, ischemic stroke, transient ischemic attack, retinal artery occlusion, mesenteric ischemia, renal vein thrombosis, cerebral venous sinus thrombosis, arterial thrombosis,Attorney Docket No. 809030.000060venous thrombosis, venous thromboembolism due to deep vein thrombosis, peripheral artery disease, and thrombosis due to artificial surfaces (e.g., catheter associated thrombosis, medical device thrombosis).

[0097] In an aspect, the disclosure provides a kit for performing any of the aforementioned methods, including: a) the RNAi agent, and b) instructions for use, and c) optionally, a means for administering the RNAi agent to the subject.

[0098] In one aspect, the disclosure provides an RNAi agent useful for inhibiting expression of Factor XI (FXI), the RNAi agent having a sense strand paired with an antisense strand having the sequence 5’-UUGAUAUAAGAAAAUCAUCCU-3’ (SEQ ID NO: 110). In some embodiments, the antisense strand including SEQ ID NO: 110 has one or more of the following chemical modifications (position 1 being the 5 ’-terminal position of the antisense strand and position numbering proceeding from 5’ to 3’): (a) the 5 ’-nucleotide at position 1 carries a vinylphosphonate modification; (b) phosphorothioate linkages are present between nucleotide positions 1-2, 2-3, 19-20, and 20-21; (c) 2’-fluoro ribonucleotides appear at positions 2, 6, 8, 9, 14, and 16; and (d) 2’-O-methyl ribonucleotides appear at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21.

[0099] In another aspect, the disclosure provides an RNAi agent having a sense strand that includes the sequence 5’-GAUGAUUUUCUUAUAUCAA-3’ (SEQ ID NO: 25) paired with an antisense strand. In some embodiments, the sense strand including SEQ ID NO: 25 has one or more of the following chemical modifications (position 1 being the 5’-terminal position of the sense strand and position numbering proceeding from 5’ to 3’): (a) phosphorothioate linkages between position pairs 1-2 and 2-3; (b) 2’-fluoro nucleotides at positions 5, 7, 8, and 9; (c) 2’-O-methyl nucleotides at positions 1, 2, 3, 4, 6, and 10-19; and (d) a triantennary N-acetylgalactosamine (GalNAc) ligand at the 3 ’-terminal nucleotide (position 19). In some embodiments, this GalNAc ligand includes a 3'-Prolinol / C12 linker bearing a trivalent GalNAc moiety.

[0100] In still another aspect, the disclosure provides an RNAi agent having a sense strand that includes the sequence 5’-gsasugAfuUfUfUfcuuauaucaa-3’ (SEQ ID NO: 195) paired with an antisense strand that includes the sequence 5’-(vinu)sUfsgauAfuAfAfgaaaAfuCfaucscsu-3’ (SEQ ID NO: 280). In some embodiments, the 3’-terminal nucleotide (position 19) of the sense strandAttorney Docket No. 809030.000060that includes SEQ ID NO: 195 further carries a triantennary GalNAc ligand. Optionally, this GalNAc ligand includes a 3'-Prolinol / C12 linker bearing a trivalent GalNAc moiety.

[0101] In another aspect, the disclosure provides an RNAi agent having a sense strand that includes SEQ ID NO: 25 with specified phosphorothioate, 2’-fluoro, and 2’-O-methyl modifications and a 3'-Prolinol / C12-linked trivalent GalNAc at position 19, paired with an antisense strand that includes SEQ ID NO: 110 and having a vinylphosphonate at position 1, phosphorothioate linkages at positions 1-2, 2-3, 19-20, and 20-21, 2’-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16, and 2’-O-methyl nucleotides at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, and 17-21.

[0102] In further aspects, the disclosure provides an RNAi agent having a sense strand paired with an antisense strand that includes the sequence 5’-UUUGAUAUAAGAAAAUCAUCC-3’ (SEQ ID NO: 111). In some embodiments, the antisense strand having SEQ ID NO: 111 includes a vinylphosphonate at position 1, phosphorothioate linkages at positions 1-2, 2-3, 19-20, and 20-21, 2’-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16, and 2’-O-methyl nucleotides at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, and 17-21.

[0103] Additional aspects provide an RNAi agent having a sense strand that includes the sequence 5’-AUGAUUUUCUUAUAUCAAA-3’ (SEQ ID NO: 26) paired with an antisense strand. In some embodiments, the sense strand that includes SEQ ID NO: 26 carries phosphorothioate linkages at positions 1-2 and 2-3, includes 2’ -fluoro nucleotides at positions 5, 7, 8, and 9, includes 2’-O-methyl nucleotides at positions 1, 2, 3, 4, 6, and 10-19, and includes at position 19 a triantennary GalNAc ligand. Optionally, this GalNAc ligand includes a 3'-Prolinol / C12 linker bearing a trivalent GalNAc moiety.

[0104] The disclosure also provides an RNAi agent having a sense strand that includes the sequence 5’-asusgaUfuUfUfCfuuauaucaaa-3’ (SEQ ID NO: 196) paired with an antisense strand having the sequence 5’-(vinu)sUfsugaUfaUfAfagaaAfaUfcauscsc-3’ (SEQ ID NO: 281). In some embodiments, the 3 ’-terminal nucleotide (position 19) of the sense strand that includes SEQ ID NO: 196 carries a triantennary GalNAc ligand. Optionally, this GalNAc ligand includes a 3'-Prolinol / C12 linker bearing a trivalent GalNAc moiety.

[0105] The disclosure further provides an RNAi agent having a sense strand that includes SEQ ID NO: 26 and an antisense strand that includes SEQ ID NO: 111, where the sense strand carries phosphorothioate linkages at positions 1-2 and 2-3, includes 2’-fluoro nucleotides atAttorney Docket No. 809030.000060positions 5, 7, 8, and 9, includes 2’-0-methyl nucleotides at positions 1-4, 6, and 10-19, and carries at the 3 ’-terminal nucleotide (position 19) a 3'-Prolinol / C12-linked trivalent GalNAc. The antisense strand that includes SEQ ID NO: 111 carries a vinylphosphonate at the 5’-terminus (position 1), phosphorothioate linkages at positions 1-2, 2-3, 19-20, and 20-21, 2’-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16, and 2’-O-methyl nucleotides at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, and 17-21.

[0106] Other aspects provide an RNAi agent having a sense strand paired with an antisense strand that includes the sequence 5’-UUUGAAGAAAGCUUUAAGUAA-3’ (SEQ ID NO: 135). In some embodiments, the antisense strand that includes SEQ ID NO: 135 carries a vinylphosphonate at position 1, includes phosphorothioate linkages at positions 1-2, 2-3, 19-20, and 20-21, includes 2’-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16, and includes 2’-O-methyl nucleotides at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, and 17-21.

[0107] The disclosure also provides an RNAi agent having a sense strand that includes the sequence 5’-ACUUAAAGCUUUCUUCAAA-3’ (SEQ ID NO: 50) paired with an antisense strand. In some embodiments, the sense strand that includes SEQ ID NO: 50 carries phosphorothioate linkages at positions 1-2 and 2-3, includes 2’ -fluoro nucleotides at positions 5, 7, 8, and 9, includes 2’-O-methyl nucleotides at positions 1-4, 6, and 10-19, and includes a triantennary GalNAc ligand at position 19, which in some embodiments is a 3'-Prolinol / C12-linked trivalent GalNAc ligand.

[0108] Further aspects provide an RNAi agent having a sense strand that includes the sequence 5’-ascsuuAfaAfGfCfuuucuucaaa-3’ (SEQ ID NO: 220) paired with an antisense strand that includes the sequence 5’-(vinu)sUfsugaAfgAfAfagcuUfuAfagusasa-3’ (SEQ ID NO: 305). In some embodiments, the sense strand that includes SEQ ID NO: 220 carries at its 3 ’-terminus (position 19) a triantennary GalNAc ligand, which in some embodiments is a 3'-Prolinol / C12-linked trivalent GalNAc ligand.

[0109] In additional aspects, the disclosure provides an RNAi agent having a sense strand that includes SEQ ID NO: 50 paired with an antisense strand that includes SEQ ID NO: 135, where the sense strand has phosphorothioate linkages at positions 1-2 and 2-3, includes 2’ -fluoro residues at positions 5, 7, 8, and 9, includes 2’-O-methyl residues at positions 1-4, 6, and 10-19, and includes a terminal 3'-Prolinol / C12-linked trivalent GalNAc ligand. In these aspects, the antisense strand having SEQ ID NO: 135 includes a vinylphosphonate at position 1, includesAttorney Docket No. 809030.000060phosphorothioate linkages at positions 1-2, 2-3, 19-20, and 20-21, includes 2’-fluoro residues at positions 2, 6, 8, 9, 14, and 16, and includes 2’-O-methyl residues at positions 1, 3-5, 7, 10-13, 15, and 17-21.

[0110] The disclosure additionally provides an RNAi agent having a sense strand paired with an antisense strand that includes the sequence 5’-UAGAAAAUCAUCCUGAAAAGACC-3’ (SEQ ID NO: 147). In some embodiments, the antisense strand that includes SEQ ID NO: 147 carries a vinylphosphonate modification at its 5 ’-terminus (position 1), phosphorothioate linkages at positions 1-2, 2-3, 21-22, and 22-23, 2’-fluoro residues at positions 2, 6, 8, 9, 14, and 16, and 2’-O-methyl residues at positions 1, 3-5, 7, 10-13, 15, 17, 18, 19, 20, 21, 22, and 23.

[0111] Additional aspects provide an RNAi agent having a sense strand that includes the sequence 5’-UCUUUUCAGGAUGAUUUUCUA-3’ (SEQ ID NO: 62) paired with an antisense strand. In some embodiments, the sense strand that includes SEQ ID NO: 62 has phosphorothioate linkages at positions 1-2 and 2-3, includes 2’-fluoro residues at positions 7, 9, 10, and 11, includes 2’-O-methyl residues at positions 1-6, 8, and 12-21, and includes at its 3’-terminus (position 21) a triantennary GalNAc ligand. In some embodiments, this GalNAc ligand includes a 3'-Prolinol / C12 linker bearing a trivalent GalNAc moiety.

[0112] The disclosure also provides an RNAi agent having a sense strand that includes the sequence 5’-uscsuuuuCfaGfGfAfugauuuucua-3’ (SEQ ID NO: 232) paired with an antisense strand that includes the sequence 5’-(vinu)sAfsgaaAfaUfCfauccUfgAfaaagascsc-3’ (SEQ ID NO: 317). In some embodiments, the sense strand that includes SEQ ID NO: 232 has at its 3’-terminus (position 21) a triantennary GalNAc ligand. Optionally, the triantennary GalNAc ligand is a 3'-Prolinol / C12 linker bearing a trivalent GalNAc moiety.

[0113] Further aspects provide an RNAi agent having a sense strand that includes SEQ ID NO: 62 with defined phosphorothioate, 2’-fluoro, and 2’-O-methyl positions and a 3'-Prolinol / C12-linked trivalent GalNAc ligand at its 3’-terminus (position 21), paired with an antisense strand that includes SEQ ID NO: 147 and carrying a vinylphosphonate at position 1, phosphorothioate linkages at positions 1-2, 2-3, 21-22, and 22-23, 2’-fluoro residues at positions 2, 6, 8, 9, 14, and 16, and 2’-O-methyl residues at positions 1, 3-5, 7, 10-13, 15, 17, 18, 19, 20, 21, 22, and 23.

[0114] The disclosure further provides an RNAi agent having a sense strand paired with an antisense strand that includes the sequence 5’-UGAUAUAAGAAAAUCAUCCUGAA-3’Attorney Docket No. 809030.000060(SEQ ID NO: 153). In some embodiments, the antisense strand that includes SEQ ID NO: 153 carries a vinylphosphonate at its 5 ’-terminus (position 1), phosphorothioate linkages at positions 1-2, 2-3, 21-22, and 22-23, 2’-fluoro residues at positions 2, 6, 8, 9, 14, and 16, and 2’-O-methyl residues at positions 1, 3-5, 7, 10-13, 15, and 17-23.

[0115] Additional aspects provide an RNAi agent having a sense strand that includes the sequence 5’-CAGGAUGAUUUUCUUAUAUCA-3’ (SEQ ID NO: 68) paired with an antisense strand. In some embodiments, the sense strand that includes SEQ ID NO: 68 has phosphorothioate linkages at positions 1-2 and 2-3, includes 2’-fluoro residues at positions 7, 9, 10, and 11, includes 2’-O-methyl residues at positions 1-6, 8, and 12-21, and includes a triantennary GalNAc ligand at its 3’-terminus (position 21). Optionally, the triantennary GalNAc ligand is a 3'-Prolinol / C12 linker bearing a trivalent GalNAc moiety.

[0116] Further aspects provide an RNAi agent having a sense strand that includes the sequence 5’-csasggauGfaUfUfUfucuuauauca-3’ (SEQ ID NO: 238) paired with an antisense strand that includes the sequence 5’-(vinu)sGfsauaUfaAfGfaaaaUfcAfuccugsasa-3’ (SEQ ID NO: 323). In some embodiments, the sense strand that includes SEQ ID NO: 238 carries a triantennary GalNAc ligand at its 3’-terminus (position 21). Optionally, the triantennary GalNAc ligand is a 3'-Prolinol / C12 linker bearing a trivalent GalNAc moiety.

[0117] The disclosure also provides an RNAi agent having a sense strand that includes SEQ ID NO: 68 with phosphorothioate linkages at positions 1-2 and 2-3, with 2’-fluoro residues at positions 7, 9, 10, and 11, with 2’-O-methyl residues at positions 1-6, 8, and 12-21, and with a 3'-Prolinol / C12-linked trivalent GalNAc ligand at its 3’ -terminus (position 21), paired with an antisense strand that includes SEQ ID NO: 153 and having a vinylphosphonate at its 5 ’-terminus (position 1), phosphorothioate linkages at positions 1-2, 2-3, 21-22, and 22-23, 2’-fluoro residues at positions 2, 6, 8, 9, 14, and 16, and 2’-O-methyl residues at positions 1, 3-5, 7, 10-13, 15, and 17-23.

[0118] Alternative or additional embodiments described herein provide a double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), comprising one or more of the features of the foregoing description or of any description elsewhere herein.Attorney Docket No. 809030.000060

[0119] Alternative or additional embodiments described herein provide a cell containing the RNAi agent disclosed herein, comprising one or more of the features of the foregoing description or of any description elsewhere herein.

[0120] Alternative or additional embodiments described herein provide a pharmaceutical composition for inhibiting expression of an FXI gene comprising the RNAi agent disclosed herein and one or more of the features of the foregoing description or of any description elsewhere herein.

[0121] Alternative or additional embodiments described herein provide a kit for performing a method comprising one or more of the features of the foregoing description or of any description elsewhere herein.

[0122] Alternative or additional embodiments described herein provide a method of inhibiting expression of Factor XI (FXI) in a cell comprising one or more of the features of the foregoing description or of any description elsewhere herein.

[0123] The disclosure provides RNAi agents for inhibiting FXI. Compositions and articles defined by the disclosure were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the disclosure will be apparent from the detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0124] A better understanding of the features and advantages of the present disclosure may be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings below. The patent application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0125] FIGs. 1A-1C provide an overview of the RNAi agent sequence screening strategy employed herein. FIG. 1A shows an exemplary embodiment of a 21 / 23mer RNAi agent duplex having a two nucleotide 3'-overhang of the antisense strand relative to the sense strand (meanwhile, the 3'-end of the sense strand and the 5'-end of the antisense strand form a blunt end). FIG. 1B shows an exemplary embodiment of a 19 / 21mer RNAi agent duplex having a two nucleotide 3'-overhang of the antisense strand relative to the sense strand (meanwhile, the 3'-end of the sense strand and the 5'-end of the antisense strand form a blunt end). FIG. 1C shows a schematic of theAttorney Docket No. 809030.000060target site positions in the FXI mRNA for a representative sample of the initial screening set of RNAi agent duplexes of the instant disclosure.

[0126] FIG. 2 shows the performance of FXI RNAi agents at 0.2nM (x-axis) and at 0.02nM (y-axis) when co-transfected with a human FXI reporter construct. These results were used to identify a highly active subset of RNAi agent duplexes (lower-left quadrant).

[0127] FIG. 3 shows the relationship of the IC50 levels observed for tested RNAi agents in primary human hepatocytes (PHH; x-axis) against primary cynomolgus hepatocytes (PCH; y-axis).

[0128] FIGs. 4A-4E show dose-response free uptake (without transfection agent) curves for selected RNAi agent duplexes in PCH cells. FIG.4A shows XD-68126MGal dose-response in PCH cells. FIG. 4B shows XD-67384MGal dose-response in PCH cells. FIG. 4C shows XD-68128MGal dose-response in PCH cells. FIG. 4D shows XD-67387MGal dose-response in PCH cells. FIG. 4E shows XD-67388MGal dose-response in PCH cells.

[0129] FIGs. 5A-5J show dose-response free uptake (without transfection reagent) curves for selected RNAi agent duplexes in PHH cells. FIG. 5A shows XD-68497MGal dose-response in PHH cells. FIG. 5B shows XD-68139MGal dose-response in PHH cells. FIG. 5C shows XD-67370MGal dose-response in PHH cells. FIG. 5D shows XD-67387MGal dose-response in PHH cells. FIG. 5E shows XD-67388MGal dose-response in PHH cells. FIG. 5F shows XD-67636MGal dose-response in PHH cells. FIG. 5G shows XD-67384MGal dose-response in PHH cells. FIG. 5H shows XD-68126MGal dose-response in PHH cells. FIG. 5I shows XD-68128MGal dose-response in PHH cells. FIG. 5J shows XD-68134MGal dose-response in PHH cells.

[0130] FIG. 6 shows hFXI knockdown performance at day 28 (D28) of RNAi agent duplex leads (modified triGalNAc RNAi agents), normalized to AAV only negative control. Each dot represents one animal.

[0131] FIG. 7 shows dose-response in vivo at 0.3, 1, and 3mg / kg for XD-67387MGal, XD-67388MGal, XD-67636MGal, XD-68128MGal, and XD-68134MGal. Values are graphed as mean and standard deviation.

[0132] FIG. 8 shows volcano plots visualizing off-target impact.

[0133] FIG. 9 shows suppression of FXI antigen by XD-67336MGal, XD-67387MGal, XD-67388MGal, XD-68128MGal, and XD-68134MGal in nonhuman primate, relative to predose.Attorney Docket No. 809030.000060

[0134] FIG. 10 shows fold elevation of aPTT (activated partial thromboplastin time) in respective individual nonhuman primates relative to predose, in response to treatment.

[0135] FIG. 11 shows fold suppression of FXI activity by XD-67336MGal, XD-67387MGal, XD-67388MGal, XD-68128MGal, and XD-68134MGal in nonhuman primates relative to predose in response to treatment.

[0136] FIG. 12 shows a schematic of the in vivo thrombosis and hemostasis models used to evaluate activity of the RNAi agent agents of the present disclosure. A ferric Chloride blood flow assay (top) illustrates thrombus formation in a mouse artery following ferric chloride-induced endothelial injury, showing progressive vessel occlusion over time. A tail bleeding assay (lower right) demonstrates assessment of bleeding duration after distal tail transection in anesthetized mice.

[0137] FIG. 13 shows the effect of mouse FXI (mFXI) RNAi agent on thrombosis and hemostasis in mice.DETAILED DESCRIPTION OF THE DISCLOSURE

[0138] The present disclosure relates, at least in part, to the discovery of double stranded ribonucleic acid interference agents (RNAi agents) capable of robust inhibition of FXI, in cells, tissues and subjects. As detailed below, FXI plays a critical role in blood coagulation, making it a molecular target for certain coagulation and / or thrombosis disorders, particularly deep vein thrombosis, renal vein thrombosis, cerebral venous sinus thrombosis, venous thromboembolism due to deep vein thrombosis, myocardial infarction, atrial fibrillation (AFib), pulmonary embolism, hematomas, ischemic stroke, transient ischemic attack, retinal artery occlusion, mesenteric ischemia, arterial thrombosis, venous thrombosis, peripheral artery disease, and thrombosis due to artificial surfaces (e.g., catheter associated thrombosis, medical device thrombosis).

[0139] Thromboembolic disease, encompassing arterial and venous thrombosis (VTE, stroke, myocardial infarction, and pulmonary embolism), represents a major global health burden contributing significantly to morbidity and mortality. Standard therapeutic interventions for the prevention and treatment of thromboembolism involve anticoagulants that generally target key components within the common pathway of the coagulation cascade, such as Factor Xa or thrombin. While effective in reducing thrombotic events, these drugs are associated with a risk ofAttorney Docket No. 809030.000060bleeding, including major hemorrhage, due to these agents’ targets being linked to both pathological thrombus formation and physiological hemostasis. Such limitation, coupled with challenges related to renal / hepatic handling, potential drug interactions, and adherence, results in a substantial unmet medical need for safer, durable antithrombotic strategies, particularly in vulnerable patients who are often undertreated or precluded from current therapy due to high bleeding risk.

[0140] To overcome the limitations of current standard of care, the present invention, at least in part, involves selectively inhibiting Factor XI (FXI). FXI is part of the coagulation pathway and functions primarily to amplify and sustain thrombin generation necessary for thrombus propagation, rather than initiating the clotting process central to normal hemostasis. Individuals with congenital FXI deficiency demonstrate a significantly reduced risk of both arterial and venous thrombosis while rarely experiencing spontaneous bleeding events. Targeting a pathway that amplifies and sustains thrombin generation and propagation aims to decouple the desired antithrombotic effect from the inherent bleeding risk.

[0141] RNAi agents disclosed herein are adapted to target such pathway by using a liver-targeted, nucleic acid-based approach. RNAi agents disclosed herein can include N-acetylgalactosamine (GalNAc)-conjugated small interfering ribonucleic acid (RNAi agent), that have been shown to specifically facilitate targeted delivery to hepatocytes by binding to the asialoglycoprotein receptor (ASGPR). Once delivered, the RNAi agents disclosed herein leverage the endogenous RNA interference (RNAi) pathway to selectively suppress hepatic expression of the gene encoding FXI ( / ' J J) via messenger ribonucleic acid (mRNA) knockdown, thereby reducing the circulating levels of FXI protein in a subject. The present disclosure thus provides highly selective and durable suppression of Factor XI synthesis, positioning the RNAi agents disclosed herein as an effective next-generation anticoagulant intended for the prevention and treatment of thrombosis with superior efficacy to conventional anticoagulants combined with a reduced incidence of bleeding.Definitions

[0142] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used inAttorney Docket No. 809030.000060this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0143] The present disclosure features one or more double-stranded RNAi agents (e.g., RNAi agent duplexes) that can modulate (e.g., inhibit) Factor XI expression. The RNAi agents of the disclosure optionally can be used in combination with modulators of other genes and / or gene products associated with the maintenance or development of diseases or disorders associated with Factor XI (e.g., clotting, ischemic stroke, deep vein thrombosis (DVT), myocardial infarction, atrial fibrillation (AFib), etc.). The RNAi agents of the disclosure modulate Factor XI RNAs such as those corresponding to the cDNA sequences referred to by GenBank Accession Nos. NM_000128.4, NC_000004.12 (human Factor XI, also known as FXI, FXIa, Factor 11, or Fll which is referred to herein generally as “FXI.” The sequence of NM_000128.4 as set forth below:

[0144] gaccatttcctacacaagctcattcagaggaggatgaagaccattttggaggaagaaaagcacccttattaagaattg cagcaagtaagccaacaaggtcttttcaggatgattttcttatatcaagtggtacatttcattttatttacttcagtttctggtgaatgtgtgactcagt tgttgaaggacacctgctttgaaggaggggacattactacggtcttcacaccaagcgccaagtactgccaggtagtctgcacttaccaccca agatgtttactcttcactttcacggcggaatcaccatctgaggatcccacccgatggtttacttgtgtcctgaaagacagtgttacagaaacact gccaagagtgaataggacagcagcgatttctgggtattctttcaagcaatgctcacaccaaataagcgcttgcaacaaagacatttatgtgga cctagacatgaagggcataaactataacagctcagttgccaagagtgctcaagaatgccaagaaagatgcacggatgacgtccactgcca ctttttcacgtacgccacaaggcagtttcccagcctggagcatcgtaacatttgtctactgaagcacacccaaacagggacaccaaccagaa taacgaagctcgataaagtggtgtctggattttcactgaaatcctgtgcactttctaatctggcttgtattagggacattttccctaatacggtgttt gcagacagcaacatcgacagtgtcatggctcccgatgcttttgtctgtggccgaatctgcactcatcatcccggttgcttgttttttaccttcttttc ccaggaatggcccaaagaatctcaaagaaatctttgtctccttaaaacatctgagagtggattgcccagtacacgcattaaaaagagcaaag ctctttctggtttcagtctacaaagctgcaggcacagcatcccagtgttctgccattcttcattttaccatgacactgatttcttgggagaagaact ggatattgttgctgcaaaaagtcacgaggcctgccagaaactgtgcaccaatgccgtccgctgccagttttttacctataccccagcccaagc atcctgcaacgaagggaagggcaagtgttacttaaagctttcttcaaacggatctccaactaaaatacttcacgggagaggaggcatctctg gatacacattaaggttgtgtaaaatggataatgagtgtaccaccaaaatcaagcccaggatcgttggaggaactgcgtctgttcgtggtgagt ggccgtggcaggtgaccctgcacacaacctcacccactcagagacacctgtgtggaggctccatcattggaaaccagtggatattaacag ccgctcactgtttctatggggtagagtcacctaagattttgcgtgtctacagtggcattttaaatcaatctgaaataaaagaggacacatctttctt tggggttcaagaaataataatccatgatcagtataaaatggcagaaagcgggtatgatattgccttgttgaaactggaaaccacagtgaattacAttorney Docket No. 809030.000060acagattctcaacgacccatatgcctgccttccaaaggagatagaaatgtaatatacactgattgctgggtgactggatgggggtacagaaa actaagagacaaaatacaaaatactctccagaaagccaagatacccttagtgaccaacgaagagtgccagaagagatacagaggacataa aataacccataagatgatctgtgccggctacagggaaggagggaaggacgcttgcaagggagattcgggaggccctctgtcctgcaaac acaatgaggtctggcatctggtaggcatcacgagctggggcgaaggctgtgctcaaagggagcggccaggtgtttacaccaacgtggtcg agtacgtggactggattctggagaaaactcaagcagtgtgaatgggttcccaggggccattggagtccctgaaggacccaggatttgctgg gagagggtgttgagttcactgtgccagcatgcttcctccacagtaacacgctgaaggggcttggtgtttgtaagaaaatgctagaagaaaac aaactgtcacaagttgttatgtccaaaactcccgttctatgatcgttgtagtttgtttgagcattcagtctctttgtttttgatcacgcttctatggagt ccaagaattaccataaggcaatatttctgaagattactatataggcagatatagcagaaaataaccaagtagtggcagtggggatcaggcag aagaactggtaaaagaagccaccataaatagatttgttcgatgaaagatgaaaactggaagaaaggagaacaaagacagtcttcaccatttt gcaggaatctacactctgcctatgtgaacacatttcttttgtaaagaaagaaattgattgcatttaatggcagattttcagaatagtcaggaattct tgtcatttccattttaaaatatatattaaaaaaaatcagttcgagtagacacgagctaagagtgaatgtgaagataacagaatttctgtgtggaag aggattacaagcagcaatttacctggaagtgataccttaggggcaatcttgaagatacactttcctgaaaaatgatttgtgatggattgtatattt atttaaaatatcttgggaggggaggctgatggagatagggagcatgctcaaacctccctaagacaagctgctgctgtgactatgggctccca aagagctagatcgtatatttatttgacaaaaatcaccatagactgcatccatactacagagaaaaaacaattagggcgcaaatggatagttaca gtaaagtcttcagcaagcagctgcctgtattctaagcactgggattttctgtttcgtgcaaatatttatctcattattgttgtgatctagttcaataac ctagaatttgaattgtcaccacatagctttcaatctgtgccaacaactatacaattcatcaagtgtg (SEQ ID NO: 1)

[0145] The below description of the various aspects and embodiments of the disclosure is provided with reference to exemplary FXI RNAs, generally referred to herein as FXI RNAs. However, such reference is meant to be exemplary only and the various aspects and embodiments of the disclosure are also directed to alternate FXI RNAs, such as mutant FXI RNAs or additional FXI splice variants. Certain aspects and embodiments are also directed to other genes involved in the intrinsic pathway of clot formation, including genes whose misregulation acts in association with that of FXI (or is affected or affects FXI regulation) to produce phenotypic effects that may be targeted for treatment (e g., stroke, deep vein thrombosis (DVT), myocardial infarction, atrial fibrillation (AFib), etc.). Factor XI (Hageman Factor, F12 Prekallikrein (PK, KLKB1), High-Molecular-Weight Kininogen (HMWK, KNGF), Factor IX (Christmas Factor, F9), Factor VIII (Antihemophilic Factor, FS), Factor X (Stuart-Prower Factor, F10), Prothrombin (Factor II, F2), and Fibrinogen (FGA, FGB, FGG) are examples of genes that interact with FXI. Such additional genes, including those of pathways that act in coordination with FXI, can be targeted using RNAi agents and the methods described herein for use of FXI-targeting RNAi agents. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.Attorney Docket No. 809030.000060

[0146] The term “FXI” refers to the protein, peptide, or polypeptide gene product of an FXI gene / transcript. In certain embodiments, the term “FXI” is also meant to include other FXI (also known as Factor 11 or Fl 1) sequences, such as FXI isoforms, mutant FXI genes, splice variants of FXI genes, and FXI gene polymorphisms. The RNAi agents of the disclosure modulate FXI expression such as that corresponding to GenBank Accession No. NP_000119.1 (miflyqvvhfilftsvsgecvtqllkdtcfeggdittvftpsakycqvvctyhprcllftftaespsedptrwftcvlkdsvtetlprvnrtaa isgysfkqcshqisacnkdiyvdldmkginynssvaksaqecqerctddvhchfftyatrqfpslehmicllkhtqtgtptritkldkvvs gfslkscalsnlacirdifpntvfadsnidsvmapdafvcgricthhpgclfftffsqewpkesqrnlcllktsesglpstrikkskalsgfsl qscrhsipvfchssfyhdtdflgeeldivaaksheacqklctnavrcqfftytpaqascnegkgkcylklssngsptkilhgrggisgytlrl ckmdnecttkikprivggtasvrgewpwqvtlhttsptqrhlcggsiignqwiltaahcfygvespkilrvysgilnqseikedtsffgvq eiiihdqykmaesgydiallklettvnytdsqrpiclpskgdrnviytdcwvtgwgyrklrdkiqntlqkakiplvtneecqkryrghkit hkmicagyreggkdackgdsggplsckhnevwhlvgitswgegcaqrerpgvytnvveyvdwilektqav (SEQ ID NO: 2))-

[0147] As used herein, an “FXI-associated disease or disorder” refers to a disease or disorder known in the art to be associated with altered FXI expression, level and / or activity, and / or treatable by altering FXI expression levels and / or activity. Exemplary FXI-associated diseases or disorders of the disclosure include, without limitation, myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, pulmonary embolism, ischemic stroke, transient ischemic attack, retinal artery occlusion, mesenteric ischemia, renal vein thrombosis, cerebral venous sinus thrombosis, venous thromboembolism due to deep vein thrombosis, arterial thrombosis, venous thrombosis, peripheral artery disease, and thrombosis due to artificial surfaces (e.g., catheter associated thrombosis, medical device thrombosis), as well as the bleeding disorders epistaxis (nosebleeds), menorrhagia, hematuria, gastrointestinal bleeding, postoperative bleeding, hemarthrosis (joint bleeding), and hematomas. An FXI RNAi agents of the disclosure is deemed to possess “FXI inhibitory activity” if a statistically significant reduction in FXI RNA (or when the FXI protein is assessed, FXI protein levels) is seen when an FXI RNAi agent of the disclosure is administered to a system (e.g., cell-free in vitro system), cell, tissue or organism, as compared to a selected control. The distribution of experimental values and the number of replicate assays performed will tend to dictate the parameters of what levels of reduction in FXI RNA (either as a % or in absolute terms) is deemed statistically significant (as assessed by standard methods of determining statistical significance known in the art). However, in certain embodiments, “FXIAttorney Docket No. 809030.000060inhibitory activity” is defined based upon a % or absolute level of reduction in the level of FXI in a system, cell, tissue or organism. For example, in certain embodiments, a dsRNA of the disclosure is deemed to possess FXI inhibitory activity if at least a 5% reduction or at least a 10% reduction in FXI RNA is observed in the presence of a dsRNA of the disclosure relative to FXI levels seen for a suitable control. (For example, in vivo FXI levels in a tissue and / or subject can, in certain embodiments, be deemed to be inhibited by a dsRNA agent of the disclosure if, e.g., a 5% or 10% reduction in FXI levels is observed relative to a control.) In certain other embodiments, a dsRNA of the disclosure is deemed to possess FXI inhibitory activity if FXI RNA levels are observed to be reduced by at least 15% relative to a selected control, by at least 20% relative to a selected control, by at least 25% relative to a selected control, by at least 30% relative to a selected control, by at least 35% relative to a selected control, by at least 40% relative to a selected control, by at least 45% relative to a selected control, by at least 50% relative to a selected control, by at least 55% relative to a selected control, by at least 60% relative to a selected control, by at least 65% relative to a selected control, by at least 70% relative to a selected control, by at least 75% relative to a selected control, by at least 80% relative to a selected control, by at least 85% relative to a selected control, by at least 90% relative to a selected control, by at least 95% relative to a selected control, by at least 96% relative to a selected control, by at least 97% relative to a selected control, by at least 98% relative to a selected control or by at least 99% relative to a selected control. In some embodiments, complete inhibition of FXI is required for a dsRNA to be deemed to possess FXI inhibitory activity. In certain models (e.g., cell culture), a dsRNA is deemed to possess FXI inhibitory activity if at least a 50% reduction in FXI levels is observed relative to a suitable control. In certain other embodiments, a dsRNA is deemed to possess FXI inhibitory activity if at least a 70% reduction in FXI levels is observed relative to a suitable control. In certain other embodiments, a dsRNA is deemed to possess FXI inhibitory activity if at least an 80% reduction in FXI levels is observed relative to a suitable control.

[0148] By way of specific example, in Example 2 below, a series of RNAi agent duplexes (RNAi agents having sense / antisense strand lengths of 19-23mers) targeting FXI were tested for the ability to reduce FXI mRNA levels in Hepal-6, HepG2, and primary human hepatocytes (PHH) cells in vitro, at 10 nM concentrations in the environment of such. Within Example 2 below, FXI inhibitory activity was ascribed to those RNAi agent duplexes that were observed to effect at least a 70% reduction of FXI mRNA levels under the assayed conditions. It is contemplated thatAttorney Docket No. 809030.000060FXI inhibitory activity could also be attributed to a dsRNA under either more or less stringent conditions than those employed for Example 2 below, even when the same or a similar assay and conditions are employed. For example, in certain embodiments, a tested dsRNA of the disclosure is deemed to possess FXI inhibitory activity if at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 75% reduction, at least an 80% reduction, at least an 85% reduction, at least a 90% reduction, or at least a 95% reduction in FXI mRNA levels is observed in a mammalian cell line in vitro at 0.1 nM dsRNA concentration or lower in the environment of a cell, relative to a suitable control.

[0149] Use of other endpoints for determination of whether a double stranded RNA of the disclosure possesses FXI inhibitory activity is also contemplated. Specifically, in one embodiment, in addition to or as an alternative to assessing FXI mRNA levels, the ability of a tested dsRNA to reduce FXI protein levels (e.g., at 7 days after contacting a mammalian cell in vitro or in vivo) is assessed, and a tested dsRNA is deemed to possess FXI inhibitory activity if at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least a 75% reduction, at least an 80% reduction, at least an 85% reduction, at least a 90% reduction, or at least a 95% reduction in FXI protein levels is observed in a mammalian cell contacted with the assayed double stranded RNA in vitro or in vivo, relative to a suitable control. Additional endpoints contemplated include, e g., assessment of a phenotype associated with reduction of FXI levels — e.g., clotting, deep vein thrombosis, ischemic stroke, as assessed directly or via assessment of appropriate markers and / or indicators of such disease or disorder.

[0150] FXI inhibitory activity can also be evaluated over time (duration) and over concentration ranges (potency), with assessment of what constitutes a dsRNA possessing FXI inhibitory activity adjusted in accordance with concentrations administered and duration of time following administration. Thus, in certain embodiments, a dsRNA of the disclosure is deemed to possess FXI inhibitory activity if at least a 50% reduction in FXI activity is observed / persists at a duration of time of 2 hours, 5 hours, 10 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days or more after administration of the dsRNA to a cell or organism. In additional embodiments, a dsRNA of theAttorney Docket No. 809030.000060disclosure is deemed to be a potent FXI inhibitory agent if FXI inhibitory activity (e.g., in certain embodiments, at least 50% inhibition of FXI) is observed at a concentration of 2 nM or less, 1 nM or less, 500 pM or less, 200 pM or less, 100 pM or less, 50 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, 2 pM or less or even 1 pM or less in the environment of a cell, for example, within an in vitro assay for FXI inhibitory activity as described herein. In certain embodiments, a potent FXI inhibitory dsRNA of the disclosure is defined as one that is capable of FXI inhibitory activity (e.g., in certain embodiments, at least 20% reduction of FXI levels) at a formulated concentration of 3 mg / kg or less when administered to a subject as a pharmaceutical composition capable of delivery (e.g., modified RNAi agent that include a targeting ligand (e.g., triGalNAc) as described herein). Preferably, a potent FXI inhibitory dsRNA of the disclosure is defined as one that is capable of FXI inhibitory activity (e.g., in certain embodiments, at least 50% reduction of FXI levels) at a formulated concentration of 3 mg / kg or less when administered to a subject in an effective delivery vehicle. More preferably, a potent FXI inhibitory dsRNA of the disclosure is defined as one that is capable of FXI inhibitory activity (e.g., in certain embodiments, at least 50% reduction of FXI levels) at a formulated concentration of 3 mg / kg or less when administered to a subject in an effective delivery vehicle. Optionally, a potent FXI inhibitory dsRNA of the disclosure is defined as one that is capable of FXI inhibitory activity (e.g., in certain embodiments, at least 50% reduction of FXI levels) at a formulated concentration of 2 mg / kg or less, or even 1 mg / kg or less, when administered to a subject in an effective delivery vehicle. Exemplary discrete formulated concentrations of FXI-targeting RNAi agents of the disclosure include about 5 mg / kg, about 4 mg / kg, about 3 mg / kg, about 2 mg / kg, about 1 mg / kg, about 500 pg / kg, about 250 pg / kg, about 100 pg / kg, about 50 pg / kg, about 25 pg / kg, about 10 pg / kg, about 5 pg / kg, about 2.5 pg / kg, about 1 pg / kg, about 500 ng / kg, about 250 ng / kg, about 100 ng / kg, about 50 ng / kg, about 25 ng / kg, about 10 ng / kg, about 5 ng / kg, about 2.5 ng / kg, about 1 ng / kg and about 500 pg / kg.

[0151] About: As used herein, the term “about” means + / - 10% of the recited value. Use of “about” is contemplated in reference to all ranges and values recited herein.

[0152] In certain embodiments, the phrase “consists essentially of’ is used in reference to the FXI RNAi agents of the disclosure. In some such embodiments, “consists essentially of’ refers to a composition that comprises a dsRNA of the disclosure which possesses at least a certain level of FXI inhibitory activity (e.g., at least 50% FXI inhibitory activity) and that also comprises one or more additional components and / or modifications that do not significantly impact the FXIAttorney Docket No. 809030.000060inhibitory activity of the dsRNA. For example, in certain embodiments, a composition “consists essentially of’ a dsRNA of the disclosure where modifications of the dsRNA of the disclosure and / or dsRNA-associated components of the composition do not alter the FXI inhibitory activity (optionally including potency or duration of FXI inhibitory activity) by greater than 3%, greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, or greater than 50% relative to the dsRNA of the disclosure in isolation. In certain embodiments, a composition is deemed to consist essentially of a dsRNA of the disclosure even if more dramatic reduction of FXI inhibitory activity (e.g., 70% reduction, 80% reduction, 90% reduction, etc. in efficacy, duration and / or potency) occurs in the presence of additional components or modifications, yet where FXI inhibitory activity is not significantly elevated (e.g., observed levels of FXI inhibitory activity are within 10% those observed for the dsRNA of the disclosure) in the presence of additional components and / or modifications.

[0153] As used herein, the term “nucleic acid” refers to deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphorami dates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs) and unlocked nucleic acids (UNAs or unlocked nucleobase analogs; see, e.g., Jensen et al. Nucleic Acids Symposium Series 52: 133-4), and derivatives thereof.

[0154] As used herein, “nucleotide” is used as recognized in the art to include those with natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and / or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, e.g., Usman and McSwiggen, supra; Eckstein, et al., International Patent Application Publication No. WO 92 / 07065; Usman et al, International Patent Application Publication No. WO 93 / 15187; UhlmanAttorney Docket No. 809030.000060& Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach, et al, Nucleic Acids Res.22:2183, 1994. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, hypoxanthine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine, thymine, and uracil at 1 ' position or their equivalents.

[0155] As used herein, “modified nucleotide” refers to a nucleotide that has one or more modifications to the nucleoside, the nucleobase, pentose ring, or phosphate group. For example, modified nucleotides exclude ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxy thy mi dine monophosphate, and deoxycytidine monophosphate. Modifications include those naturally occurring that result from modification by enzymes that modify nucleotides, such as methyltransferases. Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Synthetic or non-naturally occurring modifications in nucleotides include those with 2' modifications, e.g., 2 '-methoxy ethoxy, 2'-fluoro, 2'-allyl, 2'-0 — [2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2 — O-2'-bridge, 4'-(CH2)2 — O-2'-bridge, 2'-LNA or other bicyclic or “bridged” nucleoside analog, and 2'-0 — (N-methylcarbamate) or those comprising base analogs. In connection with 2'-modified nucleotides as described for the present disclosure, by “amino” is meant 2'-NH2 or 2'-0 — NH2, which can be modified or unmodified. Such modified groups are described, e.g., in Eckstein et al., U. S. Pat. No. 5,672,695 and Matulic-Adamic et al., U. S. Pat. No.6,248,878, which are hereby incorporated by reference in their entirety. “Modified nucleotides” of the instant disclosure can also include nucleotide analogs as described above.

[0156] In reference to the nucleic acid molecules of the present disclosure, modifications may exist upon these agents in patterns on one or both strands of the double stranded ribonucleic acid (dsRNA). As used herein, “alternating positions” refers to a pattern where every other nucleotide is a modified nucleotide or there is an unmodified nucleotide (e.g., an unmodifiedAttorney Docket No. 809030.000060ribonucleotide) between every modified nucleotide over a defined length of a strand of the dsRNA (e.g., 5'-MN NMN-3'; 3'-MNMNMN-5'; where M is a modified nucleotide and N is an unmodified nucleotide). The modification pattern starts from the first nucleotide position at either the 5' or 3' terminus according to a position numbering convention, e.g., as described herein (in certain embodiments, position 1 is designated in reference to the terminal residue of a strand of an RNAi agent agent of the disclosure, optionally after a Dicer processing event; thus, particularly for longer agents (e.g., Dicer substrate RNAi agents, as are also expressly contemplated by the instant disclosure) position 1 need not always constitute a 3' terminal or 5' terminal residue of a pre-processed agent of the disclosure). The pattern of modified nucleotides at alternating positions may run the full length of the strand, but in certain embodiments includes at least 4, 6, 8, 10, 12, 14 nucleotides containing at least 2, 3, 4, 5, 6 or 7 modified nucleotides, respectively. As used herein, “alternating pairs of positions” refers to a pattern where two consecutive modified nucleotides are separated by two consecutive unmodified nucleotides over a defined length of a strand of the dsRNA (e.g., 5'-MMNNMMNNMMNN-3'; 3'-MMNNMMNNMMNN-5'; where M is a modified nucleotide and N is an unmodified nucleotide). The modification pattern starts from the first nucleotide position at either the 5' or 3' terminus according to a position numbering convention such as those described herein. The pattern of modified nucleotides at alternating positions may run the full length of the strand, but preferably includes at least 8, 12, 16, 20, 24, 28 nucleotides containing at least 4, 6, 8, 10, 12 or 14 modified nucleotides, respectively. It is emphasized that the above modification patterns are exemplary and are not intended as limitations on the scope of the disclosure.

[0157] As used herein, “base analog” refers to a heterocyclic moiety which is located at the 1' position of a nucleotide sugar moiety in a modified nucleotide that can be incorporated into a nucleic acid duplex (or the equivalent position in a nucleotide sugar moiety substitution that can be incorporated into a nucleic acid duplex). In the dsRNAs of the disclosure, a base analog is generally either a purine or pyrimidine base excluding the common bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U). Base analogs can duplex with other bases or base analogs in dsRNAs. Base analogs include those useful in the compounds and methods of the disclosure, e.g., those disclosed in U. S. Pat. Nos. 5,432,272 and 6,001,983 to Benner and US Patent Publication No. 20080213891 to Manoharan, which are herein incorporated by reference. Nonlimiting examples of bases include hypoxanthine (I), xanthine (X), 3 -D-ribofuranosyl-(2,6-Attorney Docket No. 809030.000060diaminopyrimidine) (K), 3-P-D-ribofuranosyl-(l-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione) (P), iso-cytosine (iso-C), iso-guanine (iso-G), l-P-D-ribofuranosyl-(5-nitroindole), 1-P-D-ribofuranosyl-(3 -nitropyrrole), 5-bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, and 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl, pentacenyl, and structural derivates thereof (Schweitzer et al., J. Org. Chem., 59:7238-7242 (1994); Berger et al., Nucleic Acids Research, 28(15):2911-2914 (2000); Moran et al., J. Am. Chem. Soc., 119:2056-2057 (1997); Morales et al., J. Am. Chem. Soc., 121:2323-2324 (1999); Guckian et al., J. Am. Chem. Soc., 118:8182-8183 (1996); Morales etal., J. Am. Chem. Soc., 122(6): 1001-1007 (2000); McMinn et al., J. Am. Chem. Soc., 121:11585-11586 (1999); Guckian et al., J. Org. Chem., 63:9652-9656 (1998); Moran et al., Proc. Natl. Acad. Sci., 94:10506-10511 (1997); Das et al., J. Chem. Soc., Perkin Trans., 1:197-206 (2002); Shibata et al., J. Chem. Soc., Perkin Trans., 1: 1605-1611 (2001); Wu et al., J. Am. Chem. Soc., 122(32):7621-7632 (2000); O'Neill et al., J. Org. Chem., 67:5869-5875 (2002); Chaudhuri et al., J. Am. Chem. Soc., 117:10434-10442 (1995); and U. S. Pat. No. 6,218,108), which are hereby incorporated by reference in their entirety. Base analogs may also be a universal base.

[0158] As used herein, “universal base” refers to a heterocyclic moiety located at the T position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a nucleic acid duplex, can be positioned opposite more than one type of base” without altering the double helical structure (e.g., the structure of the phosphate backbone). Additionally, the universal base does not destroy the ability of the single stranded nucleic acid in which it resides to duplex to a target nucleic acid. The ability of a single stranded nucleic acid containing a universal base to duplex a target nucleic can be assayed by methods apparent to one in the art (e.g., UV absorbance, circular dichroism, gel shift, single stranded nuclease sensitivity, etc.). Additionally, conditions under which duplex formation is observed may be varied to determine duplex stability or formation, e g., temperature, as meltingAttorney Docket No. 809030.000060temperature (Tm) correlates with the stability of nucleic acid duplexes. Compared to a reference single stranded nucleic acid that is exactly complementary to a target nucleic acid, the single stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower Tm than a duplex formed with the complementary nucleic acid. However, compared to a reference single stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid having the mismatched base.

[0159] Some universal bases are capable of base pairing by forming hydrogen bonds between the universal base and all of the bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U) under base pair forming conditions. A universal base is not a base that forms a base pair with only one single complementary base. In a duplex, a universal base may form no hydrogen bonds, one hydrogen bond, or more than one hydrogen bond with each of G, C, A, T, and U opposite to it on the opposite strand of a duplex. Preferably, the universal bases does not interact with the base opposite to it on the opposite strand of a duplex. In a duplex, base pairing between a universal base occurs without altering the double helical structure of the phosphate backbone. A universal base may also interact with bases in adjacent nucleotides on the same nucleic acid strand by stacking interactions. Such stacking interactions stabilize the duplex, especially in situations where the universal base does not form any hydrogen bonds with the base positioned opposite to it on the opposite strand of the duplex. Non-limiting examples of universalbinding nucleotides include inosine, l-P-D-ribofuranosyl-5-nitroindole, and / or 1-P-D-ribofuranosyl-3 -nitropyrrole (US Pat. Appl. Publ. No. 20070254362 to Quay et al.; Van Aerschot et al., An acyclic 5 -nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov. 11; 23(21):4363-70; Loakes et al., 3 -Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul. 11; 23( 13 ):2361 -6; Loakes and Brown, 5-Nitroindole as an universal base analogue. Nucleic Acids Res. 1994 Oct. 11; 22(20):4039-43).

[0160] As used herein, “loop” refers to a structure formed by a single strand of a nucleic acid, in which complementary regions that flank a particular single stranded nucleotide region hybridize in a way that the single stranded nucleotide region between the complementary regions is excluded from duplex formation or Watson-Crick base pairing. A loop is a single strandedAttorney Docket No. 809030.000060nucleotide region of any length. Examples of loops include the unpaired nucleotides present in such structures as hairpins, stem loops, or extended loops.

[0161] As used herein, “extended loop” in the context of a dsRNA refers to a single stranded loop and in addition 1, 2, 3, 4, 5, 6 or up to 20 base pairs or duplexes flanking the loop. In an extended loop, nucleotides that flank the loop on the 5' side form a duplex with nucleotides that flank the loop on the 3' side. An extended loop may form a hairpin or stem loop.

[0162] As used herein, “tetraloop” in the context of a dsRNA refers to a loop (a single stranded region) consisting of four nucleotides that forms a stable secondary structure that contributes to the stability of adjacent Watson-Crick hybridized nucleotides. Without being limited to theory, a tetraloop may stabilize an adjacent Watson-Crick base pair by stacking interactions. In addition, interactions among the four nucleotides in a tetraloop include but are not limited to non -Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug. 16; 346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12; 253(5016): 191-4). A tetraloop confers an increase in the melting temperature (Tm) of an adjacent duplex that is higher than expected from a simple model loop sequence consisting of four random bases. For example, a tetraloop can confer a melting temperature of at least 55° C. in 10 mM NaHPO4 to a hairpin comprising a duplex of at least 2 base pairs in length. A tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Examples of RNA tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop. (Woese et al., Proc Natl Acad Sci USA. 1990 November; 87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 Nov. 11; 19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA)) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, the d(TNCG) family of tetraloops (e.g., d(TTCG)). (Nakano et al. Biochemistry, 41 (48), 14281-14292, 2002.; SHINJI et al. Nippon Kagakkai Keen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000).)

[0163] Where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more singleAttorney Docket No. 809030.000060stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the disclosure. As another example, a dsRNA comprising one oligonucleotide 19 nucleotides in length and another oligonucleotide 21 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 19 nucleotides that is fully complementary to the shorter oligonucleotide, may also be referred to as “fully complementary” for the purposes of the present disclosure.

[0164] The term “double-stranded RNA” or “dsRNA” or “duplex”, as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where separate RNA molecules, such dsRNA are often referred to as RNAi agent (“short interfering RNA”). Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3 '-end of one strand and the 5' end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”, “short hairpin RNA” or “shRNA”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5' end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. In some embodiments, a double stranded nucleic acid may have each strand comprise RNA, RNA analog(s) or RNA and DNA. An RNAi agent may include between 19 and 23 nucleotides. In some embodiments, an RNAi agent may include 21 nucleotides. In some embodiments an RNAi agent may include 23 nucleotides. An RNAi agent typically has 2 bp overhangs on the 3' ends of each strand such that the duplex region in the RNAi agent comprises 17-21 nucleotides. In some embodiments, a duplex region in a RNAi agent may include 21 nucleotides. In some embodiments,Attorney Docket No. 809030.000060a duplex region in a RNAi agent may include 19 nucleotides. Typically, an antisense strand of a RNAi agent is sufficiently complementary with a target sequence of the FXI antigen / RNA.

[0165] In addition, as used herein, “dsRNA” or “RNAi Agent” may include chemical modifications to ribonucleotides, intemucleoside linkages, end-groups, caps, and conjugated moieties, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNAtype molecule or other RNAi agent, are encompassed by “dsRNA” or “RNAi Agent” for the purposes of this specification and claims.

[0166] The phrase “duplex region” refers to the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the “duplex region” consists of 19 base pairs. Similarly, an oligonucleotide strand having 23 nucleotides units can base pair with another oligonucleotide of 23 nucleotides, yet only 21 bases on each strand are complementary or substantially complementary, such that the “duplex region” consists of 21 base pairs. The remaining base pairs may, for example, exist as 5' and 3' overhangs. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well know in the art. Alternatively, two strands can be synthesized and added together under biological conditions to determine if they anneal to one another. By definition, “sufficiently complementary” (contrasted with, e.g., “100% complementary”) allows for one or more mismatches to exist between a dsRNA of the disclosure and the target RNA or cDNA sequence (e.g., FXI mRNA), provided that the dsRNA possesses complementarity sufficient to trigger the destruction of the target RNA by the RNAi machinery (e.g., the RISC complex) or process. In certain embodiments, a “sufficiently complementary” dsRNA of the disclosure can harbor one, two, three or even four or more mismatches between the dsRNA sequence and the target RNA or cDNA sequence (e.g., in certain such embodiments, the antisenseAttorney Docket No. 809030.000060strand of the dsRNA harbors one, two, three, four, five or even six or more mismatches when aligned with the target RNA or cDNA sequence). Additional consideration of the preferred location of such mismatches within certain dsRNAs of the instant disclosure is considered in greater detail below.

[0167] Single-stranded nucleic acids that base pair over a number of bases are said to “hybridize.” Hybridization is typically determined under physiological or biologically relevant conditions (e.g., intracellular: pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion). Hybridization conditions generally contain a monovalent cation and biologically acceptable buffer and may or may not contain a divalent cation, complex anions, e.g. gluconate from potassium gluconate, uncharged species such as sucrose, and inert polymers to reduce the activity of water in the sample, e.g. PEG. Such conditions include conditions under which base pairs can form.

[0168] Hybridization is measured by the temperature required to dissociate single stranded nucleic acids forming a duplex, i.e., (the melting temperature; Tm). Hybridization conditions are also conditions under which base pairs can form. Various conditions of stringency can be used to determine hybridization (see, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol.152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.)=2 (# of A+T bases)+4 (# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.)=81.5+16.6 (log 10[Na+])+0.41 (% G+C)-(600 / N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1× SSC=0.165 M).

[0169] Useful variations on hybridization conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Antisense to MolecularAttorney Docket No. 809030.000060Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0170] As used herein, “oligonucleotide strand” is a single stranded nucleic acid molecule. An oligonucleotide may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2' modifications, synthetic base analogs, etc.) or combinations thereof. Such modified oligonucleotides can be preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.

[0171] As used herein, the term “ribonucleotide” encompasses natural and synthetic, unmodified and modified ribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and / or to the linkages between ribonucleotides in the oligonucleotide. As used herein, the term “ribonucleotide” specifically excludes a deoxyribonucleotide, which is a nucleotide possessing a single proton group at the 2' ribose ring position.

[0172] As used herein, the term “deoxyribonucleotide” encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and / or to the linkages between deoxyribonucleotide in the oligonucleotide. As used herein, the term “deoxyribonucleotide” also includes a modified ribonucleotide that does not permit Dicer cleavage of a dsRNA agent, e.g., a 2'-O-methyl ribonucleotide, a phosphorothioate-modified ribonucleotide residue, etc., that does not permit Dicer cleavage to occur at a bond of such a residue.

[0173] As used herein, the term “PS-NA” refers to a phosphorothioate-modified nucleotide residue. The term “PS-NA” therefore encompasses both phosphorothioate-modified ribonucleotides (“PS-RNAs”) and phosphorothioate-modified deoxyribonucleotides (“PS-DNAs”).

[0174] As used herein, “overhang” refers to unpaired nucleotides, in the context of a duplex having one or more free ends at the 5' terminus or 3' terminus of a dsRNA. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense strand or sense strand. In some embodiments, the overhang is a 3' overhang having a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. For clarity, chemicalAttorney Docket No. 809030.000060caps or non-nucleotide chemical moieties conjugated to the 3' end or 5' end of an RNAi are not considered in determining whether an RNAi has an overhang or is blunt ended. In certain embodiments, the disclosure provides a dsRNA molecule for inhibiting the expression of the FXI target gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the FXI target gene, and wherein the region of complementarity is less than 35 nucleotides in length, optionally 19-24 nucleotides in length or 25-30 nucleotides in length, and wherein the dsRNA, upon contact with a cell expressing the FXI target gene, inhibits the expression of the FXI target gene by at least 10%, 25%, or 40%.

[0175] By “homologous sequence” is meant a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and / or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or R A, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant disclosure (e g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.). Indeed, design and use of the dsRNA agents of the instant disclosure contemplates the possibility of using such dsRNA agents not only against target RNAs of FXI possessing perfect complementarity with the presently described dsRNA agents, but also against target FXI RNAs possessing sequences that are, e.g., only 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%>, 85%, 84%, 83%, 82%, 81%, 80%o etc. complementary to said dsRNA agents. Similarly, it is contemplated that the presently described dsRNA agents of the instant disclosure might be readily altered by the skilled artisan to enhance the extent of complementarity between said dsRNA agents and a target FXI RNA, e.g., of a specific allelic variant of FXI (e.g., an allele of enhanced therapeutic interest). Indeed, dsRNA agent sequences with insertions, deletions, and single point mutations relative to the target FXI sequence can also be effective for inhibition. Alternatively,Attorney Docket No. 809030.000060dsRNA agent sequences with nucleotide analog substitutions or insertions can be effective for inhibition.

[0176] Sequence identity may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions / total # of positions x 100), optionally penalizing the score for the number of gaps introduced and / or length of gaps introduced.

[0177] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). A preferred, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

[0178] In another embodiment, a gapped alignment, the alignment is optimized by introducing appropriate gaps, and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, a global alignment the alignment is optimized by introducing appropriate gaps, and percent identity is determined over the entire length of the sequences aligned, (i.e., a global alignment). A preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CAB IOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program forAttorney Docket No. 809030.000060comparing amino acid sequences, aPAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0179] Greater than 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between the dsRNA antisense strand and the portion of the FXI RNA sequence is preferred. Alternatively, the dsRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the FXI RNA (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing). Additional preferred hybridization conditions include hybridization at 70° C. in 1*SSC or 50° C. in 1 *SSC, 50% formamide followed by washing at 70° C. in 0.3*SSC or hybridization at 70° C. in 4*SSC or 50° C. in 4>< SSC, 50% formamide followed by washing at 67° C. in 1*SSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.)=2 (# of A+T bases)+4 (# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.)=81.5+16.6 (log 10[Na+])+0.41 (% G+C)-(600 / N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for l*SSC=0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4. The length of the identical nucleotide sequences may be at least 10, 12, 15, 17, 20, 22, 25, 27 or 30 bases.

[0180] By “conserved sequence region” is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism. The polynucleotide can include both coding and non-coding DNA and RNA.

[0181] By “ sense region” is meant a nucleotide sequence of a dsRNA molecule having complementarity to an antisense region of the dsRNA molecule. In addition, the sense region of a dsRNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.Attorney Docket No. 809030.000060

[0182] By “antisense region” is meant a nucleotide sequence of a dsRNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a dsRNA molecule comprises a nucleic acid sequence having complementarity to a sense region of the dsRNA molecule.

[0183] As used herein, “antisense strand” refers to a single stranded nucleic acid molecule which has a sequence complementary to that of a target RNA. When the antisense strand contains modified nucleotides with base analogs, it is not necessarily complementary over its entire length, but must at least hybridize with a target RNA.

[0184] As used herein, “sense strand” refers to a single stranded nucleic acid molecule which has a sequence complementary to that of an antisense strand. When the antisense strand contains modified nucleotides with base analogs, the sense strand need not be complementary over the entire length of the antisense strand, but must at least duplex with the antisense strand.

[0185] As used herein, “guide strand” refers to a single stranded nucleic acid molecule of a dsRNA or dsRNA-containing molecule, which has a sequence sufficiently complementary to that of a target RNA to result in RNA interference. For RNAi, or for certain dsRNA agents, the guide strand associates with RISC, binds a target RNA as a component of the RISC complex, and promotes cleavage of a target RNA by RISC. For certain longer RNAi agents, after cleavage of the RNAi agent by Dicer, a fragment of the guide strand associates with RISC, binds a target RNA as a component of the RISC complex, and promotes cleavage of a target RNA by RISC. A guide strand is an antisense strand.

[0186] As used herein, “passenger strand” refers to an oligonucleotide strand of a dsRNA or dsRNA-containing molecule, which has a sequence that is complementary to that of the guide strand. A passenger strand is a sense strand.

[0187] By “target nucleic acid” is meant a nucleic acid sequence whose expression, level or activity is to be modulated. The target nucleic acid can be DNA or RNA. For agents that target FXI, in certain embodiments, the target nucleic acid is FXI RNA, e.g., in certain embodiments, FXI mRNA. In reference to any specific RNAi agent of the instant disclosure, the term “RNA target site” for that RNAi agent refers to the sequence in FXI RNA that is complementary to the guide strand (antisense strand) of the RNAi agent. FXI RNA target sites can also interchangeably be referenced by corresponding cDNA sequences. Levels of FXI may also be targeted via targeting of upstream effectors of FXI, or the effects of modulated or misregulated FXI may also beAttorney Docket No. 809030.000060modulated by targeting of molecules downstream of FXI in the FXI signaling pathway, or within the intrinsic pathway.

[0188] As used herein, the term “seed region” refers to nucleotides two through eight (counting from the 5’ end) of the antisense or guide strand of an RNAi agent. The seed region is the primary determinant for binding to the target nucleic acid.

[0189] By “complementarity” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present disclosure, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In one embodiment, a dsRNA molecule of the disclosure comprises 12 to 30 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof.

[0190] As used herein, a dsRNA, e.g., an RNAi agent, having a sequence “sufficiently complementary” to a target RNA or cDNA sequence (e.g., FXI mRNA) means that the dsRNA has a sequence sufficient to trigger the destruction of the target RNA (where a cDNA sequence is recited, the RNA sequence corresponding to the recited cDNA sequence) by the RNAi machinery (e.g., the RISC complex) or process. For example, a dsRNA that is “sufficiently complementary” to a target RNA or cDNA sequence to trigger the destruction of the target RNA by the RNAi machinery or process can be identified as a dsRNA that causes a detectable reduction in the level of the target RNA in an appropriate assay of dsRNA activity (e.g., an in vitro assay as described in Example 2 below), or, in further examples, a dsRNA that is sufficiently complementary to aAttorney Docket No. 809030.000060target RNA or cDNA sequence to trigger the destruction of the target RNA by the RNAi machinery or process can be identified as a dsRNA that produces at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70%, at least a 75%, at least a 80%, at least a 85%, at least a 90%, at least a 95%, at least a 98% or at least a 99% reduction in the level of the target RNA in an appropriate assay of dsRNA activity. In additional examples, a dsRNA that is sufficiently complementary to a target RNA or cDNA sequence to trigger the destruction of the target RNA by the RNAi machinery or process can be identified based upon assessment of the duration of a certain level of inhibitory activity with respect to the target RNA or protein levels in a cell or organism. For example, a dsRNA that is sufficiently complementary to a target RNA or cDNA sequence to trigger the destruction of the target RNA by the RNAi machinery or process can be identified as a dsRNA capable of reducing target mRNA levels by at least 20% at least 48 hours post-administration of said dsRNA to a cell or organism. Preferably, a dsRNA that is sufficiently complementary to a target RNA or cDNA sequence to trigger the destruction of the target RNA by the RNAi machinery or process is identified as a dsRNA capable of reducing target mRNA levels by at least 40% at least 72 hours post-administration of said dsRNA to a cell or organism, by at least 40% at least four, five or seven days post-administration of said dsRNA to a cell or organism, by at least 50% at least 48 hours post-administration of said dsRNA to a cell or organism, by at least 50% at least 72 hours post-administration of said dsRNA to a cell or organism, by at least 50% at least four, five or seven days post-administration of said dsRNA to a cell or organism, by at least 80% at least 48 hours post-administration of said dsRNA to a cell or organism, by at least 80% at least 72 hours post-administration of said dsRNA to a cell or organism, or by at least 80% at least four, five or seven days post-administration of said dsRNA to a cell or organism.

[0191] In certain embodiments, a nucleic acid of the disclosure (e.g., an RNAi agent) possesses a sequence “sufficiently complementary to hybridize” to a target RNA or cDNA sequence, thereby achieving an inhibitory effect upon the target RNA. Hybridization, and conditions available for determining whether one nucleic acid is sufficiently complementary to another nucleic acid to allow the two sequences to hybridize, is described in greater detail below.

[0192] As used herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be presentAttorney Docket No. 809030.000060in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell. Within certain aspects, the term “cell” refers specifically to mammalian cells, such as human cells, that contain one or more dsRNA molecules of the present disclosure. In particular aspects, a cell processes dsRNAs or dsRNA-containing molecules resulting in RNA interference of target nucleic acids, and contains proteins and protein complexes required for RNAi, e.g., Dicer and RISC.

[0193] By “RNA” is meant a molecule comprising at least one, and preferably at least 4, 8 and 12 ribonucleotide residues. The at least 4, 8 or 12 RNA residues may be contiguous. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2' position of a 0-D-ribofuranose moiety. The terms include double-stranded RNA, single-stranded RNA, RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the dsRNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant disclosure can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0194] In certain embodiments, an RNAi agent (e.g., an siRNA) of the disclosure can be an “isolated” RNAi agent, meaning that the RNAi agent is isolated from (removed and / or purified from) a natural environment.

[0195] In some embodiments, an RNAi agent (e.g., an siRNA) of the disclosure can be a “synthetic” RNAi agent. The term “synthetic” or “non-natural” refers to an RNAi agent (e.g., an RNAi agent of the disclosure) that (i) is synthesized using a machine or (ii) that is not derived from a cell or organism that normally produces the RNAi agent.

[0196] By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the dsRNA agents of theAttorney Docket No. 809030.000060disclosure can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.

[0197] The phrase “pharmaceutically acceptable carrier” refers to a carrier for the administration of a therapeutic agent. Exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while com starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. The pharmaceutically acceptable carrier of the disclosed dsRNA compositions may be micellar structures, such as a liposomes, capsids, capsoids, polymeric nanocapsules, or polymeric microcapsules.

[0198] Polymeric nanocapsules, microcapsules, and / or lipid nanoparticles (LNPs, as well-known in the art) can facilitate transport and release of encapsulated or bound dsRNA into a cell. Polymeric nanocapsules and microcapsules include polymeric and monomeric materials, especially including polybutylcyanoacrylate. A summary of materials and fabrication methods has been published (see Kreuter, 1991). The polymeric materials which are formed from monomeric and / or oligomeric precursors in the polymerization / nanoparticle generation step, are per se known from the prior art, as are the molecular weights and molecular weight distribution of the polymeric material which a person skilled in the field of manufacturing nanoparticles may suitably select in accordance with the usual skill. In certain examples, viral vectors, such as Adeno-associated virus (AAV) may also be used for delivery of nucleic acid payloads.

[0199] Therapeutic agents of the instant disclosure commonly include a targeting ligand (e.g., triGalNAc for liver-targeted nucleic acid agents) and a range of other art-recognized nucleic acid modifications, such as 2'-O-methyl-modified nucleosides, 2'-fluoro-modified nucleosides, and phosphorothioate internucleoside linkages. Such therapeutic agents can be formulated as pharmaceutical compositions and delivered to a subject, often without the need for any nucleic acid encapsulating agent and / or other delivery vehicle (e.g., viral vector, LNP, etc.).Attorney Docket No. 809030.000060

[0200] The term “in vitro” has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts. The term “in vivo” also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and / or cells in an organism.

[0201] Treatment”, or “treating” as used herein, is defined as the application or administration of a therapeutic agent (e.g., an RNAi agent or a vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, or symptoms of the disease or disorder. The term “treatment” or “treating” is also used herein in the context of administering agents prophylactically. As used herein, the term “prevent” or “prevention”, when used in connection with the application or administration of a therapeutic agent (e.g., an RNAi agent or a vector or transgene encoding same) to a patient who has or is at risk of having a disease or disorder, refers to reducing the risk of developing the disease or disorder, or symptoms of the disease or disorder in the patient. The term “prevent” can also be used in connection with application or administration of a therapeutic agent to an isolated tissue or cell line obtained or derived from a patient, to reduce the risk of developing the disease or disorder, or symptoms of the disease or disorder in the tissue, cell line, or patient (e.g., for an ex vivo therapeutic regimen). The term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0202] Various methodologies of the instant disclosure include at least one step that involves comparing a value, level, feature, characteristic, property, etc. to a “suitable control”, referred to interchangeably herein as an “appropriate control”. A “suitable control” or “appropriate control” is a control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNAi agent of the disclosure into a cell or organism. In anotherAttorney Docket No. 809030.000060embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a “suitable control” or “appropriate control” is a predefined value, level, feature, characteristic, property, etc.

[0203] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0204] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.Factor XI

[0205] The Fll gene is located on chromosome 4q32-35 and has an open reading frame that spans approximately 23 kb and includes 15 exons. The Fll gene encodes coagulation factor XI (FXI) of the blood coagulation cascade, which is produced primarily in the liver. FXI is present in plasma as a zymogen — an inactive homodimer consisting of two identical polypeptide chains linked by disulfide bonds. FXI is activated by contact with negatively charged surfaces, leading to its conversion to Factor Xia (FXIa). During activation of FXI, an internal peptide bond is cleaved by Factor Xlla (FXIIa) in each of the two chains, resulting in activated FXIa, a serine protease composed of two heavy chains and two light chains held together by disulfide bonds. Activated FXIa then initiates the middle phase of the intrinsic pathway of blood coagulation by activating factor IX, thereby amplifying the coagulation response and facilitating the conversion of prothrombin to thrombin, ultimately resulting in fibrin formation and clot stabilization.

[0206] Factor XI plays an important role in various clotting disorders and thrombotic diseases.

[0207] Excessive activation of Factor XI is implicated in thrombotic disorders such as myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis (DVT), and pulmonary embolism (PE). In these conditions, elevated Factor XI levels can promote an inappropriate coagulation response, leading to the formation of thrombi that obstruct blood vessels. This is particularly evident in venous thromboembolism, where Factor XI plays a central role in the pathogenesis of DVT and subsequent PE. Clinical studies have demonstrated that targeting FactorAttorney Docket No. 809030.000060XI with specific inhibitors may reduce thrombotic events without significantly increasing the risk of bleeding, marking a potential therapeutic strategy for patients at high risk of thromboembolic complications. Pharmacological inhibitors of factor XI include ASUNDEXIAN™ and MILVEXIAN™, as well as the monoclonal anti-factor XI antibody ABELACIMAB™.

[0208] The association of Factor XI with conditions such as ischemic stroke and transient ischemic attacks (TIAs) further underscores its dual role in hemostasis and thrombosis. Elevated levels of Factor XI have been linked to an increased risk of ischemic events in patients with atrial fibrillation and other pro-thrombotic states. In such examples, Factor XI contributes to the formation of thrombi in cerebral arteries, leading to reduced blood flow and ischemia. Additionally, Factor XI also appears to play a role in renal vein thrombosis and cerebral venous sinus thrombosis, where its dysregulation contributes to venous occlusion, which exacerbates the risk of both local and systemic complications.

[0209] In summary, Factor XI is integral to both the hemostatic balance and the pathogenesis of various thrombotic disorders.FXI Target Sequences

[0210] In some embodiments, RNAi agents capable of inhibiting FXI expression are provided herein, that can be used to achieve a therapeutic benefit. Through examination of the FXI mRNA, including mRNAs of multiple different species (e.g., human, cynomolgus monkey, rhesus monkey, and mouse) and in vitro and in vivo testing, it has been discovered that certain sequences of FXI mRNA are useful as targeting sequences because they are more amenable than others to RNAi agent-mediated knockdown.

[0211] Accordingly, in some embodiments, RNAi agents provided herein are designed so as to have regions of complementarity to FXI mRNA (e.g., within a target sequence of FXI mRNA, see FIG. 1C) for purposes of targeting the mRNA in cells and inhibiting its expression. The region of complementary is generally of a suitable length and base content to enable annealing of the RNAi agent (or a strand thereof) to FXI mRNA for purposes of inhibiting its expression. In some embodiments, the region of complementarity is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 nucleotides in length. In some embodiments, an RNAi agent provided herein has a region of complementarity to FXI that is in the range of 12 to 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30)Attorney Docket No. 809030.000060nucleotides in length. In some embodiments, an RNAi agent provided herein has a region of complementarity toFXIthatis 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

[0212] In some embodiments, an RNAi agent disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded RNAi agent) that is at least partially complementary to FXI. In some embodiments, an RNAi agent disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded RNAi agent) that is fully complementary to FXI. In some embodiments, a region of complementarity of an RNAi agent (e.g., on an antisense strand of a double-stranded RNAi agent) is complementary to a contiguous sequence of nucleotides of FXI that is in the range of 12 to 20 nucleotides (e.g., 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 20, 14 to 18, 14 to 16, 16 to 20, 16 to 18, or 18 to 20) in length. In some embodiments, a region of complementarity of an RNAi agent (e.g., on an antisense strand of a double-stranded RNAi agent) is complementary to a contiguous sequence of nucleotides of FXI that is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides in length.

[0213] In some embodiments, a region of complementarity of an RNAi agent that is complementary to contiguous nucleotides of FXI spans the entire length of an antisense strand. In some embodiments, a region of complementarity of an RNAi agent that is complementary to contiguous nucleotides of FXI spans a portion of the entire length of an antisense strand.

[0214] In some embodiments, a region of complementarity to FXI may have one or more mismatches compared with a corresponding sequence of FXI mRNA. A region of complementarity on an RNAi agent may have up to 1, up to 2, up to 3, up to 4, up to 5, etc. mismatches provided that it maintains the ability to form complementary base pairs with FXI mRNA under appropriate hybridization conditions. Alternatively, a region of complementarity on an RNAi agent may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches provided that it maintains the ability to form complementary base pairs with FXI mRNA under appropriate hybridization conditions. In some embodiments, if there are more than one mismatches in a region of complementarity, they may be positioned consecutively (e.g., 2, 3, 4, or more in a row), or interspersed throughout the region of complementarity provided that the RNAi agent maintains the ability to form complementary base pairs with FXI mRNA under appropriate hybridization conditions.Attorney Docket No. 809030.000060

[0215] Exemplary target sequences in FXI for the RNAi agents disclosed herein include SEQ ID NOs: 343-427.RNAi Agents

[0216] Described herein are RNAi agents for inhibiting expression of the Factor XI gene encoding for FXI protein (referred to herein as FXI RNAi agents). Each FXI RNAi agent comprises a sense strand and an antisense strand. In some embodiments, an antisense strand of an RNAi agent may be referred to as a “guide strand.” For example, if an antisense strand can engage with RNA-induced silencing complex (RISC) and bind to an Argonaute protein, or engage with or bind to one or more similar factors, and direct silencing of a target gene, it may be referred to as a guide strand. In some embodiments a sense strand complementary with a guide strand may be referred to as a “passenger strand.”

[0217] For RNAi agent designs, the sense strand and antisense strand each can be 12 to 30 nucleotides in length. In some embodiments, the sense and antisense strands each can be 14 to 26 nucleotides in length. The sense and antisense strands can be either the same length or they can be different lengths. In some embodiments, the sense and antisense strands are each independently 14-21 nucleotides in length. In some embodiments, the sense and antisense strands are each 12-26 nucleotides in length. In some embodiments, the sense and antisense strands are each 19-23 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length while the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length while the antisense strand is about 23 nucleotides in length. In some embodiments, a sense strand is 23 nucleotides in length and an antisense strand is 21 nucleotides in length. In some embodiments, both the sense and antisense strands are each 21 nucleotides in length. In some embodiments, a sense strand is 22 nucleotides in length and an antisense strand is 21 nucleotides in length. In some embodiments, a sense strand is 19 nucleotides in length and an antisense strand is 21 nucleotides in length. In some embodiments, the RNAi agent sense and antisense strands are each independently 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In some embodiments, a double-stranded RNAi agent has a duplex length of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.

[0218] In some embodiments, the region of perfect or substantial complementarity between the sense strand and the antisense strand is 12-26 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20,Attorney Docket No. 809030.00006021, 22, 23, 24, 25, or 26) nucleotides in length and occurs at or near the 5' end of the antisense strand (e.g., this region may be separated from the 5' end of the antisense strand by 0, 1, 2, 3, or 4 nucleotides that are not perfectly or substantially complementary).

[0219] The sense strand and antisense strand each contain a duplex region sequence that is 12 to 23 nucleobases in length, when sense and antisense strands anneal. An antisense strand duplex region sequence is 100% (perfectly) complementary or at least about 85% (substantially) complementary to a nucleotide sequence (sometimes referred to, e.g., as a target sequence) present in the FXI mRNA target. A duplex region sequence is 100% (perfectly) complementary or at least about 85% (substantially) complementary to a duplex region sequence in the antisense strand, and thus the sense strand duplex region sequence is perfectly identical or at least about 85% identical to a nucleotide sequence (target sequence) present in the FXI mRNA target. A sense strand duplex region sequence can be the same length as a corresponding antisense duplex region sequence or it can be a different length. In some embodiments, the antisense strand duplex region sequence is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, the sense strand duplex region sequence is 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.

[0220] Examples of nucleotide sequences used in forming FXI RNAi agents are provided in Table 1 and Table 2.

[0221] The FXI RNAi agent sense and antisense strands anneal to form a duplex. A sense strand and an antisense strand of an FXI RNAi agent may be partially, substantially, or fully complementary to each other. Within the complementary duplex region, the sense strand core stretch sequence is at least 85% complementary or 100% complementary to the antisense core stretch sequence. In some embodiments, the sense strand core stretch sequence contains a sequence of at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least 85% or 100% complementary to a corresponding 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence of the antisense strand core stretch sequence (i.e., the sense and antisense core stretch sequences of an FXI RNAi agent have a region of at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least 85% base paired or 100% base paired.)

[0222] The sense strand and / or the antisense strand may optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (overhang sequence) at the 3' end, the 5' end,Attorney Docket No. 809030.000060or both the 3' and 5' ends of the duplex region sequences. The antisense strand additional nucleotides, if present, may or may not be complementary to the corresponding sequence in an FXI mRNA. The sense strand additional nucleotides, if present, may or may not be identical to the corresponding sequence in an FXI mRNA.

[0223] As used herein, an overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5' and / or 3' end of the sense strand duplex region sequence and / or antisense strand duplex region sequence. In some embodiments, an FXI RNAi agent has an antisense strand having a 3' extension (see FIG.1A, FIG. 1B) In some embodiments, an FXI RNAi agent comprises an antisense strand having a 3' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, an FXI RNAi agent comprises an antisense strand having a 3' extension of 1, 2, or 3 nucleotides in length.

[0224] In some embodiments, one or more of the antisense strand overhang nucleotides comprise uracil or thymidine nucleotides or nucleotides that are complementary to the corresponding FXI mRNA sequence. In some embodiments, a 3' antisense strand sequence region (optionally including an overhang) includes or consists of one of the following sequences, but is not limited to: AUA, UGCUU, CUG, UG, UGCC, CUGCC, CGU, CUU, UGCCUA, CUGCCU, UGCCU, UGAUU, GCCUAU, T, TT, U, UU (each listed 5'^3').

[0225] In some embodiments, the 3' end of the antisense strand can include additional abasic residues (Ab). An “abasic residue” or “abasic site” is a nucleotide or nucleoside that lacks a nucleobase at the 1 ' position of the sugar. In some embodiments, Ab or Ab Ab can be added to the 3' end of the antisense strand. In some embodiments, the abasic residue(s) can be added as inverted abasic residues (invAb). (See, e.g., F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16).

[0226] In some embodiments, a FXI RNAi agent may comprise a sense strand having a 3' extension of 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, one or more of the sense strand overhang nucleotides comprises adenosine, uracil, or thymidine nucleotides, AT dinucleotide, or nucleotides that correspond to nucleotides in the FXI mRNA sequence. In some embodiments, the 3 '-terminal region of the sense strand (optionally including an overhang) includes or consists of one of the following sequences, but is not limited to: T, UT, TT, UU, UUT, TTT, or TTTT (each listed 5' to 3').

[0227] In some embodiments, the 3' end of the sense strand may include additional abasic residues. In some embodiments, UUAb, UAb, or Ab are added to the 3' end of the sense strand. InAttorney Docket No. 809030.000060some embodiments, the one or more abasic residues added to the 3' end of the sense strand are inverted (invAb). In some embodiments, one or more inverted abasic residues or abasic sites may be inserted between the targeting ligand and the nucleobase sequence of the sense strand of the RNAi agent. In some embodiments, the inclusion of one or more inverted abasic residues or abasic sites at or near the terminal end or terminal ends of the sense strand of an RNAi agent allows for enhanced activity or other desired properties of an RNAi agent.

[0228] In some embodiments, an FXI RNAi agent may comprise a sense strand having a 5' overhang of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more of the sense strand overhang nucleotides comprise uracil or adenosine nucleotides or nucleotides that correspond to nucleotides in the FXI mRNA sequence. In some embodiments, the sense strand 5'-terminal region (optionally including an overhang) includes one of the following sequences, but is not limited to: CA, AUAGGC, AUAGG, AUAG, AUA, A, AA, AC, GCA, GGCA, GGC, UAUCA, UAUC, UCA, UAU, U, UU (each listed 5' to 3'). A sense strand can have a 3' overhang and / or a 5' overhang.

[0229] In some embodiments, the 5' end of the sense strand can include one or more additional abasic residues (e.g., (Ab) or (AbAb)). In some embodiments, the one or more abasic residues added to the 5' end of the sense strand can be inverted (e.g., invAb). In some embodiments, one or more inverted abasic residues can be inserted between the targeting ligand and the nucleobase sequence of the sense strand of the RNAi agent. In some embodiments, the inclusion of one or more inverted abasic residues at or near the terminal end or terminal ends of the sense strand of an RNAi agent may allow for enhanced activity or other desired properties of an RNAi agent. In some embodiments, an abasic (deoxyribose) residue can be replaced with a ribitol (abasic ribose) residue.

[0230] Examples of sequences used in forming FXI RNAi agents are provided in Table 1 and Table 2. In some embodiments, an FXI RNAi agent antisense strand includes a sequence of any of the sequences in Table 1 and Table 2. In some embodiments, an FXI RNAi agent antisense strand includes the sequence of nucleotides (from 5' end^-3' end) 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 3-14, 3-15, 1-16, 3-17, 3-18, 3-19, 3-20, 3-21, 3-22, 3-23, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 4-21, 4-22, 4-23, 5-16, 5-17, 5-18, 5-19, 5-20, 5-21, 5-22, 5-23, 6-18, 6-19, 6-20, 6-21, 6-22, 6-23, 7-17, 7-18, 7-19, 7-20, 7-21, 7-22, 7-23, 8-20, 8-21, 8-22, 8-23, 9-21, 9-22, or 9-23 of anyAttorney Docket No. 809030.000060of the sequences in Table 1 and Table 2. In certain embodiments, an FXI RNAi agent antisense strand comprises or consists of a sequence of any one of the modified sequences in Table 2. In some embodiments, an FXI RNAi agent antisense strand includes the sequence of any of the sequences in Table 1 and Table 2. In some embodiments, an FXI RNAi agent sense strand includes the sequence of nucleotides (from 5' end^-3' end) 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 2-12, 2-13, 2-14, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 3-21, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 4-21, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-21, 6-16, 6-17, 6-18, 6-19, 6-20, 6-21, 7-17, 7-18, 7-19, 7-20, or 7-21, of any of the sequences in Table 1 and Table 2. In certain embodiments, an FXI RNAi agent sense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 2.

[0231] In some embodiments, the antisense strand of an FXI RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 1 or Table 2. In some embodiments, the sense strand of an FXI RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 1 or Table 2.

[0232] In some embodiments an RNAi agent disclosed herein comprises an antisense strand comprising a sequence as set forth in any one of SEQ ID NOs: 88-172 or SEQ ID NOs: 258-342. In some embodiments, an RNAi agent comprises an antisense strand comprising a contiguous sequence of nucleotides that is in the range of 12 to 20 nucleotides (e.g., 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 20, 14 to 18, 14 to 16, 16 to 20, 16 to 18, or 18 to 20 nucleotides) in length of any of the sequences as set forth in any one of SEQ ID NOs: 88-172 or SEQ ID NOs: 258-342. In some embodiments, an RNAi agent comprises an antisense strand comprising a contiguous sequence of nucleotides of a sequence as set forth in any one of SEQ ID NOs: 88-172 or SEQ ID NOs: 258-342 that is 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides in length. In some embodiments, an RNAi agent comprises an antisense strand that consists of a sequence as set forth in any one of SEQ ID NOs: 88-172 or SEQ ID NOs: 258-342.

[0233] In some embodiments, an RNAi agent disclosed herein comprises a sense strand sequence as set forth in any one of SEQ ID NOs: 1-87 or SEQ ID NOs: 173-257. In some embodiments, an RNAi agent has a sense strand that comprises at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-87 or SEQ ID NOs: 173-257. In some embodiments, an RNAi agent has a sense strand that comprises aAttorney Docket No. 809030.000060contiguous sequence of nucleotides that is in the range of 7 to 36 nucleotides (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19-27, 20-36, or 15 to 36 nucleotides) in length of any of the sequences as set forth in any one of SEQ ID NOs: 1-87 or SEQ ID NOs: 173-257. In some embodiments, an RNAi agent has a sense strand that comprises a contiguous sequence of nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-87 or SEQ ID NOs: 173-257 that is 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 nucleotides in length. In some embodiments, an RNAi agent has a sense strand that consists of a sequence as set forth in any one of SEQ ID NOs: 1-87 or SEQ ID NOs: 173-257.

[0234] In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO:26 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 111. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 68 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 153. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 62 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 147. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 50 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 135. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 25 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 110. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 86 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 171. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 60 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 145. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 22 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 107. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 13 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 98. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 73 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 158.

[0235] In some embodiments, an RNAi agent provided herein comprises a sense strand comprising a sequence as set forth in any one of SEQ ID NOs: 173-257 and an antisense strand comprising a complementary sequence selected from SEQ ID NOs: 258-342, as is also arranged in Table 1, including modifications to the sense sequence and antisense sequences.Attorney Docket No. 809030.000060

[0236] In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 196 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 281. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 238 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 323. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 232 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 317. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 220 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 305. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 195 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 280. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 256 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 341. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 230 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 315. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 192 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 277. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 183 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 268. In some embodiments the sense strand comprises a sequence as set forth in SEQ ID NO: 243 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 328.

[0237] In some embodiments, the sense and antisense strands of the RNAi agents described herein contain different numbers of nucleotides. In some embodiments, the sense strand 3' end and the antisense strand 5' end of an RNAi agent form a blunt end.

[0238] In some embodiments, neither end of an RNAi agent is blunt-ended. As used herein, a blunt end refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands are complementary (form a complementary base-pair).

[0239] In alternate embodiments, the sense strand 5' end and the antisense strand 3' end of an RNAi agent form a blunt end.

[0240] In other embodiments, the sense and antisense strands of the RNAi agents described herein contain the same number of nucleotides. In some embodiments, both ends of an RNAi agent form blunt ends.Attorney Docket No. 809030.000060

[0241] Alternative RNAi agent formats than the RNAi agents specifically exemplified herein are also contemplated. For example, double-stranded RNAi agents of lengths longer or shorter than standard (generally 19-23mer) RNAi agents are contemplated, including RNAi agents having strand lengths up to 50 nucleotides, as well as RNAi agents having strand lengths as short as 12 nucleotides.

[0242] In some embodiments, the length of a duplex formed between a sense and antisense strand of an RNAi agent may be 12 to 15 or 12 to 16 nucleotides (e.g., 12 to 13, 12 to 14, 12 to 15, 12 to 16, 13 to 14, 13 to 15, 13 to 16, 14 to 15, 14 to 16, or 15 to 16 nucleotides) in length. In some embodiments, the length of a duplex formed between a sense and antisense strand of an RNAi agent is at least 12 nucleotides long (e.g., at least 12, at least 13, at least 14, or at least 15 nucleotides long). In some embodiments, the length of a duplex formed between a sense and antisense strand of an RNAi agent is 12, 13, 14, 15, or 16 nucleotides in length (see, e.g., U. S. Publication No. 2023 / 0242912).

[0243] In some embodiments, an RNAi agent provided herein comprises an antisense strand that is up to 50 nucleotides in length (e.g., up to 30, up to 27, up to 25, up to 21, or up to 19 nucleotides in length). In some embodiments, an RNAi agent provided herein comprises an antisense strand is at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, or at least 27 nucleotides in length). In some embodiments, an antisense strand of an RNAi agent disclosed herein is in the range of 12 to 50 or 12 to 30 (e.g., 12 to 30, 11 to 27, 11 to 25, 15 to 21, 15 to 27, 17 to 21, 17 to 25, 19 to 27, or 19 to 30) nucleotides in length. In some embodiments, an antisense strand of any one of the RNAi agents disclosed herein is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0244] In some embodiments, an RNAi agent may have a sense strand of up to 40 nucleotides in length (e.g., up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19 up to 17, or up to 12 nucleotides in length). In some embodiments, an RNAi agent may have a sense strand of at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, an RNAi agent may have a sense strand in a range of 12 to 50 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, an RNAiAttorney Docket No. 809030.000060agent may have a sense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, a sense strand of an RNAi agent is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides). In some embodiments, a sense strand of an RNAi agent is longer than 25 nucleotides (e.g., 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides). In some embodiments, the sense strand is 20 nucleotides in length. In some embodiments, the sense strand is 36 nucleotides in length.

[0245] Hairpin and other RNAi agent structures (e g., structures where one of the strands includes a thermodynamically-stabilizing tetraloop structure (see, e.g., U. S. Pat. Nos. 8,513,207 and 8,927,705, as well as International Patent Application Publication W02010033225, which are incorporated by reference herein for their disclosure of the structures and form of these RNAi agents) are also expressly contemplated. In some embodiments, a sense strand comprises a stemloop at its 3 '-end. In some embodiments, a sense strand comprises a stem-loop at its 5 '-end. In some embodiments, a strand comprising a stem loop is in the range of 2 to 66 nucleotides long (e.g., 2 to 66, 10 to 52, 14 to 40, 2 to 30, 4 to 26, 8 to 22, 12 to 18, 10 to 22, 14 to 26, or 14 to 30 nucleotides long). In some embodiments, a strand comprising a stem loop is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, a stem comprises a duplex of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In some embodiments, a stem-loop provides the molecule better protection against degradation (e.g., enzymatic degradation) and facilitates targeting characteristics for delivery to a target cell. For example, in some embodiments, a loop provides added nucleotides on which modification can be made without substantially affecting the gene expression inhibition activity of an RNAi agent. In certain embodiments, an RNAi agent is provided herein in which the sense strand comprises (e.g., at its 3'-end) a stem-loop set forth as: S1-L-S2, in which SI is complementary to S2, and in which L forms a loop between S I and S2 of up to 10 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length).

[0246] In some embodiments, a loop (L) of a stem-loop is a tetraloop (e g., within a nicked tetraloop structure). A tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a tetraloop has 4 to 5 nucleotides. However, in some embodiments, a tetraloop comprises or consists of 3 to 6 nucleotides, and typically consistsAttorney Docket No. 809030.000060of 4 to 5 nucleotides. In certain embodiments, a tetraloop comprises or consists of three, four, five, or six nucleotides.

[0247] Other RNAi agent designs contemplated for use with the compositions and methods disclosed herein include: 16-mer active dsRNAs (see, e.g., Nucleic Acids in Chemistry and Biology, Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., having 19 bp or shorter stems; see, e.g., Moore et al. Methods Mol. Biol., 2010, 629:141-158), blunt siRNAs (e.g., of 19 bps in length; see, e.g., Kraynack and Baker, R N A, 2006, 12:163-176), asymmetrical siRNAs (aiRNA; see, e.g., Sun et al., Nat. Biotechnol., 2008, 26:1379-1382), asymmetric shorter-duplex siRNA (see, e.g., Chang et al., Mol Then, 2009, 17(4):725-32), fork siRNAs (see, e.g., Hohjoh, FEBS Letters, 2004, 557(1-3): 193-198), single-stranded siRNAs (Elsner, Nature Biotechnology, 2012, 30:1063), dumbbell-shaped circular siRNAs (see, e.g., Abe et al., J Am Chem Soc., 2007, 129:15108-15109), and small internally segmented interfering RNA (see, e.g., Bramsen et al., Nucleic Acids Res., 2007, 35(17):5886-5897). Each of the foregoing references is incorporated by reference in its entirety for the related disclosures therein. Further non-limiting examples of RNAi agent structures that may be used in some embodiments to reduce or inhibit the expression of FXI are microRNA (miRNA), short hairpin RNA (shRNA), and short siRNA (see, e.g., Hamilton et al., EMBO J., 2002, 21(17):4671-4679; see also U. S. patent publication no.20090099115).

[0248] It should be appreciated that, in some embodiments, sequences presented in the sequence listing may be referred to in describing the structure of an RNAi agent or other nucleic acid. In such embodiments, the actual RNAi agent or other nucleic acid may have one or more alternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and / or one or more modified nucleotides and / or one or more modified internucleotide linkages and / or one or more other modification compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

[0249] In some embodiments, there is one or more (e.g., 1, 2, 3, 4, 5) mismatches between a sense and antisense strand. If there is more than one mismatch between a sense and antisense strand, they may be positioned consecutively (e.g., 2, 3 or more in a row), or interspersed throughout the region of complementarity. In some embodiments, the 3 '-terminus of the senseAttorney Docket No. 809030.000060strand contains one or more mismatches. In one embodiment, two mismatches are incorporated at the 3' terminus of the sense strand.

[0250] In some embodiments, an RNAi agent disclosed herein may include a region of complementarity comprising any one of the antisense sequences in any one of SEQ ID NOs: 88- 172. In some embodiments, an RNAi agent may include a region of complementarity consisting of any one of the antisense sequences in any one of SEQ ID NOs: 88-172.RNAi Agent Modifications

[0251] The RNAi agents disclosed herein may be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation, immunogenicity, base-paring properties, RNA distribution and cellular uptake and other features relevant to therapeutic or research use (see, e g., Bramsen et al., Nucleic Acids Res., 2009, 37:2867-2881; Bramsen and Kjems, Frontiers in Genetics, 2012, 3:1-22). Accordingly, in some embodiments, RNAi agents of the present disclosure may include one or more suitable modifications. In some embodiments, a modified nucleotide has a modification in its base (or nucleobase), the sugar (e.g., ribose, deoxyribose), or the phosphate group.The number of modifications on an RNAi agent and the positions of those nucleotide modifications may influence the properties of an RNAi agent. For example, RNAi agents maybe be delivered in vivo by conjugating them to or encompassing them in a lipid nanoparticle (LNP) or similar carrier. However, when an RNAi agent is not protected by an LNP or similar carrier, it may be advantageous for at least some of the nucleotides to be modified. Accordingly, in certain embodiments of any of the RNAi agents provided herein, all or substantially all the nucleotides of an RNAi agent are modified. In certain embodiments, more than half of the nucleotides are modified. In certain embodiments, less than half of the nucleotides are modified. Typically, with naked RNAi agent delivery, every sugar is modified at the 2'-position. These modifications may be reversible or irreversible. In some embodiments, an RNAi agent as disclosed herein has a number and type of modified nucleotides sufficient to cause the desired characteristic (e.g.,Attorney Docket No. 809030.000060protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and / or thermodynamic stability).Sugar Modifications

[0252] In some embodiments, a modified sugar (also referred herein to a sugar analog) includes a modified deoxyribose or ribose moiety, e.g., in which one or more modifications occur at the 2', 3', 4', and / or 5' carbon position of the sugar. In some embodiments, a modified sugar may also include non-natural alternative carbon structures such as those present in locked nucleic acids (“LNA”) (see, e.g., Koshkin et al., Tetrahedron, 1998, 54:3607-3630), unlocked nucleic acids (“UNA”) (see, e.g., Snead et al., Molecular Therapy — Nucleic Acids, 2013, 2, el03), and bridged nucleic acids (“BNA”) (see, e.g., Imanishi and Obika, The Royal Society of Chemistry, Chem. Commun., 2002, 16:1653-1659). Koshkin et al., Snead et al., and Imanishi and Obika are incorporated by reference herein for their disclosures relating to sugar modifications.

[0253] In some embodiments, a nucleotide modification in a sugar comprises a 2'-modifi cation. A 2'-modification may be 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2 '-O-m ethoxy ethyl, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid. Typically, the modification is 2'-fluoro or 2'-O-methyl. In some embodiments a modification in a sugar comprises a modification of the sugar ring, which may comprise modification of one or more carbons of the sugar ring. For example, a modification of a sugar of a nucleotide may comprise a 2'-oxygen if a sugar is linked to a 1 '-carbon or 4'-carbon of the sugar, or a 2'-oxygen is linked to the 1 '-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, a modified nucleotide has an acyclic sugar that lacks a 2'-carbon to 3 '-carbon bond. In some embodiments, a modified nucleotide has a thiol group, e.g., in the 4' position of the sugar.

[0254] In some embodiments, the RNAi agent described herein comprises at least one modified nucleotide (e.g., at least 1, at least 5, 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, or more). In some embodiments, the sense strand of the RNAi agent comprises at least one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more). In some embodiments, the antisense strand of the RNAi agent comprises at least one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).

[0255] In some embodiments, all the nucleotides of the sense strand of the RNAi agent are modified. In some embodiments, all the nucleotides of the antisense strand of the RNAi agent areAttorney Docket No. 809030.000060modified. Tn some embodiments, all the nucleotides of the RNAi agent (i.e., both the sense strand and the antisense strand) are modified. In some embodiments, the modified nucleotide comprises a 2'-modification (e.g., a 2'-fluoro or 2'-O-methyl).

[0256] The present disclosure provides RNAi agents having different modification patterns. In some embodiments, the modified RNAi agents comprise a sense strand sequence having a sequence as set forth in any one of SEQ ID NOs: 173-257, and an antisense strand sequence having a sequence as set forth in any one of SEQ ID NOs: 258-342. In some embodiments, for these RNAi agents, one or more of positions 1-4, 6, and 10-19 of the sense strand, or one or more of positions 1-6, 8, and 12-21 of the sense strand, and / or one or more of positions 1, 3-5, 7, 10-13, 15, and 17-21, or one or more of positions 1, 3-5, 7, 10-13, 15, and 17-23 of the antisense strand are modified with a 2'-O-methyl. In some embodiments, all of positions 1-4, 6, and 10-19 of the sense strand, and all of positions 1, 3-5, 7, 10-13, 15, and 17-21 of the antisense strand are modified with a 2'-O-methyl. In some embodiments, all of positions 1-6, 8, and 12-21 of the sense strand, and all of positions 1, 3-5, 7, 10-13, 15, and 17-23 of the antisense strand are modified with a 2'-O-methyl. In some embodiments, one or more of positions 5 and 7-9 of the sense strand, or one or more of positions 7, and 9-11 of the sense strand, and / or one or more of positions 2, 6, 8, 9, 14, and 16 of the antisense strand are modified with a 2'-fluoro. In some embodiments, all of positions 5 and 7-9 of the sense strand, and all of positions 2, 6, 8, 9, 14, and 16 of the antisense strand are modified with a 2'-fluoro. In some embodiments, all of positions 7, and 9-11 of the sense strand, all of positions 2, 6, 8, 9, 14, and 16 of the antisense strand are modified with a 2'-fluoro. In some embodiments, all of positions 1-4, 6, and 10-19 of the sense stand and all of positions 1, 3-5, 7, 10-13, 15, and 17-21 of the antisense strand are modified with a 2'-O-methyl, and all of positions 5 and 7-9 of the sense strand, and all of positions 2, 6, 8, 9, 14, and 16 of the antisense strand are modified with a 2'-fluoro. In some embodiments, all of positions 1-6, 8, and 12-21 of the sense stand and all of positions 1, 3-5, 7, 10-13, 15, and 17-23 of the antisense strand are modified with a 2'-O-methyl, and all of positions 7 and 9-11 of the sense strand, and all of positions 2, 6, 8, 9, 14, and 16 of the antisense strand are modified with a 2'-fluoro.

[0257] In certain embodiments, the 5'-terminal nucleotide of the antisense strand is a vinyl phosphonate-modified nucleotide, e.g., a vinyl-phosphonate 2'-OMe-uracil (“vinu” modification).Attorney Docket No. 809030.000060

[0258] In some embodiments, all or substantially all of the nucleotides of an RNAi agent are modified nucleotides. As used herein, an RNAi agent wherein substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides (i.e., unmodified). As used herein, a sense strand wherein substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides. As used herein, an antisense sense strand wherein substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides. In some embodiments, one or more nucleotides of an RNAi agent is a ribonucleotide.

[0259] In some embodiments, the terminal 3 '-end group (e.g., a 3 '-hydroxyl) can be modified with a phosphate group or other group, which can be used, for example, to attach linkers, adapters or labels or for the direct ligation of an oligonucleotide to another nucleic acid.5' Terminal Phosphates

[0260] In some embodiments, 5 '-terminal phosphate groups of RNAi agents enhance the interaction with Argonaute 2 or Argonaute 3. However, RNAi agents comprising a 5'-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo. In some embodiments, RNAi agents include analogs of 5' phosphates that are resistant to such degradation. In some embodiments, a phosphate analog may be oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 5' end of an oligonucleotide strand of an RNAi agent is attached to chemical moiety that mimics the electrostatic and steric properties of a natural 5 '-phosphate group (“phosphate mimic”) (see, e.g., Prakash et al., Nucleic Acids Res., 2015, 43(6):2993-3011, the contents of which relating to phosphate analogs are incorporated herein by reference). Many phosphate mimics have been developed that can be attached to the 5' end (see, e.g., U. S. Pat. No. 8,927,513, the contents of which relating to phosphate analogs are incorporated herein by reference). Other modifications have been developed for the 5' end of oligonucleotides (see, e.g., International Patent Application Publication No. WO2011133871, the contents of which relating to phosphate analogs are incorporated herein by reference). In certain embodiments, a hydroxyl group is attached to the 5' end of the oligonucleotide.Attorney Docket No. 809030.000060

[0261] In some embodiments, an RNAi agent has a phosphate analog at a 4'-carbon position of the sugar (referred to as a “4'-phosphate analog”) (see, e.g., International Patent Application Publication No. WO2018045317, entitled 4'-Phosphate Analogs and Oligonucleotides Comprising the Same, which content relating to phosphate analogs is incorporated herein by reference). In some embodiments, an RNAi agent provided herein comprises a 4'-phosphate analog at a 5 '-terminal nucleotide. In some embodiments, a phosphate analog is an oxymethylphosphonate in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its d'carbon) or analog thereof. In other embodiments, a 4'-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bound to the 4'-carbon of the sugar moiety or analog thereof. In certain embodiments, a 4'-phosphate analog is an oxymethylphosphonate. In some embodiments, an oxymethylphosphonate is represented by the formula — O — CH2 — PO(OH)2 or — O — CH2 — PO(OR)2, in which R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3, or a protecting group. In certain embodiments, the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3.

[0262] In certain embodiments, a phosphate analog attached to the RNAi agent is a methoxy phosphonate (MOP). In certain embodiments, a phosphate analog attached to the RNAi agent is a 5' mono-methyl protected MOP. In some embodiments, the following uridine nucleotide comprising a phosphate analog may be used, e.g., at the first position of a guide (antisense) strand, which modified nucleotide is referred to as [MePhosphonate-4O-mU] or 5 '-Methoxy, Phosphonate-4’oxy-2'-O-methyluridine.Modified Internucleotide Linkages

[0263] In some embodiments, phosphate modifications or substitutions may result in an RNAi agent comprising at least one (e.g., at least 1, at least 2, at least 3 or at least 5) modified internucleotide linkage. In some embodiments, any one of the RNAi agents disclosed herein comprises 1 to 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3 or 1 to 2) modified internucleotide linkages. In some embodiments, any one of the RNAi agents disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.Attorney Docket No. 809030.000060

[0264] A modified internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage or a boranophosphate linkage. In some embodiments, at least one modified intemucleotide linkage of any one of the RNAi agents as disclosed herein is a phosphorothioate linkage.

[0265] In some embodiments, the RNAi agent described herein has a phosphorothioate linkage between one or more of positions 1 and 2 of the sense strand, positions 2 and 3 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 19 and 20 of the antisense strand, positions 20 and 21 of the antisense strand, positions 21 and 22 of the antisense strand, and positions 22 and 23 of the antisense strand. In some embodiments, the RNAi agent described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, positions 2 and 3 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 19 and 20 of the antisense strand, and positions 20 and 21 of the antisense strand. In other embodiments, the RNAi agent described herein has a phosphorothioate linkage between each of: positions 1 and 2 of the sense strand, positions 2 and 3 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 21 and 22 of the antisense strand, and positions 22 and 23 of the antisense strand.

[0266] In some embodiments, one or more nucleotides of an FXI RNAi agent are linked by non-standard linkages or backbones (i.e., modified internucleoside linkages or modified backbones). Modified internucleoside linkages or backbones include, but are not limited to, 5'-phosphorothioate groups (represented herein as a lower case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3 '-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, a modified internucleoside linkage or backbone lacks a phosphorus atom. Modified intemucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-Attorney Docket No. 809030.000060sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some embodiments, modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.

[0267] In some embodiments, a sense strand of an FXI RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of an FXI RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, a sense strand of an FXI RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, an antisense strand of an FXI RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.

[0268] In some embodiments, an FXI RNAi agent sense strand contains at least two phosphorothioate internucleoside linkages. In some embodiments, the at least two phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, the at least two phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6-8 from the 5' end of the sense strand. In some embodiments, an FXI RNAi agent antisense strand contains four phosphorothioate intemucleoside linkages. In some embodiments, the four phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the antisense strand and between the nucleotides at positions 19-21, 20-22, or 21-23 from the 5' end. In some embodiments, an FXI RNAi agent contains at least two phosphorothioate intemucleoside linkages in the sense strand and three or four phosphorothioate intemucleoside linkages in the antisense strand.

[0269] In some embodiments, an FXI RNAi agent contains one or more modified nucleotides and one or more modified intemucleoside linkages. In some embodiments, a 2'-modified nucleoside is combined with modified intemucleoside linkage.Attorney Docket No. 809030.000060Base Modifications

[0270] In some embodiments, RNAi agents provided herein have one or more modified nucleobases. In some embodiments, modified nucleobases (also referred to herein as base analogs) are linked at the 1' position of a nucleotide sugar moiety. In certain embodiments, a modified nucleobase is a nitrogenous base. In certain embodiments, a modified nucleobase does not contain nitrogen atom (see, e.g., U. S. Patent Application Publication No. 20080274462). In some embodiments, a modified nucleotide comprises a universal base. However, in certain embodiments, a modified nucleotide does not contain a nucleobase (abasic).

[0271] In some embodiments a universal base is a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a duplex, can be positioned opposite more than one type of base without substantially altering structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., oligonucleotide) that is fully complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower Tm than a duplex formed with the complementary nucleic acid. However, in some embodiments, compared to a reference singlestranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid comprising the mismatched base.

[0272] Non-limiting examples of universal -binding nucleotides include inosine, 1- -D-ribofuranosyl-5-nitroindole, and / or l-P-D-ribofuranosyl-3-nitropyrrole (U. S. patent publication no. 20070254362; Van Aerschot et al., Nucleic Acids Res. 1995, 23(21):4363-70; Loakes et al., Nucleic Acids Res. 1995, 23(13):2361-6; Loakes and Brown, Nucleic Acids Res., 1994, 22(20):4039-43. Each of the foregoing is incorporated by reference herein for their disclosures relating to base modifications).Reversible Modifications

[0273] While certain modifications to protect an RNAi agent from the in vivo environment before reaching target cells can be made, they can reduce the potency or activity of the RNAi agentAttorney Docket No. 809030.000060once it reaches the cytosol of the target cell. Reversible modifications can be made such that the molecule retains desirable properties outside of the cell, which are then removed upon entering the cytosolic environment of the cell. Reversible modification can be removed, for example, by the action of an intracellular enzyme or by the chemical conditions inside of a cell (e.g., through reduction by intracellular glutathione).

[0274] In some embodiments, a reversibly modified nucleotide comprises a glutathionesensitive moiety. Typically, nucleic acid molecules have been chemically modified with cyclic disulfide moieties to mask the negative charge created by the intemucleotide diphosphate linkages and improve cellular uptake and nuclease resistance (see, e.g., U. S. patent publication 20110294869, International Patent Application Publication No. WO2015188197; Meade et al., Nature Biotechnology, 2014, 32:1256-1263; International Patent Application Publication No. W02014088920; each of which are incorporated by reference for their disclosures of such modifications). This reversible modification of the internucleotide diphosphate linkages is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g., glutathione). Earlier examples include neutralizing phosphotriester modifications that were reported to be cleavable inside cells (Dellinger et al., J. Am. Chem. Soc., 2003, 125:940-950).

[0275] In some embodiments, such a reversible modification allows protection during in vivo administration (e.g., transit through the blood and / or lysosomal / endosomal compartments of a cell) where the RNAi agent will be exposed to nucleases and other harsh environmental conditions (e.g., pH). When released into the cytosol of a cell where the levels of glutathione are higher compared to extracellular space, the modification is reversed, and the result is a cleaved RNAi agent oligonucleotide. Using reversible, glutathione sensitive moieties, it is possible to introduce sterically larger chemical groups into the RNAi agent of interest as compared to the options available using irreversible chemical modifications. This is because these larger chemical groups will be removed in the cytosol and, therefore, should not interfere with the biological activity of the RNAi agent inside the cytosol of a cell. As a result, these larger chemical groups can be engineered to confer various advantages to the nucleotide or RNAi agent, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the glutathione-sensitive moiety can be engineered to modify the kinetics of its release.Attorney Docket No. 809030.000060

[0276] In some embodiments, a glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, a glutathione-sensitive moiety is attached to the 2'carbon of the sugar of a modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5 '-carbon of a sugar, particularly when the modified nucleotide is the 5 '-terminal nucleotide of an oligonucleotide of the RNAi agent. In some embodiments, the glutathione-sensitive moiety is located at the 3 '-carbon of sugar, particularly when the modified nucleotide is the 3 '-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group (see, e.g., International Patent Application Publication No. WO2018039364, the contents of which are incorporated by reference herein for its relevant disclosures).

[0277] In some embodiments, the FXI RNAi agents disclosed herein target an FXI gene. In some embodiments, the antisense strand of an FXI RNAi agent disclosed herein includes a sequence or subsequence that is fully, substantially, or at least partially complementary to a target FXI. For the RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (from 5' end— >3' end) can be perfectly complementary to the FXI gene, or can be non-complementary to the FXI gene. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5' end— >3' end) is a U, A, or dT. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5' end 3' end) forms an A: U or U: A base pair with the sense strand.

[0278] In some embodiments, an FXI RNAi agent antisense strand comprises the sequence of nucleotides (from 5' end— >3' end) of any of the antisense strand sequences in Table 1 or Table 2. In some embodiments, an FXI RNAi sense strand comprises the sequence of nucleotides (from 5' end— >3' end) of any of the sense strand sequences in Table 1 or Table 2.

[0279] In some embodiments, an FXI RNAi agent is comprised of (i) an antisense strand comprising the sequence of nucleotides (from 5' end^-3' end) of any of the antisense strand sequences in Table 1 or Table 2, and (ii) a sense strand comprising the sequence of nucleotides (from 5' end— >-3' end) of any of the sense strand sequences in Table 1 or Table 2.

[0280] In some embodiments, the FXI RNAi agents include 21 / 23-mer and 19 / 21-mer nucleotide sequences shown in Table 1 and Table 2.

[0281] The FXI RNAi agent sense strands and antisense strands that comprise or consist of the nucleotide sequences in Table 1 can be modified nucleotides or unmodified nucleotides. In some embodiments, the FXI RNAi agents having the sense and antisense strand sequences thatAttorney Docket No. 809030.000060comprise or consist of any of the nucleotide sequences in Table 2 are all or substantially all modified nucleotides.

[0282] In some embodiments, the antisense strand of an FXI RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 1 or Table 2. In some embodiments, the sense strand of an FXI RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 1 or Table 2.

[0283] Certain modified FXI RNAi agent sense and antisense strands are provided in Table 1. Modified FXI RNAi agent antisense strands, as well as their underlying unmodified nucleobase sequences, are provided in Table 2 and Table 1, respectively. Modified FXI RNAi agent sense strands, as well as their underlying unmodified sequences, are provided in Table 2 and Table 1, respectively.

[0284] In some embodiments, an FXI RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 1 or Table 2.

[0285] In some embodiments, an FXI RNAi agent comprises or consists of a duplex having the nucleobase sequences of the sense strand and the antisense strand of any of the sequences in Table 1 or Table 2.

[0286] Examples of antisense strands containing modified nucleotides are provided in Table 1. Examples of sense strands containing modified nucleotides are provided in Table 2.

[0287] As used in Table 2, the following notations are used to indicate modified nucleotides, targeting groups, and linking groups. As the person of ordinary skill in the art would readily understand, unless otherwise indicated by the sequence, that when present in an oligonucleotide, the monomers are mutually linked by 5 '-3 '-phosphodiester bonds:A=adenosine-3 '-phosphate;C=cytidine-3 '-phosphate;G=guanosine-3 '-phosphate;U=uridine-3 '-phosphaten=any 2'-0Me modified nucleotidea=2'-O-methyladenosine-3 '-phosphateas =2'-O-methyladenosine-3 '-phosphorothioatec=2'-O-methylcytidine-3 '-phosphatecs=2'-O-methylcytidine-3 '-phosphorothioateAttorney Docket No. 809030.000060g=2'-O-methylguanosine-3 '-phosphategs=2'-O-methylguanosine-3'-phosphorothioatet=2'-O-methyl-5-methyluridine-3 '-phosphatets=2'-O-methyl-5-methyluridine-3 '-phosphorothioateu=2'-O-methyluridine-3 '-phosphateus=2'-O-methyluridine-3 '-phosphorothioateNf=any 2'-fluoro modified nucleotideAf=2 '-fl uoroadenosine-3 '-phosphateAfs=2'-fluoroadenosine-3'-phosporothioateCf=2'-fluorocytidine-3 '-phosphateCfs=2'-fluorocytidine-3 '-phosphorothioateGf=2 '-fluoroguanosine-3 '-phosphateGfs=2 '-fluoroguanosine-3 '-phosphorothioateTf=2'-fluoro-5'-methyluridine-3 '-phosphateTfs=2'-fluoro-5'-methyluridine-3 '-phosphorothioateUf=2'-fluorouridine-3 ’-phosphateUfs=2'-fluorouridine-3 '-phosphorothioatedN=any 2'-deoxyribonucleotidedT=2'-deoxythymidine-3 '-phosphates=phosphorothioate linkage(vinu)=vinyl-phosphonate 2'-0Me-uracil

[0288] The person of ordinary skill in the art would readily understand that the terminal nucleotide at the 3' end of a given oligonucleotide sequence would typically have a hydroxyl ( — OH) group at the respective 3' position of the given monomer instead of a phosphate moiety ex vivo. Unless expressly indicated otherwise herein, such understandings of the person of ordinary skill in the art are used when describing the FXI RNAi agents and compositions of FXI RNAi agents disclosed herein.

[0289] In some embodiments, an FXI RNAi agent comprises any of the duplexes represented by any of the Duplex ID Nos. presented herein. In some embodiments, an FXI RNAi agent consists of any of the duplexes represented by any of the Duplex ID Nos. presented herein. In some embodiments, an FXI RNAi agent comprises the sense strand and antisense strandAttorney Docket No. 809030.000060nucleotide sequences of any of the duplexes represented by any of the Duplex ID Nos. presented herein. In some embodiments, an FXI RNAi agent includes the sense strand and antisense strand nucleotide sequences of any of the duplexes represented by any of the Duplex ID Nos. presented herein and a targeting group and / or linking group, wherein the targeting group and / or linking group is covalently linked (i.e., conjugated) to the sense strand or the antisense strand. In some embodiments, an FXI RNAi agent includes the sense strand and antisense strand modified nucleotide sequences of any of the duplexes represented by any of the Duplex ID Nos. presented herein. In some embodiments, an FXI RNAi agent comprises the sense strand and antisense strand modified nucleotide sequences of any of the duplexes represented by any of the Duplex ID Nos. presented herein and a targeting group and / or linking group, wherein the targeting group and / or linking group is covalently linked to the sense strand or the antisense strand.

[0290] In some embodiments, an FXI RNAi agent comprises, consists of, or consists essentially of, the duplex structure of any of the duplexes in Table 1 and Table 2.Synthesis of RNAi Agents

[0291] The FXI RNAi agent sense strands and antisense strands can be synthesized and / or modified by methods known in the art. In particular, RNAi agents of the disclosure may be obtained using a number of techniques known to those of skill in the art. For example, the RNAi agents disclosed herein may be chemically synthesized or may be encoded by plasmid (e.g., transcribed as sequences that fold into duplexes having hairpin loops.). RNAi agents may also be generated by cleavage of longer dsRNAs (e.g., a dsRNA >25 nucleotides in length) by either E. coli RNase II or Dicer. These enzymes process the dsRNA into biologically active RNAi agent (see e.g., Byron et al. Ambion Tech Notes; 10 (1 ):4-6 (2009); Calegari et al. PNAS USA 99:14236 (2002); Kawaski et al., Nucleic Acids Res., 31:981-987 (2003); Knight and Bass, Science, 293:2269-2271 (2001); Roberston et al., J. Biol. Chem 243:82(1969); and Yang et al., PNAS USA 99:9942-9947 (2002).

[0292] In embodiments, RNAi agents of the disclosure may be chemically synthesized. Oligonucleotides (e.g., modified oligonucleotides or portions thereof lacking ribonucleotides) may be synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99 / 54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and U. S. Pat. No.Attorney Docket No. 809030.0000606,001,311- The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 '-end, and phosphoramidites at the 3 '-end.

[0293] RNAi agent molecules without modifications may be synthesized using procedures as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433. These syntheses make use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 '-end, and phosphoramidites at the 3 '-end that can be used for certain RNAi agent molecules of the disclosure.

[0294] In certain embodiments, the RNAi agent molecules of the disclosure may be synthesized, deprotected, and analyzed according to methods described in U. S. Pat. Nos.6,995,259, 6,686,463, 6,673,918, 6,649,751, 6,989,442, and 7,205,399.

[0295] In a non-limiting synthesis example, small scale syntheses may be conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 pmol scale protocol with a 2.5 min coupling step for 2'-O-methylated nucleotides and a 45 second coupling step for 2'-deoxy nucleotides or 2'-deoxy -2 '-fluoro nucleotides.

[0296] Alternatively, the RNAi agent molecules of the present disclosure may be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International Paten Application Publication No. WO 93 / 23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and / or deprotection.

[0297] For each oligonucleotide of a target sequence, the two individual, complementary strands of the RNAi agent are synthesized separately using solid phase synthesis, then purified separately by reversed phase solid phase extraction (SPE). The complementary strands are annealed to form the double strand (duplex RNAi agent) and delivered in the desired concentration and buffer of choice.

[0298] In embodiments, single strand oligonucleotides are synthesized using phosphoramidite chemistry on an automated solid-phase synthesizer, using procedures as are generally known in the art (see for example U. S. Patent Application Publication No. US20090176725). A synthesis column is packed with solid support derivatized with the first nucleoside residue (natural or chemically modified). Synthesis is initiated by detritylation of the acid labile 5'-O-dimethoxytrityl group to release the 5 '-hydroxyl. A suitably protectedAttorney Docket No. 809030.000060phosphorami dite and a suitable activator in acetonitrile are delivered simultaneously to the synthesis column resulting in coupling of the amidite to the 5'-hydroxyl. The column is then washed with a solvent, such as acetonitrile. An oxidizing solution, such as an iodine solution is pumped through the column to oxidize the phosphite triester linkage P(III) to its phosphotriester P(V) analog. Unreacted 5 '-hydroxyl groups are capped using reagents such as acetic anhydride in the presence of 2,6-lutidine and N-methylimidazole. The elongation cycle is resumed with the detritylation step for the next phosphoramidite incorporation. This process is repeated until the desired sequence is synthesized. The synthesis concludes with the final 5 '-terminus protecting group (trityl or 5'-O-dimethoxytrityl).

[0299] Upon completion of the synthesis, the solid-support and associated oligonucleotide may be dried under argon pressure or vacuum. Aqueous base may be added and the mixture heated to effect cleavage of the succinyl linkage, removal of the cyanoethyl phosphate protecting group, and deprotection of the exocyclic amine protection.

[0300] The techniques herein provide that the following process may be performed on single strands that do not contain ribonucleotides. After treating the solid support with the aqueous base, the mixture may be filtered to separate the solid support from the deprotected crude synthesis material. The solid support may be then rinsed with water, which may be combined with the filtrate. The resultant basic solution allows for retention of the 5'-O-dimethoxytrityl group to remain on the 5' terminal position (trityl-on).

[0301] For single strands that contain ribonucleotides, the following process may be performed. After treating the solid support with the aqueous base, the mixture may be filtered to separate the solid support from the deprotected crude synthesis material. The solid support may be then rinsed with dimethyl sulfoxide (DMSO), which may be combined with the filtrate. Fluoride reagent, such as triethylamine trihydrofluoride, is added to the mixture, and the solution may be heated. The reaction is quenched with suitable buffer to provide a solution of crude single strand with the 5'-O-dimethoxytrityl group on the final 5' terminal position.

[0302] The trityl-on solution of each crude single strand may be purified using chromatographic purification, such as SPE RPC purification. The hydrophobic nature of the trityl group permits stronger retention of the desired full-length oligo than the non-tritylated truncated failure sequences. The failure sequences may be selectively washed from the resin with a suitable solvent, such as low percent acetonitrile. Retained oligonucleotides may be then detritylated on-Attorney Docket No. 809030.000060column with trifluoroacetic acid to remove the acid-labile trityl group. Residual acid is washed from the column, a salt exchange is performed, and a final desalting of the material commenced. The full-length oligo is recovered in a purified form with an aqueous-organic solvent. The final product may be then analyzed for purity (HPLC), identity (Maldi-TOF MS), and yield (UV A260). The oligos are dried via lyophilization or vacuum condensation.

[0303] Annealing: Based on the analysis of the product, the dried oligos may be dissolved in appropriate buffers followed by mixing equal molar amounts (calculated using the theoretical extinction coefficient) of the sense and antisense oligonucleotide strands. The solution may be then analyzed for purity of duplex by chromatographic methods and desired final concentration. If the analysis indicates an excess of either strand, then the additional non-excess strand may be titrated until duplexing is complete. When analysis indicates that the target product purity has been achieved the material may be delivered and ready for use.

[0304] Various RNAi agent molecules of the disclosure may also be synthesized using the teachings of Scaringe et al., U. S. Pat. Nos. 5,889,136; 6,008,400; 6,111,086; and 8,394,628. The FXI RNAi agents described herein are ultimately formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 1 or Table 2, can be hybridized to any antisense strand containing a sequence listed in Table 1 or Table 2, provided the two sequences have a region of at least 85% complementarity over a contiguous 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence.

[0305] In some embodiments, a FXI RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. In some embodiments, a FXI RNAi agent is prepared as a sodium salt. Such forms are within the scope of the disclosures disclosed herein.Targeting Ligands

[0306] In some embodiments, it may be desirable to target the RNAi agents of the disclosure to one or more cells or one or more organs. Such a strategy may help to avoid undesirable effects in other organs or may avoid undue loss of the RNAi agent to cells, tissue or organs that would not benefit for the RNAi agent. Accordingly, in some embodiments, RNAi agents disclosed herein may be modified to facilitate targeting of a particular tissue, cell or organ, e.g., to facilitate delivery of the RNAi agent to the liver. In certain embodiments, RNAi agents disclosed herein may be modified to facilitate delivery of the RNAi agent to the hepatocytes of theAttorney Docket No. 809030.000060liver. In some embodiments, an RNAi agent comprises a nucleotide that is conjugated to one or more targeting ligand.

[0307] A targeting ligand may comprise a carbohydrate, amino sugar, cholesterol, peptide, polypeptide, protein or part of a protein (e.g., an antibody or antibody fragment) or lipid. In some embodiments, a targeting ligand is an aptamer.

[0308] In some embodiments, it is desirable to target an oligonucleotide that reduces the expression of FXI to the hepatocytes of the liver of the subject. Any suitable hepatocyte targeting moiety may be used for this purpose. In some embodiments, the present disclosure provides methods of reducing the expression of a target gene in a liver tissue. For example, a liver tissue can include a hepatocyte expressing a target gene. In some embodiments, the present disclosure may include an hepatocyte, where the hepatocyte is a primary hepatocyte, such as, e.g., a mature liver cell that has been isolated directly from living liver tissue of a subject (e.g., a donor).

[0309] GalNAc is a high affinity ligand for asialoglycoprotein receptor (ASGPR), which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to RNAi agents of the instant disclosure may be used to target these RNAi agents to the ASGPR expressed on these hepatocyte cells, as previously disclosed for nucleic acid payloads, e.g., in U. S. Patent No. 6,906,182; U. S. Patent No. 8,106,022; and U. S. Patent Application Publication No. 2005 / 0020525, among others (e.g., the following patent documents describe methods for conjugating nucleic acids like siRNAs to GalNAc, as well as teaching exemplary GalNAc structures: U. S. Patent No. 8,575,123; U. S. Patent Application Publication No. 2009 / 0239814; and U. S. Patent No. 9,708,607. Each of the aforementioned patent documents is incorporated by reference herein in its entirety. In some cases, the GalNAc is a derivative disclosed in U. S. Patent Application Publication No. 2020 / 0270611, the contents of which are incorporated herein by reference in their entirety.)

[0310] In some embodiments, an RNAi agent of the instant disclosure is conjugated directly or indirectly to a monovalent GalNAc. In some embodiments, the RNAi agent is conjugated directly or indirectly to more than one monovalent GalNAc (e.g., is conjugated to 2, 3, or 4 monovalent GalNAc moieties, and is typically conjugated to 3 or 4 monovalent GalNAcAttorney Docket No. 809030.000060moieties). In some embodiments, an RNAi agent of the instant disclosure is conjugated to a one or more bivalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties.

[0311] In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of an RNAi agent are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of the RNAi agent are each conjugated to a separate GalNAc.

[0312] It is expressly contemplated that a triantennary GalNAc structure can be attached to any of the sense strand or antisense strand oligonucleotides of the RNAi agents presented herein. In certain embodiments, a triantennary GalNAc (which can be represented as “(triGalNAc)” in, e.g., Table 2) is added at the 3'-terminal nucleotide residue of the sense strand of an RNAi agent structure disclosed herein. An exemplary triantennary GalNAc moiety of the instant disclosure is shown in the following schematic, attached to the 3'-terminal nucleotide residue of an RNAi agent:

[0313] It is contemplated that other triantennary GalNAc-type modifications as known in the art could also or alternatively be used with an RNAi agent of the instant disclosure. Exemplary such alternative triantennary GalNAc-type modifications are those encompassed by the following formula:Attorney Docket No. 809030.000060wherein X comprises an RNAi agent of the instant disclosure; W comprises a linker molecule or chemical linkage that can be present or absent, each R7 independently comprises an acyl group that can be present or absent, for example an acetyl group, and each n is independently an integer from about 1 to about 20. In another embodiment, W is selected from the group consisting of amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester linkage.

[0314] Other exemplary GalNAc modifications known in the art include, without limitation:3'-(l+l+l) trivalent GalNAcAttorney Docket No. 809030.000060 AcHN5'-triantennary GalNAc3' or 5'-(l+l+l) trivalent C3 Linker GalNAcAttorney Docket No. 809030.0000605'-Simple GalNAc Phosphoramidite5'-C3 Linker GalNAc PhosphoramiditeOAc-C3 Linker GalNAc CPGAttorney Docket No. 809030.0000603' or 5'-(l+l+l) trivalent C3 Linker GalNAcH0... OH H O AGHN HQ. OH AcHN Q HOZOH,, Z L\ n H H HoS^S^°-^XX^-vNX,x~-x„ N' '^0AcHN M3'-Prolinol / C12-linker with attached trivalent GalNAc

[0315] Conjugation can be performed on the sense strand, e.g., 5' end and / or 3' end of the sense strand of the RNAi agent. Conjugation can also be performed on the antisense strand, e.g., 3'end of the antisense strand. In some cases, conjugation can also be performed at the 5' end of the antisense strand. In some embodiments, a conjugate is linked to a nucleotide of an RNAi agent directly, for example, via a phosphodiester bond. In some embodiments, a linker is used for conjugating an RNAi agent to one or more conjugates (e.g., GalNAc). The conjugated RNAi agent may have a formula of A-L-B (A= siRNA; L=linker and B= conjugate). In some embodiments, the linker is a bivalent Ci-Cso saturated or unsaturated, straight or branched alkyl, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1-C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-,Attorney Docket No. 809030.000060S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C:- C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1-C4 alkyl), -C(O)N(cycloalkyl), aryl, heteroaryl, cycloalkyl, or cycloalkenyl. In some embodiments, the linker is a non-cleavable linker. In other embodiments, the linker is a cleavable linker. The linker described herein can be a non-polymeric linker. A non-polymeric linker refers to a linker that does not contain a repeating unit of monomers generated by a polymerization process. Exemplary non-polymeric linkers include, but are not limited to, C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or Cl alkyl group), homobifunctional cross linkers, heterobifunctional cross linkers, peptide linkers, traceless linkers, self-immolative linkers, maleimide-based linkers, or combinations thereof. In some cases, the non-polymeric linker comprises a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or Cl alkyl group), a homobifunctional cross linker, a heterobifunctional cross linker, a peptide linker, a traceless linker, a self-immolative linker, a maleimide-based linker, or a combination thereof. In additional cases, the non-polymeric linker does not comprise more than two of the same type of linkers, e g., more than two homobifunctional cross linkers, or more than two peptide linkers. In further cases, the non-polymeric linker optionally comprises one or more reactive functional groups. In some embodiments, the linker is an aminohexyl linker (e.g., -(CH2)6NH-).

[0316] Appropriate methods or chemistry (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. W02016100401, the contents of which relating to such linkers are incorporated herein by reference. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. A “labile linker” refers to a linker that can be cleaved, e.g., by acidic pH. A “stable linker” refers to a linker that cannot be cleaved.

[0317] Exemplary U. S. Patents that teach the preparation of oligonucleotide 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; 5,486,603; 5,512,439; 4,824,941; 4,835,263; 5,112,963; 5,214,136; 5,578,717; 5,580,731; 5,578,718; 5,608,046; 4,876,335; 4,904,582; 5,245,022; 5,254,469; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 4,587,044; 4,605,735;Attorney Docket No. 809030.0000604,667,025; 4,762,779; 4,789,737; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5; 5,597,696; 5,599,923; 5,599,928;5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are hereby incorporated herein by reference. See also, e.g., U. S. Patent No. 10,975,388, the entire contents of which are also hereby incorporated herein by reference.

[0318] In some embodiments, a duplex extension (e.g., of up to 3, 4, 5, or 6 base pairs in length) is provided between a targeting ligand (e.g., a GalNAc moiety) and a double-stranded oligonucleotide.

[0319] According to some embodiments of the present disclosure, provided is a double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises a sense strand and an antisense strand, wherein said sense strand comprises at least 14 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 3-87, wherein a substitution of a uracil for any thymine in SEQ ID NOs: 3-87 does not count as a difference that contributes to said differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs 3-87; and wherein said antisense strand comprises at least 14 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 88-172, wherein a substitution of a uracil for any thymine in SEQ ID NOs: 88-172 does not count as a difference that contributes to said differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 88-172, wherein at least one of said sense strand and said antisense strand comprises one or more tris(GalNAc) moieties conjugated to one or more terminal nucleotide position.Assessment of FXI Levels

[0320] In certain embodiments, RNAi agent-mediated inhibition of a FXI target sequence is assessed. In such embodiments, FXI RNA levels can be assessed by art-recognized methods (e.g., qPCR, RT-PCR, Northern blot, expression array, etc.), optionally via comparison of FXI levels in the presence of an FXI RNAi agent of the disclosure relative to the absence of such an FXI RNAi agent. In certain embodiments, FXI levels in the presence of an FXI RNAi agent areAttorney Docket No. 809030.000060compared to those observed in the presence of vehicle alone, in the presence of an RNAi agent directed against an unrelated target RNA, or in the absence of any treatment.

[0321] It is also recognized that levels of FXI protein can be assessed and that FXI protein levels are, under different conditions, either directly or indirectly related to FXI RNA levels and / or the extent to which an RNAi agent inhibits FXI expression, thus art-recognized methods of assessing FXI protein levels (e.g., Western blot, immunoprecipitation, other antibody-based methods, etc.) can also be employed to examine the inhibitory effect of an RNAi agent of the disclosure.

[0322] In certain embodiments, potency of an RNAi agent of the disclosure is determined in reference to the number of copies of an RNAi agent present in the cytoplasm of a target cell that are required to achieve a certain level of target gene knockdown. For example, in certain embodiments, a potent RNAi agent is one capable of causing 50% or greater knockdown of a target mRNA when present in the cytoplasm of a target cell at a copy number of 1000 or fewer RISC-loaded antisense strands per cell. More preferably, a potent RNAi agent is one capable of producing 50% or greater knockdown of a target mRNA when present in the cytoplasm of a target cell at a copy number of 500 or fewer RISC-loaded antisense strands per cell. Optionally, a potent RNAi agent is one capable of producing 50% or greater knockdown of a target mRNA when present in the cytoplasm of a target cell at a copy number of 300 or fewer RISC-loaded antisense strands per cell.

[0323] In certain embodiments, an RNAi agent of the disclosure is administered in an amount sufficient to reduce FXI target mRNA expression when the RNAi agent is introduced into a mammalian cell. In exemplary embodiments, reduction of FXI target mRNA expression is assessed to have occurred if FXI target mRNA levels are decreased by at least 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, as compared to a corresponding mammalian cell administered an appropriate control that does not include the FXI-targeting RNAi agent of the disclosure. In an exemplary embodiment, the mammalian cell used to determine whether reduction of FXI target mRNA expression has occurred relative to an appropriate control is a Hepal-6 cell, and the FXI-targeting RNAi agent of the disclosure is optionally administered via transfection (e.g., using Lipofectamine™) at a concentration of at or below 1 nM in the environment of a cell, at or belowAttorney Docket No. 809030.000060100 pM in the environment of a cell, at or below 10 pM in the environment of a cell, at or below 1 nM in the environment of a cell, in an in vitro assay as described herein.

[0324] FXI inhibitory levels and / or FXI levels may also be assessed indirectly, e.g., measurement of one or more disease- or disorder-associated phenotypes (e.g., phenotypes of myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, pulmonary embolism, ischemic stroke, transient ischemic attack, retinal artery occlusion, mesenteric ischemia, renal vein thrombosis, cerebral venous sinus thrombosis, arterial thrombosis, venous thrombosis, venous thromboembolism due to deep vein thrombosis, peripheral artery disease, and thrombosis due to artificial surfaces (e.g., catheter associated thrombosis, medical device thrombosis), among other phenotypes) and / or biomarkers in a subject may be used to assess FXI levels and / or FXI inhibitory efficacy of an RNAi agent of the instant disclosure. Without wishing to be bound by theory, in an aspect of the disclosure, an activated partial thromboplastin time (aPTT) test may be used to assess FXI levels and / or FXI inhibitory efficacy of an RNAi agent of the instant disclosure.

[0325] An in vitro assay that recapitulates RNAi in a cell-free system can be used to evaluate RNAi agent constructs targeting FXI RNA sequence(s), and thus to assess FXI-specific gene inhibitory activity (also referred to herein as FXI inhibitory activity) of an RNAi agent. The assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with RNAi agents directed against FXI RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from a selected FXI expressing plasmid using T7 RNA polymerase or via chemical synthesis. Sense and antisense strands of the RNAi agent (for example, 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol / vol], RNA (10-50 pM final concentration), and 10% [vol / vol] lysis buffer containing RNAi agent (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug / mlAttorney Docket No. 809030.000060creatine phosphokinase, 100 urn GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U / uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25° C. for 10 minutes before adding RNA, then incubated at 25° C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25x Passive Lysis Buffer (PROMEGA®). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which RNAi agent is omitted from the reaction.

[0326] Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [a-32P] CTP, passed over a G50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5'-32P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by PHOSPHOR IMAGER® (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without RNAi agent and the cleavage products generated by the assay.

[0327] In one embodiment, this assay is used to determine target sites in the FXI RNA target for RNAi agent-mediated RNAi cleavage, wherein a plurality of RNAi agent constructs are screened for RNAi-mediated cleavage of the FXI RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.

[0328] In certain embodiments, an RNAi agent of the disclosure is deemed to possess FXI inhibitory activity if, e.g., a 50% reduction in FXI RNA levels is observed in a system, cell, tissue or organism, relative to a suitable control. Additional metes and bounds for determination of FXI inhibitory activity of an RNAi agent of the disclosure are described supra.Cell Culture

[0329] The RNAi agents of the disclosure can be tested for cleavage activity in vivo, for example, using the following procedure. The nucleotide sequences of the disclosure are shown in Table 1 and Table 2.

[0330] The RNAi agents of the disclosure can be tested in cell culture using Hepal-6 or other mammalian cells (e g., primary human cells, human cell lines Huh7, Hep3B, HepG2, DU145,Attorney Docket No. 809030.000060Calu3, SW480, T84, PL45, HeLa etc., and mouse cell lines AML12, Neuro2a, etc.) to determine the extent of FXI RNA and FXI protein inhibition. FXI RNA inhibition is measured after delivery of these reagents by a suitable transfection agent or by free uptake to, for example, cultured Hepal-6 cells or other transformed or non-transformed mammalian cells in culture. Relative amounts of target FXI RNA are measured versus an appropriate control using real-time PCR monitoring of amplification (e.g., ABI 7700 TAQMAN®). A comparison is made to the activity of oligonucleotide sequences made to unrelated targets or to a randomized RNAi agent control with the same overall length and chemistry, or simply to appropriate vehicle-treated or untreated controls. Primary and secondary lead reagents are chosen for the target and optimization performed.

[0331] TAQMAN® (Real-Time PCR Monitoring of Amplification) and LIGHTCYCLER™ Quantification of mRNA

[0332] Total RNA is prepared from cells, tissues, and / or subjects following RNAi agent delivery, for example, using AMBION® Rnaqueous 4-PCR purification kit for large scale extractions, or Promega SV96 for 96-well assays. For TAQMAN® analysis, dual-labeled probes are synthesized with, for example, the reporter dyes FAM or VIC covalently linked at the 5 '-end and the quencher dye TAMRA conjugated to the 3 '-end. PCR amplifications are performed on, for example, an ABI PRISM™ 7700 Sequence detector using 50 uL reactions consisting of 10 uL total RNA, 100 nM forward primer, 100 mM reverse primer, 100 nM probe, lxTAQMAN® PCR reaction buffer (PE-APPLIED BIOSYSTEMS®), 5.5 mM MgC12, 100 uM each dATP, dCTP, dGTP and dTTP, 0.2 U RNase Inhibitor (PROMEGA®), 0.025 U AMPLITAQ GOLD™ (PE-APPLIED BIOSYSTEMS®) and 0.2 U M-MLV Reverse Transcriptase (PROMEGA®). The thermal cycling conditions can consist of 30 minutes at 48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. Quantitation of target FXI mRNA level is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 30, 10 ng / rxn) and normalizing in either parallel or same tube TaqMan reactions.Western Blotting

[0333] Cellular protein extracts can be prepared using a standard micro preparation technique (for example using RIPA buffer), or preferably, by extracting nuclear proteins by a method such as the NE-PER Nuclear and Cytoplasmic Extraction kit (THERMO-FISHERAttorney Docket No. 809030.000060SCIENTIFIC®). Cellular protein extracts are run on Tris-Glycine polyacrylamide gel and transferred onto membranes. Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hours at 4° C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected on a VERSADOC™ imaging system.

[0334] In several cell culture systems (including in, e.g., the primary human hepatocyte (PHH) and primary cynomologus hepatocyte (PCH) systems exemplified herein), cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In one embodiment, RNAi agents of the disclosure are complexed with cationic lipids for cell culture experiments. RNAi agent and cationic lipid mixtures are prepared in serum-free OPTIMMEM™ (INVITROGEN®) immediately prior to addition to the cells. OPTIMEM™ is warmed to room temperature (about 20-25° C.) and cationic lipid is added to the final desired concentration. RNAi agents are added to OPTIMEM™to the desired concentration and the solution is added to the diluted RNAi agents and incubated for 15 minutes at room temperature. In dose response experiments, the RNA agent is serially diluted into OPTIMEM™prior to addition of the cationic lipid.Animal Models

[0335] The efficacy of FXI RNAi agents may be evaluated in an animal model. Animal models of FXI-related diseases, conditions, or disorders as are known in the art can be used for evaluation of the efficacy, potency, toxicity, etc. of FXI RNAi agents. These animal models may be used as a source cells or tissue for assays of the compositions of the disclosure. Such models can also be used or adapted for use for pre-clinical evaluation of the efficacy of RNAi agent compositions of the disclosure in modulating FXI gene expression toward therapeutic use, and / or for assessment of the toxicity, or side effects of treatment with an RNAi agent. Alternatively, an RNAi agent can be used in an animal model to determine the mechanism of action of such an agent.

[0336] Such models and / or wild-type mice can be used in evaluating the efficacy of RNAi agents of the disclosure to inhibit FXI levels, expression, development of FXI-associated phenotypes, diseases or disorders, etc. These models, wild-type mice and / or other models canAttorney Docket No. 809030.000060similarly be used to evaluate the safety / toxicity and efficacy of RNAi agent molecules of the disclosure in a pre-clinical setting.

[0337] Specific examples of animal model systems useful for evaluation of the FXI-targeting RNAi agents of the disclosure include C57 / BL6 mice. In an exemplary in vivo experiment, RNAi agents of the disclosure are subcutaneously injected (alternatively or additionally, intravenous or subcutaneous injection of RNAi agents of the disclosure is also contemplated) into such mice at doses ranging from 0.3 to 3 mg / kg or, alternatively, repeated doses are administered at single-dose IC50 levels, and organ samples (e.g., liver, but may also include prostate, kidney, lung, pancreas, colon, skin, spleen, bone marrow, lymph nodes, mammary fat pad, etc.) are harvested 24 hours after administration of the final dose. Such organs are then evaluated for mouse and / or human FXI levels, depending upon the model used. Duration of action can also be examined at, e.g., 1, 4, 7, 14, 21 or more days after final RNAi agent administration.Delivery of FXI RNAi Agents

[0338] In certain embodiments, the present disclosure relates to a method for treating a subject having a FXI-associated disease or disorder, or at risk of developing a FXI-associated disease or disorder. In such embodiments, the RNAi agent can act as novel therapeutic agents for controlling the FXI-associated disease or disorder. The method comprises administering a pharmaceutical composition of the disclosure to the patient (e.g., human), such that the expression, level and / or activity of a FXI RNA is reduced. The expression, level and / or activity of a polypeptide encoded by a FXI RNA might also be reduced by an RNAi agent of the instant disclosure, even where said RNAi agent is directed against a non-coding region of the FXI transcript (e.g., a targeted 5' UTR or 3' UTR sequence). Because of their high specificity, the RNAi agents of the present disclosure can specifically target FXI sequences of cells and tissues, optionally in an allele-specific manner where polymorphic alleles exist within an individual and / or population.

[0339] In the treatment of an FXI-associated disease or disorder, the RNAi agent can be brought into contact with the cells or tissue of a subject, e.g., the cells or tissue of a subject exhibiting disregulation of FXI and / or otherwise targeted for reduction of FXI levels. For example, RNAi agent substantially identical to all or part of a FXI RNA sequence, may be brought into contact with or introduced into such a cell, either in vivo or in vitro. Similarly, RNAi agentAttorney Docket No. 809030.000060substantially identical to all or part of a FXI RNA sequence may be administered directly to a subject having or at risk of developing an FXI-associated disease or disorder.

[0340] Therapeutic use of the RNAi agents of the instant disclosure can involve use of formulations of RNAi agents comprising multiple different RNAi agent sequences. For example, two or more, three or more, four or more, five or more, etc. of the presently described agents can be combined to produce a formulation that, e.g., targets multiple different regions of the FXI RNA, or that not only target FXI RNA but also target, e.g., cellular target genes associated with a FXI-associated disease or disorder. An RNAi agent of the instant disclosure may also be constructed such that either strand of the RNAi agent independently targets two or more regions of FXI RNA, or such that one of the strands of the RNAi agent targets a cellular target gene of FXI known in the art.

[0341] Use of multifunctional RNAi agent molecules that target more than one region of a target nucleic acid molecule can also provide potent inhibition of FXI RNA levels and expression. Additionally, and / or alternatively, single or multifunctional agents of the disclosure can be designed to selectively target one splice variant of FXI over another.

[0342] Thus, the RNAi agents of the instant disclosure, individually, or in combination or in conjunction with other drugs, can be used to treat, inhibit, reduce, or prevent a FXI-associated disease or disorder. For example, the RNAi agents can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

[0343] The RNAi agents also can be used in combination with other known treatments to treat, inhibit, reduce, or prevent an FXI-associated disease or disorder in a subject or organism. For example, the described molecules could be used in combination with one or more known compounds, treatments, or procedures to treat, inhibit, reduce, or prevent a FXI-associated disease or disorder in a subject or organism as are known in the art.

[0344] An RNAi agent of the disclosure can be conjugated (e.g., at its 5' or 3' terminus of its sense or antisense strand) or unconjugated to another moiety (e.g. a non-nucleic acid moiety such as a peptide), or an organic compound (e.g., a dye, cholesterol, or the like). Modifying RNAi agents in this way may improve cellular uptake or enhance cellular targeting activities of the resulting RNAi agent derivative as compared to the corresponding unconjugated RNAi agent, areAttorney Docket No. 809030.000060useful for tracing the RNAi agent derivative in the cell, or improve the stability of the RNAi agent derivative compared to the corresponding unconjugated RNAi agent.Methods of Introducing Nucleic Acids, Vectors, and Host Cells

[0345] RNAi agents of the disclosure may be directly introduced into a cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced.

[0346] The RNAi agents of the disclosure can be introduced using nucleic acid delivery methods known in art including injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and the like. The nucleic acid may be introduced along with other components that perform one or more of the following activities: enhance nucleic acid uptake by the cell or otherwise increase inhibition of the target FXI RNA.

[0347] A cell having a target FXI RNA may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The cell may be a stem cell or a differentiated cell. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands.

[0348] Depending on the particular target FXI RNA sequence and the dose of RNAi agent material delivered, this process may provide partial or complete loss of function for the FXI RNA. A reduction or loss of RNA levels or expression (either FXI RNA expression or encoded polypeptide expression) in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targeted cells is exemplary. Inhibition of FXI RNA levels or expression refers to the absence (or observable decrease) in the level of FXI RNA or FXI RNA-encoded protein. Specificity refers to the abilityAttorney Docket No. 809030.000060to inhibit the FXI RNA without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS). Inhibition of target FXI RNA sequence(s) by the RNAi agents of the disclosure also can be measured based upon the effect of administration of such RNAi agents upon development / progression of an FXI-associated disease or disorder, either in vivo or in vitro.

[0349] The RNAi agent may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of material may yield more effective inhibition; lower doses may also be useful for specific applications.Lipid Nanoparticles (LNPs)

[0350] In certain embodiments, a lipid solution containing a mixture of lipids suitable to form lipid nanoparticles may be used for encapsulation of an RNAi agent. In some embodiments, a suitable lipid solution is ethanol based. For example, a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (e.g., 100% ethanol). In another embodiment, a suitable lipid solution is isopropyl alcohol based. In another embodiment, a suitable lipid solution is dimethylsulfoxide-based. In another embodiment, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol and dimethylsulfoxide.

[0351] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. For example, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of or greater than about 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml, 5.0 mg / ml, 6.0 mg / ml, 7.0 mg / ml, 8.0 mg / ml, 9.0 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, or 100 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration ranging from about 0.1- 100 mg / ml, 0.5-90 mg / ml, 1.0-80 mg / ml, 1.0-70 mg / ml, 1.0-60 mg / ml, 1.0-50 mg / ml, 1.0-40 mg / ml, 1.0-30 mg / ml, 1.0-20 mg / ml, 1.0-15 mg / ml, 1.0-10 mg / ml, 1.0-9 mg / ml, 1.0-8 mg / ml, 1.0-7 mg / ml, 1.0-6 mg / ml, or 1.0-5 mg / ml. In some embodiments, a suitable lipid solution may containAttorney Docket No. 809030.000060a mixture of desired lipids at a total concentration up to about 100 mg / ml, 90 mg / ml, 80 mg / ml, 70 mg / ml, 60 mg / ml, 50 mg / ml, 40 mg / ml, 30 mg / ml, 20 mg / ml, or 10 mg / ml.

[0352] Any desired lipids may be mixed at any ratios suitable for encapsulating RNAi agents. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including cationic lipids, helper lipids (e.g. non cationic lipids and / or cholesterol lipids) and / or PEGylated lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including one or more cationic lipids, one or more helper lipids (e.g. non cationic lipids and / or cholesterol lipids) and one or more PEGylated lipids.

[0353] An exemplary mixture of lipids for use with the disclosure is composed of four lipid components: a cationic lipid, a non-cationic lipid (e.g., DSPC, DPPC, DOPE or DEPE), a cholesterol-based lipid (e.g., cholesterol) and a PEG-modified lipid (e.g., DMG-PEG2K). In some embodiments, the molar ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) may be between about 20-50:25-35:20-50:1-5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 20:30:48.5:1.5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:20: 10, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 50:25:20:5.

[0354] In some embodiments, a mixture of lipids for use with the disclosure may comprise no more than three distinct lipid components. In some embodiments, one distinct lipid component in such a mixture is a cholesterol-based or imidazol-based cationic lipid. An exemplary mixture of lipids may be composed of three lipid components: a cationic lipid (e.g., a cholesterol-based or imidazol-based cationic lipid such as ICE, HGT4001 or HGT4002), a non-cationic lipid (e.g., DSPC, DPPC, DOPE or DEPE) and a PEG-modified lipid (e g., DMG-PEG2K). The molar ratio of cationic lipid to non-cationic lipid to PEG-modified lipid may be between about 55-65:30-40:1-15, respectively. In some embodiments, a molar ratio of cationic lipid (e.g., a cholesterol -based or imidazol-based lipid such as ICE, HGT4001 or HGT4002) to non-cationic lipid (e.g., DSPC,Attorney Docket No. 809030.000060DPPC, DOPE or DEPE) to PEG-modified lipid (e g., DMG-PEG2K) of 60:35:5 is particularly suitable for use with the disclosure.Cationic Lipids

[0355] As used herein, the phrase “cationic lipids” refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available. Particularly suitable cationic lipids for use in the compositions and methods of the disclosure include those described in International Patent Application Publication Nos. WO 2010 / 053572 and WO 2012 / 170930, both of which are incorporated herein by reference. In certain embodiments, cationic lipids suitable for the compositions and methods of the disclosure include an ionizable cationic lipid described in U. S. Patent 11,999,675, filed Mar. 29, 2012 (incorporated herein by reference), such as, e.g., (15Z, 18Z) — N, N-dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien-l-yl)tetracosa-15,18-dien-l-amine (HGT5000), (15Z, 18Z)— N, N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-l-yl)tetracosa-4,15,18-trien-l-amine (HGT5001), and (15Z,18Z) — N, N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-l-yl)tetracosa-5, 15, 18-trien-l -amine (HGT5002).

[0356] In some embodiments, cationic lipids suitable for the compositions and methods of the disclosure include cationic lipids such as 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien- 1 -yl)amino)butyl)piperazine-2, 5 -dione (OF-02).

[0357] In some embodiments, cationic lipids suitable for the compositions and methods of the disclosure include a cationic lipid described in International Patent Application Publication No. WO 2015 / 184256 A2 entitled “Biodegradable lipids for delivery of nucleic acids” which is incorporated by reference herein such as 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-l,4-dioxane-2, 5-dione (Target 23), 3-(5-(bi s(2 -hydroxy dodecyl)amino)pentan-2-yl)-6-(5 -((2 -hydroxy dodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-l,4-dioxane-2, 5-dione (Target 24).

[0358] In some embodiments, cationic lipids suitable for the compositions and methods of the disclosure include a cationic lipid described in International Patent Application Publication No. WO 2013 / 063468 and in U. S. Pat. No. 11,890,377, both of which are incorporated by reference herein.Attorney Docket No. 809030.000060

[0359] In some embodiments, one or more cationic lipids suitable for the present disclosure may be N-[l-(2,3-dioleyloxy)propyl]-N, N, N-trimethylammonium chloride or “DOTMA”. (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U. S. Pat. No. 4,897,355). Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or “DOGS,” 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N, N-dimethyl-l-propanaminium or “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U. S. Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propane or “DODAP”, l,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”.

[0360] Additional exemplary cationic lipids also include l,2-distearyloxy-N, N-dimethyl-3 -aminopropane or “DSDMA”, l,2-dioleyloxy-N, N-dimethyl-3 -aminopropane or “DODMA”, l,2-dilinoleyloxy-N, N-dimethyl-3 -aminopropane or “DLinDMA”, l,2-dilinolenyloxy-N, N-dimethyl-3 -aminopropane or “DLenDMA”, N-dioleyl-N, N-dimethylammonium chloride or “DODAC”, N, N-distearyl-N, N-dimethylammonium bromide or “DDAB”, N-(l,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxyethyl ammonium bromide or “DMRIE”, 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane or “CLinDMA”, 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9', l-2'-octadecadienoxy)propane or “CpLinDMA”, N, N-dimethyl-3,4-di oleyloxybenzylamine or “DMOBA”, l,2-N, N'-dioleylcarbamyl-3 -dimethylaminopropane or “DOcarbDAP”, 2,3-Dilinoleoyloxy-N, N-dimethylpropylamine or “DLinDAP”, 1,2-N, N'-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”, l,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”, 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane or “DLin-DMA”, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane or “DLin-K-XTC2-DMA”, and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-l-yl)-l,3-dioxolan-4-yl)-N, N-dimethylethanamine (DLin-KC2-DMA)) (see, WO 2010 / 042877; Semple et al., Nature Biotech.28: 172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT Publication WO2005 / 121348A1). In some embodiments, one or more of the cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.

[0361] In some embodiments, one or more cationic lipids may be chosen from XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane), MC3 (((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)-N, N-dimethyl-Attorney Docket No. 809030.0000602,2-di((9Z, 12Z)-octadeca-9, 12-dienyl)tetrahydro-3aH-cyclopenta[d] [1,3]dioxol-5-amine)), NC98-5 (4,7, 13 -tri s(3 -oxo-3 -(undecylamino)propyl)-N 1, N16-diundecyl-4,7, 10,13-tetraazahexadecane-l,16-diamide), DODAP (l,2-dioleyl-3 -dimethylammonium propane), HGT4003 (WO 2012 / 170889, the teachings of which are incorporated herein by reference in their entirety), ICE (WO 2011 / 068810, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U. S. Pat. No. 11,999,675, the teachings of which are incorporated herein by reference in their entirety) or HGT5001 (cis or trans) (U. S. Pat. No. 11,999,675), aminoalcohol lipidoids such as those disclosed in International Patent Application Publication No. W02010 / 053572, DOTAP (l,2-dioleyl-3-trimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3 -trimethylammonium propane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids” J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al. “Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech. 2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materials for low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869), N1GL, N2GL, V1GL and combinations thereof.

[0362] In some embodiments, the one or more cationic lipids are amino lipids. Amino lipids suitable for use in the disclosure include those described in WO2017180917, which is hereby incorporated by reference. Exemplary aminolipids in W02017180917 include those described at paragraph

[0744] such as DLin-MC3-DMA (MC3), (13Z,16Z) — N, N-dimethyl-3-nonyldocosa-13,16-dien-l-amine (L608), and Compound 18. Other amino lipids include Compound 2, Compound 23, Compound 27, Compound 10, and Compound 20. Further amino lipids suitable for use in the disclosure include those described in WO2017112865, which is hereby incorporated by reference. Exemplary amino lipids in International Patent Application Publication No. WO2017112865 include a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), and (20-1), and compounds of paragraphs

[0185] ,

[0201] ,

[0276] , In some embodiments, cationic lipids suitable for use in the disclosure include those described in International Patent Application Publication No. WO2016118725, which is hereby incorporated by reference. Exemplary cationic lipids in WO2016118725 include those such as KL22 and KL25. In some embodiments, cationic lipids suitable for use in the disclosure include those described in International Patent Application Publication No. WO2016118724, which isAttorney Docket No. 809030.000060hereby incorporated by reference. Exemplary cationic lipids in WO2016118725 include those such asKLIO, l,2-dilinoleyloxy-N, N-dimethylaminopropane (DLin-DMA), andKL25.

[0363] In some embodiments, cationic lipids constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipids in a suitable lipid solution by weight or by molar. In some embodiments, cationic lipid(s) constitute(s) about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 3540%) of the total lipid mixture by weight or by molar.Non-Cationic / Helper Lipids

[0364] As used herein, the phrase “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase “anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), di oleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), di oleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), l,2-dierucoyl-sn-glycero-3 -phosphoethanolamine (DEPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), or a mixture thereof. In some embodiments, a mixture of lipids for use with the disclosure may include DSPC as a non-cationic lipid component. In some embodiments, a mixture of lipids for use with the disclosure may include DPPC as a non-cationic lipid component. In some embodiments, a mixture of lipids for use with the disclosure may include DOPE as a non-cationic lipid component. In other embodiments, a mixture of lipids for use with the disclosure may include DEPE as a non-cationic lipid component.

[0365] In some embodiments, non-cationic lipids may constitute at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% of the total lipids in a suitable lipid solution by weight or by molar. In some embodiments, non-cationic lipid(s)Attorney Docket No. 809030.000060constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution by weight or by molar.Cholesterol-Based Lipids

[0366] In some embodiments, a suitable lipid solution includes one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi (N, N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U. S. Pat. No. 5,744,335), or ICE. In some embodiments, cholesterol-based lipid(s) constitute(s) at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total lipids in a suitable lipid solution by weight or by molar. In some embodiments, cholesterol-based lipid(s) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution by weight or by molar.PEGylated Lipids

[0367] In some embodiments, a suitable lipid solution includes one or more PEGylated lipids. For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present disclosure. Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 2 kDa, up to 3 kDa, up to 4 kDa or up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. For example, a suitable lipid solution may include a PEG-modified lipid such as l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K). In some embodiments, particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or Cl 8).

[0368] PEG-modified phospholipid and derivatized lipids may constitute at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total lipids in a suitable lipid solution by weight or by molar. In some embodiments, the PEG-modified phospholipid and derivatized lipids constitute about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 1.5% to about 5% of the total lipid present in the liposomal transfer vehicle. In some embodiments, oneAttorney Docket No. 809030.000060or more PEG-modified lipids constitute about 1.5%, about 2%, about 3% about 4% or about 5% of the total lipids by molar ratio. In some embodiments, PEGylated lipid(s) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution by weight or by molar.

[0369] Various combinations of lipids, i.e., cationic lipids, non-cationic lipids, PEG-modified lipids and optionally cholesterol, that can used to prepare, and that are comprised in, preformed lipid nanoparticles are described in the literature and herein. For example, a suitable lipid solution may contain cKK-E12, DOPE, cholesterol, and DMG-PEG2K; C 12-200, DOPE, cholesterol, and DMG-PEG2K; HGT5000, DOPE, cholesterol, and DMG-PEG2K; HGT5001, DOPE, cholesterol, and DMG-PEG2K; cKK-E12, DPPC, cholesterol, and DMG-PEG2K; C12-200, DPPC, cholesterol, and DMG-PEG2K; HGT5000, DPPC, chol, and DMG-PEG2K; HGT5001, DPPC, cholesterol, and DMG-PEG2K; or ICE, DOPE and DMG-PEG2K. Additional combinations of lipids are described in the art, e.g., U. S. Pat. No. 11,013,812, U. S. Pat. No.11,013,812, and U. S. Pat. No. 11,013,812, the disclosures of which are included here in their full scope by reference. The selection of cationic lipids, non-cationic lipids and / or PEG-modified lipids which comprise the lipid mixture as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s) and the nature of the and the characteristics of the RNAi agent to be encapsulated. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus the molar ratios may be adjusted accordingly.RNAi agent-LNP Formation

[0370] The process of forming LNPs encapsulating RNAi agents (RNAi agent-LNPs) by mixing an RNAi agent solution with a lipid solution as described above, to yield a LNP formation solution suitable for RNAi agent-LNP formation has been described previously.Exemplified RNAi Agent Modification Pattern and Formulation

[0371] In certain embodiments, the present disclosure provides a double-stranded ribonucleic acid (RNAi agent) molecule adapted to selectively inhibit expression of Factor XI messenger RNA. In some embodiments, the double-stranded RNAi agent may include a 23-nucleotide antisense strand having a 5'-end modified with a vinylphosphonate group, and a 21-Attorney Docket No. 809030.000060nucleotide sense strand conjugated at its 3 '-end to a triantennary N-acetylgalactosamine (GalNAc) ligand, to facilitate targeted delivery to hepatocytes. In some embodiments, each nucleotide of the double-stranded RNAi agent may be chemically modified with 2'-fluoro (2'-F) and 2'-O-methyl (2'-0Me) ribose substitutions to enhance nuclease resistance and to reduce immunostimulatory potential. The skilled artisan will recognize that different patterns of modifications may be included upon effective RNAi agents. In certain exemplified embodiments, a RNAi agent of the disclosure includes (i) a 23-nucleotide antisense strand having a 5'-end modified with a vinylphosphonate group, including phosphorothioate linkages between each of the following adjacent pairs of residues, respectively: 1-2, 2-3, 21-22, and 22-23, 2’-fluoro-modified nucleotides at each of positions 2, 6, 8, 9, 14, and 16, and 2’-O-methyl modified nucleotides at all positions not having a 2’-fluoro modification (i.e., 2’-O-methyl modified nucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23), and (ii) a 21-nucleotide sense strand conjugated at its 3 '-end to a triantennary N-acetylgalactosamine (GalNAc) ligand, including phosphorothioate linkages between each of the following adjacent pairs of residues, respectively: 1-2 and 2-3, 2’-fluoro-modified nucleotides at each of positions 7, 9, 10, and 11, and 2’-O-methyl modified nucleotides at all positions not having a 2’-fluoro modification (i.e., 2’-O-methyl modified nucleotides at each of positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21). In other exemplified embodiments, a RNAi agent of the disclosure includes (i) a 21-nucleotide antisense strand having a 5 '-end modified with a vinylphosphonate group, including phosphorothioate linkages between each of the following adjacent pairs of residues, respectively: 1-2, 2-3, 19-20, and 20-21, 2’-fluoro-modified nucleotides at each of positions 2, 6, 8, 9, 14, and 16, and 2’-O-methyl modified nucleotides at all positions not having a 2’-fluoro modification (i.e., 2’-O-methyl modified nucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21), and (ii) a 19-nucleotide sense strand conjugated at its 3'-end to a triantennary N-acetylgalactosamine (GalNAc) ligand, including phosphorothioate linkages between each of the following adjacent pairs of residues, respectively: 1-2 and 2-3, 2’-fluoro-modified nucleotides at each of positions 5, 7, 8, and 9, and 2’-O-methyl modified nucleotides at all positions not having a 2’-fluoro modification (i.e., 2’-O-methyl modified nucleotides at each of positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19). In some embodiments, a double-stranded RNAi agent may be provided in sodium salt form, such as, e.g., including forty-one, forty-three, or forty-five sodium (Na+) counter ions replacing a proton (H+) on phosphorus atoms present in a free acid form.Attorney Docket No. 809030.000060

[0372] In some embodiments, a double-stranded RNAi agent attached to a targeting ligand (e.g., a GalNAc) may be formulated as a drug product. In some embodiments, a double-stranded RNAi agent may be formulated as a drug product that is a sodium salt. In some embodiments, a sodium salt of the drug product may have a concentration as measured in free acid form in water (e.g., 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 225 mg / mL, 250 mg / mL, 275 mg / mL, 300 mg / mL, or any value therebetween). In some embodiments, the drug product may include a pharmaceutically acceptable excipient and / or an additive, such as, e.g., a pH modifier (e.g., sodium hydroxide, phosphoric acid, and / or the like), such as to achieve a desired pH (e.g., approximately 7.0). In some embodiments, the drug product may be stored as a solution (e.g., an aqueous solution).Pharmaceutical Compositions

[0373] In certain embodiments, the present disclosure provides for a pharmaceutical composition comprising the RNAi agents of the present disclosure. The RNAi agent sample can be suitably formulated and introduced into the environment of the cell by any means that allows for a sufficient portion of the sample to enter the cell to induce gene silencing, if it is to occur. Many formulations for dsRNA are known in the art and can be used so long as the dsRNA gains entry to the target cells so that it can act. See, e.g., U. S. Patent Application Publication Nos.2004 / 0203145 Al and 2005 / 0054598 Al. For example, the RNAi agent of the instant disclosure can be formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids. Formulations of RNAi agent with cationic lipids can be used to facilitate transfection of the RNAi agent into cells. For example, cationic lipids, such as lipofectin (U. S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (published PCT International Patent Application Publication No. WO 97 / 30731), can be used. Suitable lipids include OLIGOFECTAMINE®, LIPOFECTAMINE® (LIFE TECHNOLOGIES™), NC388 (RIBOZYME® Pharmaceuticals, Inc., Boulder, Colo), or FUGENE 6™ (ROCHE®) all of which can be used according to the manufacturer’s instructions.

[0374] Such compositions typically include the nucleic acid molecule and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents,Attorney Docket No. 809030.000060isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0375] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0376] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF™, Parsippany, N. J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositionsAttorney Docket No. 809030.000060can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0377] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0378] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0379] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U. S. Pat. No.6,468,798.

[0380] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.Attorney Docket No. 809030.000060For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0381] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0382] The compounds can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J. Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst. Pharm. 53(3), 325 (1996).

[0383] The compounds can also be administered by a method suitable for administration of nucleic acid agents, such as a DNA vaccine. These methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U. S. Pat. No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U. S. Pat. No. 6,168,587. Additionally, intranasal delivery is possible, as described in, inter alia, Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U. S. Pat. No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U. S. Pat. No. 6,471,996).

[0384] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U. S. Pat. No. 4,522,811.

[0385] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determiningAttorney Docket No. 809030.000060the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0386] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a compound used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0387] As defined herein, a therapeutically effective amount of a nucleic acid molecule (i.e., an effective dosage) depends on the nucleic acid selected. For instance, single dose amounts of an RNAi agent (or, e g., a construct(s) encoding for such RNAi agent) in the range of approximately 1 pg to 1000 mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 mg may be administered. In some embodiments, 1-5 g of the compositions can be administered. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a nucleic acid (e.g., an RNAi agent), protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.Attorney Docket No. 809030.000060

[0388] The nucleic acid molecules of the disclosure can be inserted into expression constructs, e.g., viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, e.g., using methods known in the art, including but not limited to those described in Xia et al., (2002), supra. Expression constructs can be delivered to a subject by, for example, inhalation, orally, intravenous injection, local administration (see U. S. Pat. No. 5,328,470) or by subcutaneous injection or stereotactic injection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 3054-3057). The pharmaceutical preparation of the delivery vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the delivery vehicle is imbedded. Alternatively, where the complete delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0389] The expression constructs may be constructs suitable for use in the appropriate expression system and include, but are not limited to retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or Hl RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the RNAi agent. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct, e.g., Tuschl (2002, Nature Biotechnol 20: 500-505).

[0390] It can be appreciated that the method of introducing RNAi agents into the environment of the cell will depend on the type of cell and the make up of its environment. For example, when the cells are found within a liquid, one preferable formulation is with a lipid formulation such as in lipofectamine and the RNAi agents can be added directly to the liquid environment of the cells. Lipid formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art. When the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable. Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used. In some instances, it may be preferable to formulate RNAi agents in a buffer or saline solution and directly inject the formulated RNAi agents into cells, as in studies with oocytes. TheAttorney Docket No. 809030.000060direct injection of RNAi agent duplexes may also be done. For suitable methods of introducing dsRNA (e.g., RNAi agents), see U. S. published patent application No. 2004 / 0203145 Al.

[0391] Suitable amounts of an RNAi agent must be introduced and these amounts can be empirically determined using standard methods. Typically, effective concentrations of individual RNAi agent species in the environment of a cell will be 50 nanomolar or less, 10 nanomolar or less, or compositions in which concentrations of 1 nanomolar or less can be used. In another embodiment, methods utilizing a concentration of 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less, and even a concentration of 10 picomolar or less, 5 picomolar or less, 2 picomolar or less or 1 picomolar or less can be used in many circumstances.

[0392] The method can be carried out by addition of the RNAi agent compositions to an extracellular matrix in which cells can live provided that the RNAi agent composition is formulated so that a sufficient amount of the RNAi agent can enter the cell to exert its effect. For example, the method is amenable for use with cells present in a liquid such as a liquid culture or cell growth media, in tissue explants, or in whole organisms, including animals, such as mammals and especially humans.

[0393] The level or activity of a FXI RNA can be determined by a suitable method now known in the art or that is later developed. It can be appreciated that the method used to measure a target RNA and / or the expression of a target RNA can depend upon the nature of the target RNA. For example, where the target FXI RNA sequence encodes a protein, the term “expression” can refer to a protein or the FXI RNA / transcript derived from the FXI gene (either genomic or of exogenous origin). In such instances the expression of the target FXI RNA can be determined by measuring the amount of FXI RNA / transcript directly or by measuring the amount of FXI protein. Protein can be measured in protein assays such as by staining or immunoblotting or, if the protein catalyzes a reaction that can be measured, by measuring reaction rates. All such methods are known in the art and can be used. Where target FXI RNA levels are to be measured, art-recognized methods for detecting RNA levels can be used (e.g., RT-PCR, Northern Blotting, etc.). In targeting FXI RNAs with the RNAi agents of the instant disclosure, it is also anticipated that measurement of the efficacy of an RNAi agent in reducing levels of FXI RNA or protein in a subject, tissue, in cells, either in vitro or in vivo, or in cell extracts can also be used to determine the extent of reduction of FXI-associated phenotypes (e.g., disease or disorders, e g., chronic liver disease, liver inflammation, cirrhosis, liver fibrosis, and / or hepatocellular carcinoma, etc ). The aboveAttorney Docket No. 809030.000060measurements can be made on cells, cell extracts, tissues, tissue extracts or other suitable source material.

[0394] The determination of whether the expression of a FXI RNA has been reduced can be by a suitable method that can reliably detect changes in RNA levels. Typically, the determination is made by introducing into the environment of a cell undigested dsRNA such that at least a portion of that RNAi agent enters the cytoplasm, and then measuring the level of the target RNA. The same measurement is made on identical untreated cells and the results obtained from each measurement are compared.

[0395] The RNAi agent can be formulated as a pharmaceutical composition which comprises a pharmacologically effective amount of an RNAi agent and pharmaceutically acceptable carrier. A pharmacologically or therapeutically effective amount refers to that amount of an RNAi agent effective to produce the intended pharmacological, therapeutic or preventive result. The phrases “pharmacologically effective amount” and “therapeutically effective amount” or simply “effective amount” refer to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 20% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 20% reduction in that parameter.

[0396] Suitably formulated pharmaceutical compositions of this disclosure can be administered by means known in the art such as by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In some embodiments, the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection.

[0397] In general, a suitable dosage unit of dsRNA will be in the range of 0.001 to 0.25 milligrams per kilogram body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kilogram body weight per day, or in the range of 0.001 to 5 micrograms per kilogram of body weight per day, or in the range of 1 to 500 nanograms per kilogram of body weight per day, or in the range of 0.01 to 10 micrograms per kilogram body weight per day, or in the range of 0.10 to 5 micrograms per kilogram body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram body weight per day. A pharmaceutical composition comprising theAttorney Docket No. 809030.000060dsRNA can be administered once daily. However, the therapeutic agent may also be dosed in dosage units containing two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage unit. The dosage unit can also be compounded for a single dose over several days, e.g., using a conventional sustained release formulation which provides sustained and consistent release of the dsRNA over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose. Regardless of the formulation, the pharmaceutical composition must contain dsRNA in a quantity sufficient to inhibit expression of the target gene in the animal or human being treated. The composition can be compounded in such a way that the sum of the multiple units of dsRNA together contain a sufficient dose.

[0398] Data can be obtained from cell culture assays and animal studies to formulate a suitable dosage range for humans. The dosage of compositions of the disclosure lies within a range of circulating concentrations that include the ED50 (as determined by known methods) with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a compound used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels of dsRNA in plasma may be measured by standard methods, for example, by high performance liquid chromatography.

[0399] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.Methods of Treatment

[0400] The present disclosure provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disease or disorder caused, in whole or in part, by FXI (e.g., misregulation and / or elevation of FXI transcript and / or FXI protein levels), or treatable via selective targeting of FXI.Attorney Docket No. 809030.000060

[0401] In certain aspects, the disclosure provides a method for preventing in a subject, a disease or disorder as described herein (including, e.g., prevention of the commencement of transforming events within a subject via inhibition of FXI expression), by administering to the subject a therapeutic agent (e.g., an RNAi agent or vector or transgene encoding same). Subjects at risk for the disease can be identified by, for example, one or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the detection of, e.g., myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, pulmonary embolism, ischemic stroke, transient ischemic attack, retinal artery occlusion, mesenteric ischemia, renal vein thrombosis, cerebral venous sinus thrombosis, arterial thrombosis, venous thrombosis, venous thromboembolism due to deep vein thrombosis, peripheral artery disease, or thrombosis due to artificial surfaces (e.g., catheter associated thrombosis, medical device thrombosis), among other diseases and / or disorders, in the subject.

[0402] Another aspect of the disclosure pertains to methods of treating subjects therapeutically, i.e., altering the onset of symptoms of the disease or disorder. These methods can be performed in vitro (e.g., by culturing the cell with the RNAi agent) or, alternatively, in vivo (e.g., by administering the RNAi agent to a subject).

[0403] With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient’s “drug response phenotype”, or “drug response genotype”). Thus, another aspect of the disclosure provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the target FXI RNA molecules of the present disclosure or target FXI RNA modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[0404] The subject is administered a therapeutically effective amount of any one or more of the FXI RNAi agents described herein. The subject can be a human, patient, or human patient.Attorney Docket No. 809030.000060The subject may be an adult, adolescent, child, or infant. Administration of a pharmaceutical composition described herein can be to a human being or animal.

[0405] In some embodiments, the FXI RNAi agents described herein are used to treat a subject that would benefit from a reduction and / or inhibition of FXI gene expression. In some embodiments, the described FXI RNAi agents are used to treat (including prophylactically) at least one symptom or pathological mediated at least in part by FXI expression. The subject is administered a therapeutically effective amount of any one or more of the described RNAi agents. In some embodiments, the subject is administered a prophylactically effective amount of any one or more of the described RNAi agents, thereby preventing the at least one symptom.

[0406] In certain embodiments, the present disclosure provides methods for treatment of diseases, disorders, conditions, or pathological states mediated at least in part by FXI expression, in a patient in need thereof, wherein the methods include administering to the patient any of the FXI RNAi agents described herein.

[0407] In some embodiments, the FXI RNAi agents are used to treat or manage a pathological state (such as a condition or disease) of a subject, for which the pathological state is mediated at least in part by FXI expression. The subject is administered a therapeutically effective amount of one or more of the FXI RNAi agents or FXI RNAi agent-containing compositions described herein. In some embodiments, the method comprises administering a composition comprising an FXI RNAi agent described herein to a subject to be treated.

[0408] In some embodiments, the gene expression level and / or mRNA level of an FXI gene in a subject to whom a described FXI RNAi agent is administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the FXI RNAi agent or to a subject not receiving the FXI RNAi agent. The gene expression level and / or mRNA level in the subject is reduced in a cell, group of cells, and / or tissue of the subject.

[0409] In some embodiments, the protein level of FXI in a subject to whom a described FXI RNAi agent has been administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the FXI RNAi agent or to a subject not receiving the FXI RNAi agent. The protein level in the subject is reduced in a cell, group of cells, tissue, blood, and / or other fluid of the subject.Attorney Docket No. 809030.000060

[0410] A reduction in FXI gene expression, FXI mRNA, or FXI protein levels can be assessed and quantified by general methods known in the art. The Examples disclosed herein set forth generally known methods for assessing inhibition of FXI gene expression and reduction in FXI protein levels. The reduction or decrease in FXI mRNA level and / or protein level are collectively referred to herein as a reduction or decrease in FXI or inhibiting or reducing the expression of FXI.Cells, Tissues, and Non-Human Organisms

[0411] Cells, tissues, and non-human organisms that include at least one of the FXI RNAi agents described herein is contemplated. The cell, tissue, or non-human organism is made by delivering the RNAi agent to the cell, tissue, or non-human organism.Kits

[0412] In some embodiments, the disclosure provides a kit comprising an RNAi agent as disclosed herein, and instructions for use. In some embodiments, the kit comprises an RNAi agent as disclosed herein, and a package insert containing instructions for use of the kit and / or any component thereof. In some embodiments, the kit comprises, in a suitable container, an RNAi agent as disclosed herein, one or more controls, and various buffers, reagents, enzymes and other standard ingredients well known in the art. In some embodiments, the container comprises at least one vial, well, test tube, flask, bottle, syringe or other container means, into which the RNAi agent is placed, and in some instances, suitably aliquoted. In some embodiments where an additional component is provided, the kit contains additional containers into which this component is placed. The kits can also include a means for containing the RNAi agent and any other reagent in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Containers and / or kits can include labeling with instructions for use and / or warnings.

[0413] In some embodiments, a kit comprises an RNAi agent as disclosed herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the RNAi agent and instructions for treating or delaying progression of a disease, disorder or condition associated with FXI expression in a subject in need thereof.Attorney Docket No. 809030.000060

[0414] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-FV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ, of Oregon Press, Eugene, 2000).

[0415] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.Attorney Docket No. 809030.000060

[0416] The above provided embodiments and items are now illustrated with the following, non-limiting examples.EXAMPLES

[0417] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the disclosure, and are not intended to limit the scope of the disclosure.Example 1: Sequence Strategy

[0418] Small interfering RNA (RNAi agent) duplexes (i.e., a pair of oligonucleotide strands having a double-stranded region of strand-strand complementarity and including sense and antisense strands, as defined in reference to a target sequence, e.g., a target sequence within a naturally occurring FXI RNA) having either 19 / 21 (sense strand sequence of 19 nucleotides in length / antisense strand of 21 nucleotides in length) or 21 / 23 (sense strand sequence of 21 nucleotides in length / antisense strand of 23 nucleotides in length) formats were initially designed (FIGs. 1A and IB)

[0419] As described herein, the present disclosure relates to Factor XI (also known as FXI, Factor 11 or Fll') and the modulation of mammalian (e.g., human) FXI mRNA (NCB1 GenBank Sequence for the human FXI mRNA employed was NM_000128.4). Initially, RNAi agent candidates were identified, and their locations were mapped along the FXI transcript (FIG. 1C).Sense and antisense strands for selected RNAi agents were identified; as the first antisense nucleotide is not believed to hybridize with target sequence, nucleotide U was used for antisense position 1, with corresponding A included at position 21 of the sense strand (see Table 1). Table 1 further identifies the seed region. Generally, the seed region includes nucleotides in positions 2-8 of the antisense strand (again noting the inclusion of a U at position 1 in the antisense strands shown in Table 1, which was then modified to a vinyl-phosphonate 2'-OMe-uracil at position 1 of the antisense strands shown in Table 2) and is responsible for target site recognition. Without wishing to be bound by theory, the 5' most U (included at position 1 of the antisense strand) is included to facilitate binding with the RISC complex.Attorney Docket No. 809030.000060Table 1RNAi Agent SequencesDuplex ID Sense Sense Strand SEQ Antisense Strand SEQ Seed Start Sequence ID IDNO: NO:XD-67358 17 AGCUCAUUCAG 3 UUCCUCCUCUGAAUGAG 88 UCCUCCU AGGAGGAA CUUG XD-67359 18 GCUCAUUCAGA 4 UAUCCUCCUCUGAAUGA 89 AUCCUCC GGAGGAUA GCUU XD-67360 19 CUCAUUCAGAG 5 UCAUCCUCCUCUGAAUG 90 CAUCCUC GAGGAUGA AGCU XD-67362 60 GCACCCUUAUU 6 UAAUUCUUAAUAAGGGU 91 AAUUCUU AAGAAUUA GCUU XD-67363 61 CACCCUUAUUA 7 UCAAUUCUUAAUAAGGG 92 CAAUUCU AGAAUUGA UGCU XD-67365 63 CCCUUAUUAAG 8 UUGCAAUUCUUAAUAAG 93 UGCAAUU AAUUGCAA GGUG XD-67366 64 CCUUAUUAAGA 9 UCUGCAAUUCUUAAUAA 94 CUGCAAU AUUGCAGA GGGU XD-67367 65 CUUAUUAAGAA 10 UGCUGCAAUUCUUAAUA 95 GCUGCAA UUGCAGCA AGGG XD-67368 66 UUAUUAAGAAU 11 UUGCUGCAAUUCUUAAU 96 UGCU GCA UGCAGCAA AAGG XD-67369 67 UAUUAAGAAUU 12 UUUGCUGCAAUUCUUAA 97 UUGCUGC GCAGCAAA UAAG XD-67370 91 CAACAAGGUCU 13 UCCUGAAAAGACCUUGU 98 CCUGAAA UUUCAGGA UGGC XD-67372 93 ACAAGGUCUUU 14 UAUCCUGAAAAGACCUU 99 AUCCUGA UCAGGAUA GUUG XD-67375 96 AGGUCUUUUCA 15 UAUCAUCCUGAAAAGAC 100 AUCAUCC GGAUGAUA CUUG XD-67376 97 GGUCUUUUCAG 16 UAAUCAUCCUGAAAAGA 101 AAUCAUC GAUGAUUA CCUU XD-67378 99 UCUUUUCAGGA 17 UAAAAUCAUCCUGAAAA 102 AAAAUCA UGAUUUUA GACC XD-67379 100 CUUUUCAGGAU 18 UGAAAAUCAUCCUGAAA 103 GAAAAUC GAUUUUCA AGAC XD-67380 101 UUUUCAGGAUG 19 UAGAAAAUCAUCCUGAA 104 AGAAAAU AUUUUCUA AAGA XD-67382 103 UUCAGGAUGAU 20 UUAAGAAAAUCAUCCUG 105 UAAGAAA UUUCUUAA AAAA XD-67383 104 UCAGGAUGAUU 21 UAUAAGAAAAUCAUCCU 106 AUAAGAA UUCUUAUA GAAA XD-67384 105 CAGGAUGAUUU 22 UUAUAAGAAAAUCAUCC 107 UAUAAGA UCUUAUAA UGAA XD-67385 106 AGGAUGAUUUU 23 UAUAUAAGAAAAUCAUC 108 AUAUAAG CUUAUAUA CUGA XD-67386 107 GGAUGAUUUUC 24 UGAUAUAAGAAAAUCAU 109 GAUAUAA UUAUAUCA CCUG XD-67387 108 GAUGAUUUUCU 25 UUGAUAUAAGAAAAUCA 110 UGAUAUA UAUAUCAA UCCU XD-67388 109 AUGAUUUUCUU 26 UUUGAUAUAAGAAAAUC 111 UUGAUAUAUAUCAAA AUCCAttorney Docket No. 809030.000060XD-67389 150 UUCAGUUUCUG 27 UCAUUCACCAGAAACUG 112 CAUUCAC GUGAAUGA AAGU XD-67390 151 UCAGUUUCUGG 28 UACAUUCACCAGAAACU 113 ACAUUCA UGAAUGUA GAAG XD-67391 152 CAGUUUCUGGU 29 UCACAUUCACCAGAAAC 114 CACAUUC GAAUGUGA UGAA XD-67392 153 AGUUUCUGGUG 30 UACACAUUCACCAGAAA 115 ACACAUU AAUGUGUA CUGA XD-67393 154 GUUUCUGGUGA 31 UCACACAUUCACCAGAA 116 CACACAU AUGUGUGA ACUG XD-67396 157 UCUGGUGAAUG 32 UAGUCACACAUUCACCA 117 AGUCACA UGUGACUA GAAA XD-67397 158 CUGGUGAAUGU 33 UGAGUCACACAUUCACC 118 GAGUCAC GUGACUCA AGAA XD-67436 391 AUUUCUGGGUA 34 UGAAAGAAUACCCAGAA 119 GAAAGAA UUCUUUCA AUCG XD-67440 395 CUGGGUAUUCU 35 UGCUUGAAAGAAUACCC 120 GCUUGAA UUCAAGCA AGAA XD-67441 396 UGGGUAUUCUU 36 UUGCUUGAAAGAAUACC 121 UGCUUGA UCAAGCAA CAGA XD-67442 397 GGGUAUUCUUU 37 UUUGCUUGAAAGAAUAC 122 UUGCUUG CAAGCAAA CCAG XD-67443 398 GGUAUUCUUUC 38 UAUUGCUUGAAAGAAUA 123 AUUGCUU AAGCAAUA CCCA XD-67446 401 AUUCUUUCAAG 39 UAGCAUUGCUUGAAAGA 124 AGCAUUG CAAUGCUA AUAC XD-67449 404 CUUUCAAGCAA 40 UGUGAGCAUUGCUUGAA 125 GUGAGCA UGCUCACA AGAA XD-67450 405 UUUCAAGCAAU 41 UUGUGAGCAUUGCUUGA 126 UGUGAGC GCUCACAA AAGA XD-67453 408 CAAGCAAUGCU 42 UUGGUGUGAGCAUUGCU 127 UGGUGUG CACACCAA UGAA XD-67454 409 AAGCAAUGCUC 43 UUUGGUGUGAGCAUUGC 128 UUGGUGU ACACCAAA UUGA XD-67528 540 CCACUGCCACUU 44 UGUGAAAAAGUGGCAGU 129 GUGAAAA UUUCACA GGAC XD-67537 864 AAAUCUUUGUC 45 UUUAAGGAGACAAAGAU 130 UUAAGGA UCCUUAAA uucuXD-67571 925 AAAGCUCUUUC 46 UGAAACCAGAAAGAGCU 131 GAAACCA UGGUUUCA UUGC XD-67629 1086 CUGCCAGUUUU 47 UUAGGUAAAAAACUGGC 132 UAGGUAA UUACCUAA AGCG XD-67631 1143 GUGUUACUUAA 48 UGAAAGCUUUAAGUAAC 133 GAAAGCU AGCUUUCA ACUU XD-67634 1146 UUACUUAAAGC 49 UGAAGAAAGCUUUAAGU 134 GAAGAAA UUUCUUCA AACA XD-67636 1148 ACUUAAAGCUU 50 UUUGAAGAAAGCUUUAA 135 UUGAAGA UCUUCAAA GUAA XD-67637 1149 CUUAAAGCUUU 51 UUUUGAAGAAAGCUUUA 136 UUUGAAG CUUCAAAA AGUA XD-67643 1172 CUCCAACUAAA 52 UGAAGUAUUUUAGUUGG 137 GAAGUAU AUACUUCA AGAU XD-67657 1226 UGUGUAAAAUG 53 U CAU U AU CCAU U U U ACA 138 CAUUAUCGAUAAUGA CAACAttorney Docket No. 809030.000060XD-67783 1445 GUGGCAUUUUA 54 UAUUGAUUUAAAAUGCC 139 AUUGAUU AAUCAAUA ACUG XD-68016 2119 AGAAGAAAACA 55 UGACAGUUUGUUUUCUU 140 GACAGUU AACUGUCA CUAG XD-68111 2662 ACACUUUCCUG 56 UCAUUUUUCAGGAAAGU 141 CAUUUUU AAAAAUGA GUAU XD-68115 61 CACCCUUAUUA 57 UUGCAAUUCUUAAUAAG 142 UGCAAUU AGAAUUGCAA GGUGCU XD-68119 65 CUUAUUAAGAA 58 UUUGCUGCAAUUCUUAA 143 UUGCUGC UUGCAGCAAA UAAGGG XD-68123 94 CAAGGUCUUUU 59 UAUCAUCCUGAAAAGAC 144 AUCAUCC CAGGAUGAUA CUUGUU XD-68126 97 GGUCUUUUCAG 60 UAAAAUCAUCCUGAAAA 145 AAAAUCA GAUGAUUUUA GACCUU XD-68127 98 GUCUUUUCAGG 61 UGAAAAUCAUCCUGAAA 146 GAAAAUC AUGAUUUUCA AGACCU XD-68128 99 UCUUUUCAGGA 62 UAGAAAAUCAUCCUGAA 147 AGAAAAU UGAUUUUCUA AAGACC XD-68129 100 CUUUUCAGGAU 63 UAAGAAAAUCAUCCUGA 148 AAGAAAA GAUUUUCUUA AAAGAC XD-68130 101 UUUUCAGGAUG 64 UUAAGAAAAUCAUCCUG 149 UAAGAAA AUUUUCUUAA AAAAGA XD-68131 102 UUUCAGGAUGA 65 UAUAAGAAAAUCAUCCU 150 AUAAGAA UUUUCUUAUA GAAAAG XD-68132 103 UUCAGGAUGAU 66 UUAUAAGAAAAUCAUCC 151 UAUAAGA UUUCUUAUAA UGAAAA XD-68133 104 UCAGGAUGAUU 67 UAUAUAAGAAAAUCAUC 152 AUAUAAG UUCUUAUAUA CUGAAA XD-68134 105 CAGGAUGAUUU 68 UGAUAUAAGAAAAUCAU 153 GAUAUAA UCUUAUAUCA CCUGAA XD-68135 106 AGGAUGAUUUU 69 UUGAUAUAAGAAAAUCA 154 UGAUAUA CUUAUAUCAA UCCUGA XD-68136 107 GGAUGAUUUUC 70 UUUGAUAUAAGAAAAUC 155 UUGAUAU UUAUAUCAAA AUCCUG XD-68137 150 UUCAGUUUCUG 71 UCACAUUCACCAGAAAC 156 CACAUUC GUGAAUGUGA UGAAGU XD-68138 151 UCAGUUUCUGG 72 UACACAUUCACCAGAAA 157 ACACAUU UGAAUGUGUA CUGAAG XD-68139 152 CAGUUUCUGGU 73 UCACACAUUCACCAGAA 158 CACACAU GAAUGUGUGA ACUGAA XD-68141 154 GUUUCUGGUGA 74 UGUCACACAUUCACCAG 159 GUCACAC AUGUGUGACA AAACUG XD-68142 155 UUUCUGGUGAA 75 UAGUCACACAUUCACCA 160 AGUCACA UGUGUGACUA GAAACU XD-68143 156 UUCUGGUGAAU 76 UGAGUCACACAUUCACC 161 GAGUCAC GUGUGACUCA AGAAAC XD-68172 389 CGAUUUCUGGG 77 UGAAAGAAUACCCAGAA 162 GAAAGAA UAUUCUUUCA AUCGCU XD-68178 395 CUGGGUAUUCU 78 UUUGCUUGAAAGAAUAC 163 UUGCUUG UUCAAGCAAA CCAGAA XD-68185 402 UUCUUUCAAGC 79 UGUGAGCAUUGCUUGAA 164 GUGAGCA AAUGCUCACA AGAAUA XD-68186 403 UCUUUCAAGCA 80 UUGUGAGCAUUGCUUGA 165 UGUGAGCAUGCUCACAA AAGAAUAttorney Docket No. 809030.000060XD-68259 539 UCCACUGCCACU 81 UCGUGAAAAAGUGGCAG 166 CGUGAAA UUUUCACGA UGGACG XD-68265 864 AAAUCUUUGUC 82 UUUUUAAGGAGACAAAG 167 UUUUAAG UCCUUAAAAA AUUUCU XD-68350 1144 UGUUACUUAAA 83 UGAAGAAAGCUUUAAGU 168 GAAGAAA GCUUUCUUCA AACACU XD-68352 1146 UUACUUAAAGC 84 UUUGAAGAAAGCUUUAA 169 UUGAAGA UUUCUUCAAA GUAACA XD-68353 1147 UACUUAAAGCU 85 UUUUGAAGAAAGCUUUA 170 UUUGAAG UUCUUCAAAA AGUAAC XD-68497 1453 UUAAAUCAAUC 86 UUUUAUUUCAGAUUGAU 171 UUUAUUU UGAAAUAAAA UUAAAA XD-68518 1493 GGGUUCAAGAA 87 UGAUUAUUAUUUCUUGA 172 GAUUAUUAUAAUAAUCA ACCCCA

[0420] In further designs, the sequences were chemically modified. For certain RNAi agents of the disclosure, the chemical modifications included at least one vinyl phosphonate-modified nucleotide, which was positioned in the presently exemplified RNAi agents at the 5'-terminus of the antisense strand. In related designs, the sense strand sequences were conjugated with a triantennary N-acetylgalactosamine (GalNAc) ligand - notably, for any of the below modified sense strand sequences of below Table 2 below, inclusion of a 3'-terminal triantennary GalNAc moiety (e.g., the triGalNAc ligand as defined below and elsewhere herein) for targeted delivery to liver cells is expressly contemplated. As used herein, Duplex IDs annotated with “M” (e.g., XD-6735M) represent RNAi agents of the disclosure including one or more chemical modifications. Further, as used herein, Duplex IDs annotated with “MGal” (e.g., XD-6735MGal) represent RNAi agents of the disclosure including one or more chemical modifications and further including a 3'-terminal triantennary GalNAc moiety.Table 2Modified RNAi Agent SequencesDuplex ID Sense Strand Sequence Modified SEQ ID Antisense Strand Modified SEQ ID NO: N...

Claims

Attorney Docket No. 809030.000060What is claimed is:

1. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises a sense strand and an antisense strand, and wherein said antisense strand comprises a region of complementarity which comprises at least 14 contiguous nucleotides differing by no more than 3 nucleotides from any one of SEQ ID NOs: 88-172.

2. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises a sense strand and an antisense strand, wherein said sense strand comprises at least 14 contiguous nucleotides differing by no more than 3 nucleotides from any one of SEQ ID NOs: 3-87; and wherein said antisense strand comprises at least 14 contiguous nucleotides differing by no more than 3 nucleotides from any one of SEQ ID NOs: 88-172.

3. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises a sense strand and an antisense strand, wherein said antisense strand comprises a region of complementarity which comprises at least 14 contiguous nucleotides differing by no more than 3 nucleotides from the complement of any one of SEQ ID NOs: 343-427.

4. The RNAi agent of any one of claims 1-3, wherein at least one of the sense strand and the antisense strand comprises a targeting ligand.

5. The RNAi agent of claim 4, wherein the targeting ligand targets a receptor which mediates delivery to a liver tissue.

6. The RNAi agent of claim 4, wherein the targeting ligand enhances hepatocyte cell delivery of the RNAi agent.

7. The RNAi agent of claim 4, wherein the targeting ligand binds the Asialoglycoprotein receptor (ASGPR) on hepatocyte cells.

8. The RNAi agent of claim 4, wherein the targeting ligand is one or more GalNAc moieties attached through a bivalent or trivalent branched linker.Attorney Docket No. 809030.0000609. The RNAi agent of claim 4, wherein the ligand is attached to the 3' end, the 5' end, or the 3' and 5' end of the sense strand.

10. The RNAi agent of claim 4, wherein the targeting ligand is a triantennary GalNAc ligand.

11. The RNAi agent of claim 4, wherein the targeting ligand is attached to the RNAi agent at the 3 '-terminal nucleotide residue of the sense strand.

12. The RNAi agent of claim 4, wherein the targeting ligand comprises a structure selected from the group consisting ofAttorney Docket No. 809030.000060Attorney Docket No. 809030.000060Attorney Docket No. 809030.000060HO... OH AcHN HO HCL. OH K n H AcHN A HQ OH CL A H H 1N NAcHN A13. The RNAi agent of any one of claims 4-12, wherein the targeting ligand is conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

14. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises a sense strand and an antisense strand, wherein said sense strand comprises at least 14 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 3-87, wherein a substitution of a uracil for any thymine in SEQ ID NOs: 3-87 does not count as a difference that contributes to said differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs 3-87; and wherein said antisense strand comprises at least 14 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences ofAttorney Docket No. 809030.000060SEQ ID NOs: 88-172, wherein a substitution of a uracil for any thymine in SEQ ID NOs: 88-172 does not count as a difference that contributes to said differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 88-172, wherein at least one of said sense strand and said antisense strand comprises one or more tris(GalNAc) moieties conjugated to one or more terminal nucleotide position, optionally via a linker or carrier.

15. The RNAi agent of any one of claims 1-14, wherein the antisense strand comprises a region of complementarity having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 110, 111, 135, 147, and 153.

16. The RNAi agent of any one of claims 1-15, wherein the sense strand comprises a region having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 25, 26, 50, 62, or 68.

17. The RNAi agent of any one of claims 1-16, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 3-87.

18. The RNAi agent of any one of claims 1-17, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 88-172.

19. The RNAi agent of any one of claims 1-18, wherein the double stranded RNAi agent comprises at least one modified nucleotide.

20. The RNAi agent of any one of claims 1-19, wherein all of the nucleotides of the sense strand are modified nucleotides.

21. The RNAi agent of any one of claims 1-20, wherein substantially all of the nucleotides of the antisense strand are modified nucleotides.

22. The RNAi agent of any of one of claims 1-21, wherein all of the nucleotides of the sense strand are modified nucleotides.Attorney Docket No. 809030.00006023. The RNAi agent of any of one of claims 1 -22, wherein all of the nucleotides of the antisense strand are modified nucleotides.

24. The RNAi agent of any one of claims 1-23, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

25. The RNAi agent of any one of claims 17-24, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy -nucleotide, a 3 '-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a T-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2'-hydroxly-modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, 3'-terminal deoxy-thymine nucleotides (dT), a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a nucleotide comprising a 5'-methylphosphonate group, a nucleotide comprising a 5' phosphate or 5' phosphate mimic, a nucleotide comprising vinyl phosphonate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA) S-Isomer, a terminal nucleotide linked to a cholesteryl derivative, a deoxyribonucleotide, and a dodecanoic acid bisdecylamide group.

26. The RNAi agent of claim 25, wherein the modifications on the nucleotides are 2'-O-methyl and 2'-fluoro modifications.

27. The RNAi agent of claim 25, further comprising at least one phosphorothioate internucleotide linkage.

28. The RNAi agent of claim 27, wherein the double stranded RNAi agent comprises 6-8 phosphorothioate intemucleotide linkages.Attorney Docket No. 809030.00006029. The RNAi agent of claim 1, wherein the region of complementarity is at least 17 nucleotides in length.

30. The RNAi agent of claim 1, wherein the region of complementarity is 19-23 nucleotides in length.

31. The RNAi agent of claim 1, wherein the region of complementarity is 19 nucleotides in length.

32. The RNAi agent of any one of the preceding claims, wherein each strand is no more than 30 nucleotides in length.

33. The RNAi agent of any one of the preceding claims, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

34. The RNAi agent of any one of the preceding claims, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

35. The RNAi agent of claim 1, wherein the region of complementarity comprises any one of the antisense sequences in any one of SEQ ID NOs: 88-172.

36. The RNAi agent of claim 1, wherein the region of complementarity consists of any one of the antisense sequences in any one of SEQ ID NOs: 88-172.

37. The RNAi agent of any one of the preceding claims, further comprising a phosphate or phosphate mimic at the 5 '-end of the antisense strand.

38. The RNAi agent of claim 37, wherein the phosphate mimic is a vinyl -phosphonate 2'-OMe-uracil (vinu).

39. The RNAi agent of any one of the preceding claims, wherein the RNAi agent comprises at least one modified nucleotide selected from the group consisting of a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, and a nucleotide comprising vinyl phosphonate, optionally wherein the RNAi agent comprises at least one of each of the following modifications:Attorney Docket No. 809030.0000602'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a deoxyribonucleotide, and a nucleotide comprising vinyl phosphonate.

40. The RNAi agent of any one of the preceding claims, wherein the RNAi agent comprises a pattern of modified nucleotides as shown in SEQ ID NOs: 173-342.

41. The RNAi agent of claim 40, wherein the double stranded region is 15-30 nucleotide pairs in length.

42. The RNAi agent of claim 41, wherein the double stranded region is 17-23 nucleotide pairs in length.

43. The RNAi agent of claim 41, wherein the double stranded region is 17-25 nucleotide pairs in length.

44. The RNAi agent of claim 41, wherein the double stranded region is 23-27 nucleotide pairs in length.

45. The RNAi agent of claim 41, wherein the double stranded region is 19-21 nucleotide pairs in length.

46. The RNAi agent of claim 40, wherein the double stranded region is 21-23 nucleotide pairs in length.

47. The RNAi agent of claim 40, wherein each strand has 15-30 nucleotides.

48. The RNAi agent of claim 40, wherein each strand has 19-30 nucleotides.

49. The RNAi agent of claim 40, wherein the modifications on the nucleotides are selected from the group consisting of LNA, glycol nucleic acid (GNA), HNA, CeNA, 2'-methoxy ethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof, preferably wherein the modifications on nucleotides are selected from the group consisting of 2'-O-m ethyl, 2 '-fluoro, and combinations thereof.

50. The RNAi agent of claim 49, wherein the modifications on the nucleotides are 2'-O-methyl or 2'-fluoro modifications.Attorney Docket No. 809030.00006051. The RNAi agent of claim 40, wherein the base pair at the 1 position of the 5'-end of the antisense strand of the duplex is an AU base pair.

52. The RNAi agent of claim 51, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

53. The RNAi agent of claim 51, wherein the sense strand has a total of 19 nucleotides and the antisense strand has a total of 21 nucleotides.

54. The RNAi agent of claim 40, wherein said RNAi agent is selected from the group of RNAi agents listed in any one of SEQ ID NOs: 173-342.

55. The RNAi agent of claim 40, wherein all of the nucleotides of said sense strand and all of the nucleotides of said antisense strand comprise a modification.

56. A cell containing the RNAi agent of any one of claims 1-55.

57. A pharmaceutical composition for inhibiting expression of an FXI gene comprising the RNAi agent of any one of claims 1-55.

58. The pharmaceutical composition of claim 57, wherein the RNAi agent is administered in an unbuffered solution.

59. The pharmaceutical composition of claim 58, wherein said unbuffered solution is saline or water.

60. The pharmaceutical composition of claim 57, wherein said RNAi agent is administered with a buffer solution.

61. The pharmaceutical composition of claim 60, wherein said buffer solution comprises acetate, citrate, prolamine, carbonate, phosphate, or any combination thereof.

62. The pharmaceutical composition of claim 60, wherein said buffer solution is phosphate buffered saline (PBS).Attorney Docket No. 809030.00006063. A method of inhibiting expression of Factor XT (FXT) in a cell, the method comprising: (a) contacting the cell with the RNAi agent of any one of claims 1-55 or the pharmaceutical composition of any one of claims 57-62; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an FXI gene, thereby inhibiting expression of FXI in the cell.

64. The method of claim 63, wherein said cell is within a subject.

65. The method of claim 64, wherein the subject is a human.

66. The method of claim 64, wherein the subject is selected from the group consisting of a rhesus monkey, a cynomolgus monkey, a mouse, and a rat.

67. The method of claim 64, wherein the human subject suffers from a disease or disorder selected from the group consisting of myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, pulmonary embolism, ischemic stroke, transient ischemic attack, retinal artery occlusion, mesenteric ischemia, renal vein thrombosis, cerebral venous sinus thrombosis, and peripheral artery disease.

68. The method of claim 67, wherein the disease or disorder is myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, pulmonary embolism or ischemic stroke.

69. The method of any one of claims 63-68, wherein the FXI expression is inhibited by at least about 30%.

70. A method of treating a subject having a disorder that would benefit from a reduction in FXI expression, comprising administering to the subject a therapeutically effective amount of the RNAi agent of any one of claims 1-55 or the pharmaceutical composition of any one of claims 57-62, thereby treating the subject.

71. The method of claim 70, wherein the subject suffers from an FXI-associated disorder.

72. The method of claim 70, wherein the subject is a human.Attorney Docket No. 809030.00006073. The method of claim 71, wherein the FXI-associated disorder is myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, and ischemic stroke.

74. The method of any one of claims 70-73, wherein the FXI expression is inhibited by at least about 30%, optionally wherein the FXI expression is inhibited in human hepatocytes from about 30% to about 90%.

75. The method of any one of claims 70-74, further comprising administering a pharmaceutically acceptable excipient to the subject, optionally wherein the pharmaceutically acceptable excipient is an additive (e.g., a pH modifier).

76. The method of any one of claims 70-75, wherein the RNAi agent is administered at a dose of about 0.01 mg / kg to about 50 mg / kg.

77. The method of any one of claims 70-76, wherein the RNAi agent is administered to the subject intravenously or subcutaneously.

78. The method of claim 70, wherein the method reduces the expression of a target gene in a liver tissue, optionally a target gene expressed by a hepatocyte, optionally wherein the hepatocyte is a primary hepatocyte.

79. A method of inhibiting the expression of FXI in a subject, the method comprising: administering to said subject a therapeutically effective amount of the RNAi agent of any one of claims 1-55 or the pharmaceutical composition of any one of claims 57-62, thereby inhibiting the expression of FXI in said subject.

80. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises a sense strand and an antisense strand, and wherein said antisense strand comprises a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense strand nucleobase sequences of a duplex selected from the group consisting of SEQ ID NOs: 88-172.

81. The RNAi agent of claim 80, wherein the RNAi agent comprises one or more modifications selected from the group consisting of a 2'-O-methyl modified nucleotide, a 2'-fluoro modifiedAttorney Docket No. 809030.000060nucleotide, a 2 '-C -alkyl -modified nucleotide, a nucleotide comprising a glycol nucleic acid (GNA), a phosphorothioate (PS), a deoxyribonucleotide, and a vinyl-phosphonate 2'-0Me-uracil (vinu), optionally wherein said RNAi agent comprises at least one of each modification selected from the group consisting of a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-C-alkylmodified nucleotide, a nucleotide comprising a glycol nucleic acid (GNA), a phosphorothioate and a vinyl-phosphonate 2'-0Me-uracil (vinu).

82. The RNAi agent of claim 80 or claim 81, wherein the RNAi agent comprises four or more PS modifications, optionally six to ten PS modifications, optionally six PS modifications.

83. A method for treating or preventing a FXI-associated disease or disorder in a subject, the method comprising administering to said subject a therapeutically effective amount of the RNAi agent of any one of claims 1-55 or 80-82 or the pharmaceutical composition of any one of claims 57-62, thereby treating or preventing a FXI-associated disease or disorder in the subject.

84. The method of claim 83, wherein the FXI-associated disease or disorder is selected from the group consisting of myocardial infarction, atrial fibrillation (AFib), deep vein thrombosis, pulmonary embolism, ischemic stroke, transient ischemic attack, retinal artery occlusion, mesenteric ischemia, renal vein thrombosis, cerebral venous sinus thrombosis, and peripheral artery disease.

85. A kit for performing the method of any one of claims 70-79, comprising a) the RNAi agent, and b) instructions for use, and c) optionally, a means for administering the RNAi agent to the subject.

86. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand and (ii) an antisense strand comprising 5’-UUGAUAUAAGAAAAUCAUCCU-3’ (SEQ ID NO: 110).

87. The RNAi agent of claim 86, wherein the antisense strand comprising SEQ ID NO: 110 further comprises the following modifications (position 1 being the 5’-terminal position of the antisense strand and position numbering proceeding from 5’ to 3’):(a) the 5 ’-end (position 1) is modified with a vinylphosphonate group;Attorney Docket No. 809030.000060(b) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2, 2-3, 19-20, and 20-21;(c) 2’-fluoro modified ribonucleotides at each of positions 2, 6, 8, 9, 14, and 16; and (d) 2’-O-methyl modified ribonucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21.

88. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand comprising 5’-GAUGAUUUUCUUAUAUCAA-3’ (SEQ ID NO: 25) and (ii) an antisense strand.

89. The RNAi agent of claim 88, wherein the sense strand comprising SEQ ID NO: 25 further comprises the following modifications (position 1 being the 5’-terminal position of the sense strand and position numbering proceeding from 5’ to 3’):(a) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2 and 2-3;(b) 2’ -fluoro modified ribonucleotides at each of positions 5, 7, 8, and 9;(c) 2’-O-methyl modified ribonucleotides at each of positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19; and(d) the 3 ’-end (position 19) is modified with a triantennary N-acetylgalactosamine (GalNAc) ligand.

90. The RNAi agent of claim 89, wherein the triantennary N-acetylgalactosamine (GalNAc) ligand is a 3'-Prolinol / C12-linker with attached trivalent GalNAc:HO OHl<.nH HAcHN g QHO HO, OH OAcHN J O OJ HOHO, OHIXnH H lHO -v-r— V O N N ' 'QAcHN AAttorney Docket No. 809030.00006091. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand comprising 5’-gsasugAfuUfUfUfcuuauaucaa-3’ (SEQ ID NO: 195) and (ii) an antisense strand comprising 5’-(vinu)sUfsgauAfuAfAfgaaaAfuCfaucscsu-3’ (SEQ ID NO: 280).

92. The RNAi agent of claim 91, wherein the 3 ’-end of the sense strand (position 19) is modified with a triantennary N-acetylgalactosamine (GalNAc) ligand, optionally wherein the triantennary N-acetylgalactosamine (GalNAc) ligand is a 3'-Prolinol / C12-linker with attached trivalent GalNAc:HO.. OHNNV-°AcHN A 1° > HOHO. OH Ql£. H H: O ( \x-^N V0^AcHN I ¥ " J N ~ ~ Uo o 0HoHO. OHi. S;..n H H lHO N.~x^x N " AAcHN A93. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises:(i) a sense strand comprising 5’-GAUGAUUUUCUUAUAUCAA-3’ (SEQ ID NO: 25) and the following modifications (position 1 being the 5 ’-terminal position of the sense strand and position numbering proceeding from 5’ to 3’):(a) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2 and 2-3;(b) 2’-fluoro modified ribonucleotides at each of positions 5, 7, 8, and 9;(c) 2’-O-methyl modified ribonucleotides at each of positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19; andAttorney Docket No. 809030.000060(d) the 3’-end (position 19) is modified with a 3'-Prolinol / C12-linker with attached tri valent GalNAc:HO -OHiS H HAcHN JI I„, 'l HOHO OH Q„ „ 7"“\HO& A-'0A SX-X. N,^.0ACHN T HQ. OH.,AcHN £.and (ii) an antisense strand comprising 5’-UUGAUAUAAGAAAAUCAUCCU-3’ (SEQ ID NO: 110) and the following modifications (position 1 being the 5’-terminal position of the antisense strand and position numbering proceeding from 5’ to 3’):(e) the 5 ’-end (position 1) is modified with a vinylphosphonate group;(f) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2, 2-3, 19-20, and 20-21;(g) 2’-fluoro modified ribonucleotides at each of positions 2, 6, 8, 9, 14, and 16; and(h) 2’-O-methyl modified ribonucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21.

94. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand and (ii) an antisense strand comprising 5’-UUUGAUAUAAGAAAAUCAUCC-3’ (SEQ ID NO: 111).

95. The RNAi agent of claim 94, wherein the antisense strand comprising SEQ ID NO: 111 further comprises the following modifications (position 1 being the 5 ’-terminal position of the antisense strand and position numbering proceeding from 5’ to 3’):(a) the 5 ’-end (position 1) is modified with a vinylphosphonate group;(b) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2, 2-3, 19-20, and 20-21;(c) 2’-fluoro modified ribonucleotides at each of positions 2, 6, 8, 9, 14, and 16; andAttorney Docket No. 809030.000060(d) 2’-0-methyl modified ribonucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21.

96. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand comprising 5’-AUGAUUUUCUUAUAUCAAA-3’ (SEQ ID NO: 26) and (ii) an antisense strand.

97. The RNAi agent of claim 96, wherein the sense strand comprising SEQ ID NO: 26 further comprises the following modifications (position 1 being the 5’-terminal position of the sense strand and position numbering proceeding from 5’ to 3’):(a) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2 and 2-3;(b) 2’ -fluoro modified ribonucleotides at each of positions 5, 7, 8, and 9;(c) 2’-O-methyl modified ribonucleotides at each of positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19; and(d) the 3 ’-end (position 19) is modified with a triantennary N-acetylgalactosamine (GalNAc) ligand.

98. The RNAi agent of claim 97, wherein the triantennary N-acetylgalactosamine (GalNAc) ligand is a 3'-Prolinol / C12-linker with attached trivalent GalNAc:AcHNO N A HO, OHHOAcHN99. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand comprisingAttorney Docket No. 809030.0000605’-asusgaUfuUfUfCfuuauaucaaa-3’ (SEQ ID NO: 196) and (ii) an antisense strand comprising 5’-(vinu)sUfsugaUfaUfAfagaaAfaUfcauscsc-3’ (SEQ ID NO: 281).

100. The RNAi agent of claim 99, wherein the 3 ’-end of the sense strand (position 19) is modified with a triantennary N-acetylgalactosamine (GalNAc) ligand, optionally wherein the triantennary N-acetylgalactosamine (GalNAc) ligand is a 3'-Prolinol / C12-linker with attached tri valent GalNAc:KnH HN N-°AcHN 1 I0''I HOHOZOH oK n H H 3 0 4 \ „£ d O-JH 1HOPHurJHO & X--70^- n0 s / X / V M N ^x^x S NtA,AcHN A101. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises:(i) a sense strand comprising 5’-AUGAUUUUCUUAUAUCAAA-3’ (SEQ ID NO: 26) and the following modifications (position 1 being the 5 ’-terminal position of the sense strand and position numbering proceeding from 5’ to 3’):(a) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2 and 2-3;(b) 2’-fluoro modified ribonucleotides at each of positions 5, 7, 8, and 9;(c) 2’-O-methyl modified ribonucleotides at each of positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19; andAttorney Docket No. 809030.000060(d) the 3’-end (position 19) is modified with a 3'-Prolinol / C12-linker with attached tri valent GalNAc:HO -OHiS H HAcHN JI I„, 'l HOHO OH Q„ „ 7"“\HO& A-'0A SX-X. N,^.0ACHN T HQ. OH.,AcHN £.and (ii) an antisense strand comprising 5’-UUUGAUAUAAGAAAAUCAUCC-3’ (SEQ ID NO: 111) and the following modifications (position 1 being the 5’-terminal position of the antisense strand and position numbering proceeding from 5’ to 3’):(e) the 5 ’-end (position 1) is modified with a vinylphosphonate group;(f) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2, 2-3, 19-20, and 20-21;(g) 2’-fluoro modified ribonucleotides at each of positions 2, 6, 8, 9, 14, and 16; and(h) 2’-O-methyl modified ribonucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21.

102. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand and (ii) an antisense strand comprising 5’-UUUGAAGAAAGCUUUAAGUAA-3’ (SEQ ID NO: 135).

103. The RNAi agent of claim 102, wherein the antisense strand comprising SEQ ID NO: 135 further comprises the following modifications (position 1 being the 5 ’-terminal position of the antisense strand and position numbering proceeding from 5’ to 3’):(a) the 5 ’-end (position 1) is modified with a vinylphosphonate group;(b) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2, 2-3, 19-20, and 20-21;(c) 2’-fluoro modified ribonucleotides at each of positions 2, 6, 8, 9, 14, and 16; andAttorney Docket No. 809030.000060(d) 2’-0-methyl modified ribonucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21.

104. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand comprising 5’-ACUUAAAGCUUUCUUCAAA-3’ (SEQ ID NO: 50) and (ii) an antisense strand.

105. The RNAi agent of claim 104, wherein the sense strand comprising SEQ ID NO: 50 further comprises the following modifications (position 1 being the 5’-terminal position of the sense strand and position numbering proceeding from 5’ to 3’):(a) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2 and 2-3;(b) 2’ -fluoro modified ribonucleotides at each of positions 5, 7, 8, and 9;(c) 2’-O-methyl modified ribonucleotides at each of positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19; and(d) the 3 ’-end (position 19) is modified with a triantennary N-acetylgalactosamine (GalNAc) ligand.

106. The RNAi agent of claim 105, wherein the triantennary N-acetylgalactosamine (GalNAc) ligand is a 3'-Prolinol / C12-linker with attached trivalent GalNAc:AcHNO N A HO, OHHOAcHN107. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand comprisingAttorney Docket No. 809030.0000605’-ascsuuAfaAfGfCfuuucuucaaa-3’ (SEQ ID NO: 220) and (ii) an antisense strand comprising 5’-(vinu)sUfsugaAfgAfAfagcuUfuAfagusasa-3’ (SEQ ID NO: 305).

108. The RNAi agent of claim 107, wherein the 3’-end of the sense strand (position 19) is modified with a triantennary N-acetylgalactosamine (GalNAc) ligand, optionally wherein the triantennary N-acetylgalactosamine (GalNAc) ligand is a 3'-Prolinol / C12-linker with attached tri valent GalNAc:KnH HN N-°AcHN 1 I0''I HOHOZOH oK n H H t O; [ \£ d O-JH 1HOPHurJHO V X-7-o^-n0 ^xx-s, H N ^Xx H N iAcHN A109. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises:(i) a sense strand comprising 5’-ACUUAAAGCUUUCUUCAAA-3’ (SEQ ID NO: 50) and the following modifications (position 1 being the 5 ’-terminal position of the sense strand and position numbering proceeding from 5’ to 3’):(a) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2 and 2-3;(b) 2’-fluoro modified ribonucleotides at each of positions 5, 7, 8, and 9;(c) 2’-O-methyl modified ribonucleotides at each of positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19; andAttorney Docket No. 809030.000060(d) the 3’-end (position 19) is modified with a 3'-Prolinol / C12-linker with attached tri valent GalNAc:HO -OHiS H HAcHN JI I„, 'l HOHO OH Q„ „ 7"“\HO& A-'0A SX-X. N,^.0ACHN T HQ. OH.,AcHN £.and (ii) an antisense strand comprising 5’-UUUGAAGAAAGCUUUAAGUAA-3’ (SEQ ID NO: 135) and the following modifications (position 1 being the 5’-terminal position of the antisense strand and position numbering proceeding from 5’ to 3’):(e) the 5 ’-end (position 1) is modified with a vinylphosphonate group;(f) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2, 2-3, 19-20, and 20-21;(g) 2’-fluoro modified ribonucleotides at each of positions 2, 6, 8, 9, 14, and 16; and(h) 2’-O-methyl modified ribonucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21.

110. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand and (ii) an antisense strand comprising 5’-UAGAAAAUCAUCCUGAAAAGACC-3’ (SEQ ID NO: 147).

111. The RNAi agent of claim 110, wherein the antisense strand comprising SEQ ID NO: 147 further comprises the following modifications (position 1 being the 5 ’-terminal position of the antisense strand and position numbering proceeding from 5’ to 3’):(a) the 5 ’-end (position 1) is modified with a vinylphosphonate group;(b) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2, 2-3, 21-22, and 22-23;(c) 2’-fluoro modified ribonucleotides at each of positions 2, 6, 8, 9, 14, and 16; andAttorney Docket No. 809030.000060(d) 2’-0-methyl modified ribonucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23.

112. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand comprising 5’-UCUUUUCAGGAUGAUUUUCUA-3’ (SEQ ID NO: 62) and (ii) an antisense strand.

113. The RNAi agent of claim 112, wherein the sense strand comprising SEQ ID NO: 62 further comprises the following modifications (position 1 being the 5’-terminal position of the sense strand and position numbering proceeding from 5’ to 3’):(a) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2 and 2-3;(b) 2’-fluoro modified ribonucleotides at each of positions 7, 9, 10, and 11;(c) 2’-O-methyl modified ribonucleotides at each of positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21; and(d) the 3 ’-end (position 21) is modified with a triantennary N-acetylgalactosamine (GalNAc) ligand.

114. The RNAi agent of claim 113, wherein the triantennary N-acetylgalactosamine (GalNAc) ligand is a 3'-Prolinol / C12-linker with attached trivalent GalNAc:AcHNO N A HO, OHHOAcHN115. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand comprisingAttorney Docket No. 809030.0000605’-uscsuuuuCfaGfGfAfugauuuucua-3’ (SEQ ID NO: 232) and (ii) an antisense strand comprising 5’-(vinu)sAfsgaaAfaUfCfauccUfgAfaaagascsc-3’ (SEQ ID NO: 317).

116. The RNAi agent of claim 115, wherein the 3’-end of the sense strand (position 21) is modified with a triantennary N-acetylgalactosamine (GalNAc) ligand, optionally wherein the triantennary N-acetylgalactosamine (GalNAc) ligand is a 3'-Prolinol / C12-linker with attached tri valent GalNAc:KnH HN N-°AcHN 1 I0''I HOHOZOH oK n H H 3 0 4 \ „£ d O-JH 1HOPHuHO V A^--0v- n0 MNb N-AAcHN A117. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises:(i) a sense strand comprising 5’-UCUUUUCAGGAUGAUUUUCUA-3’ (SEQ ID NO: 62) and the following modifications (position 1 being the 5 ’-terminal position of the sense strand and position numbering proceeding from 5’ to 3’):(a) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2 and 2-3;(b) 2’-fluoro modified ribonucleotides at each of positions 7, 9, 10, and 11;(c) 2’-O-methyl modified ribonucleotides at each of positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21; andAttorney Docket No. 809030.000060(d) the 3’-end (position 21) is modified with a 3'-Prolinol / C12-linker with attached tri valent GalNAc:HO -OHiS H HAcHN JI I„, ''I HOHO OH Q„ „ 7"“\HO& A-'0A SX-X. N,^.0ACHN T HQ. OH.,AcHN £.and (ii) an antisense strand comprising 5’-UAGAAAAUCAUCCUGAAAAGACC-3’ (SEQ ID NO: 147) and the following modifications (position 1 being the 5 ’-terminal position of the antisense strand and position numbering proceeding from 5’ to 3’):(e) the 5 ’-end (position 1) is modified with a vinylphosphonate group;(f) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2, 2-3, 21-22, and 22-23;(g) 2’-fluoro modified ribonucleotides at each of positions 2, 6, 8, 9, 14, and 16; and(h) 2’-O-methyl modified ribonucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23.

118. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand and (ii) an antisense strand comprising 5’-UGAUAUAAGAAAAUCAUCCUGAA-3’ (SEQ ID NO: 153).

119. The RNAi agent of claim 118, wherein the antisense strand comprising SEQ ID NO: 153 further comprises the following modifications (position 1 being the 5 ’-terminal position of the antisense strand and position numbering proceeding from 5’ to 3’):(a) the 5 ’-end (position 1) is modified with a vinylphosphonate group;(b) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2, 2-3, 21-22, and 22-23;(c) 2’-fluoro modified ribonucleotides at each of positions 2, 6, 8, 9, 14, and 16; andAttorney Docket No. 809030.000060(d) 2’-0-methyl modified ribonucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23.

120. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand comprising 5’-CAGGAUGAUUUUCUUAUAUCA-3’ (SEQ ID NO: 68) and (ii) an antisense strand.

121. The RNAi agent of claim 120, wherein the sense strand comprising SEQ ID NO: 68 further comprises the following modifications (position 1 being the 5’-terminal position of the sense strand and position numbering proceeding from 5’ to 3’):(a) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2 and 2-3;(b) 2’-fluoro modified ribonucleotides at each of positions 7, 9, 10, and 11;(c) 2’-O-methyl modified ribonucleotides at each of positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21; and(d) the 3 ’-end (position 21) is modified with a triantennary N-acetylgalactosamine (GalNAc) ligand.

122. The RNAi agent of claim 121, wherein the triantennary N-acetylgalactosamine (GalNAc) ligand is a 3'-Prolinol / C12-linker with attached trivalent GalNAc:AcHNO N A HO, OHHOAcHN123. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises (i) a sense strand comprisingAttorney Docket No. 809030.0000605’-csasggauGfaUfUfUfucuuauauca-3’ (SEQ ID NO: 238) and (ii) an antisense strand comprising 5’-(vinu)sGfsauaUfaAfGfaaaaUfcAfuccugsasa-3’ (SEQ ID NO: 323).

124. The RNAi agent of claim 123, wherein the 3’-end of the sense strand (position 21) is modified with a triantennary N-acetylgalactosamine (GalNAc) ligand, optionally wherein the triantennary N-acetylgalactosamine (GalNAc) ligand is a 3'-Prolinol / C12-linker with attached tri valent GalNAc:KnH HN N-°AcHN 1 I0''I HOHOZOH oK n H H t O; [ \£ d O-JH 1HOPHurJHO V N-r-o^-n0 ^XX-S, H N ^Xx H N iAcHN A125. A double stranded ribonucleic acid interference agent (RNAi agent) for inhibiting expression of Factor XI (FXI), wherein said RNAi agent comprises:(i) a sense strand comprising 5’-CAGGAUGAUUUUCUUAUAUCA-3’ (SEQ ID NO: 68) and the following modifications (position 1 being the 5 ’-terminal position of the sense strand and position numbering proceeding from 5’ to 3’):(a) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2 and 2-3;(b) 2’-fluoro modified ribonucleotides at each of positions 7, 9, 10, and 11;(c) 2’-O-methyl modified ribonucleotides at each of positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21; andAttorney Docket No. 809030.000060(d) the 3’-end (position 21) is modified with a 3'-Prolinol / C12-linker with attached trivalent GalNAc:HO,0HH H, NAcHN 11O HO OHAcHNHQ. OHAcHN £; and (ii) an antisense strand comprising 5’-UGAUAUAAGAAAAUCAUCCUGAA-3’ (SEQ ID NO: 153) and the following modifications (position 1 being the 5’-terminal position of the antisense strand and position numbering proceeding from 5’ to 3’):(e) the 5 ’-end (position 1) is modified with a vinylphosphonate group;(f) phosphorothioate linkages are present between each of the following adjacent pairs of residues: 1-2, 2-3, 21-22, and 22-23;(g) 2’-fluoro modified ribonucleotides at each of positions 2, 6, 8, 9, 14, and 16; and(h) 2’-O-methyl modified ribonucleotides at each of positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23.

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