Modified double stranded RNA agents

Abstract

The disclosure relates to modified double-stranded ribonucleic acid (dsRNA) targeting a target gene, and methods of using the modified dsRNA to inhibit expression of the target gene.

Description

Attorney Docket No.: BCR-007WO MODIFIED DOUBLE STRANDED RNA AGENTS CROSS-REFERENCE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 445,669, filed February 14, 2023, U.S. Provisional Patent Application No.63 / 445,673, filed February 14, 2023, and U.S. Provisional Patent Application No.63 / 445,676, filed, February 14, 2023, each of which is incorporated by reference in its entirety herein. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing XML which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML copy, created on Month XX, 20XX, is named XXXXX, and is XXX,XXX bytes in size. FIELD

[0003] The disclosure relates to nucleotide modifications in double-stranded ribonucleic acids (dsRNA) targeting genes in a genome, and methods of using the modified dsRNA to inhibit expression of genes in a genome. SUMMARY

[0004] The present disclosure is based, in part, upon the development of modified double- stranded ribonucleic acid (dsRNA) targeting mRNA transcripts of target genes in a genome, pharmaceutical compositions comprising the dsRNAs targeting target genes and methods of using the dsRNA to inhibit expression of target genes in a cell.

[0005] In some aspects, the disclosure provides for a modified double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a target gene comprising a sense strand and an antisense strand each 15 to 30 nucleotides in length.

[0006] In some embodiments, the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end; a 2'-fluoro nucleotide at positions 2, 4, 6, 8, and 14 from the 5’ end; or a 2'-fluoro nucleotide at positions 2, 4, 6, 8, 14, and 16 from the 5’ end. In some embodiments, the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 12, 11, and 9 from the 3’ end; a 2'-fluoro nucleotide at positions 15, 13, 11, and 9 from the 3’ end; a 2'- fluoro nucleotide at positions 13, 12, and 11 from the 3’ end; a 2'-fluoro nucleotide at positions 13, 12, 11, and 9 from the 3’ end; or a 2'-fluoro nucleotide at positions 15, 13, 12, and 9 from the 3’ end.

[0007] In some embodiments, the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 12, 11, and 9 from the 3’ end; a 2'-fluoro nucleotide at positions 15, 13, 11, and 9 from the 3’Attorney Docket No.: BCR-007WO end; a 2'-fluoro nucleotide at positions 13, 12, and 11 from the 3’ end; a 2'-fluoro nucleotide at positions 13, 12, 11, and 9 from the 3’ end; or a 2'-fluoro nucleotide at positions 15, 13, 12, and 9 from the 3’ end.

[0008] In some embodiments, the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 12, 11, and 9 from the 3’ end; the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 13, 12, 11, and 9 from the 3’ end; antisense strand comprises a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 12, and 9 from the 3’ end; the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 4, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 11, and 9 from the 3’ end; or the antisense strand comprises a 2'- fluoro nucleotide at positions 2, 4, 6, 8, 14, and 16 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 13, 12, and 11 from the 3’ end.

[0009] In some embodiments, the antisense strand has a 3’ end nucleotide overhang compared to the sense strand. In some embodiments, the 3’ end nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand. In some embodiments, the antisense and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. In some embodiments, the antisense strand and the sense strand are at least 80% complementary. In some embodiments, the antisense strand and the sense strand comprise at least one, at least two, at least three, or at least four mismatched nucleotides. In some embodiments, the antisense strand comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a target mRNA corresponding to the target gene. In some embodiments, the antisense strand of the dsRNA comprises at least 80% complementarity to a target mRNA corresponding to the target gene. In some embodiments, the antisense strand of the dsRNA comprises one, two, three, or four mismatches to a target mRNA corresponding to the target gene.

[0010] In some embodiments, at least one nucleotide of the dsRNA is a modified nucleotide. In some embodiments, the modified nucleotide is at least one of: a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide; an inverted abasic nucleotide, a thymidine-glycol nucleic acid (GNA) S-Isomer; an inosine, and inverted deoxyribonucleotide (3'-3' linked nucleotide), and a thymidine-glycol nucleic acid (GNA) S-Isomer. In some embodiments, the modified nucleotide is at least one of: 5’-vinyl phosphonate nucleotide, a 5’-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2’-MOE (methoxyethyl)nucleotide, and / or a 2’-arabino fluoro (2’-araF) nucleotide. InAttorney Docket No.: BCR-007WO some embodiments, the antisense strand comprises a phosphate mimic at the 5’ end; optionally wherein the phosphate mimic is a 5'-E-Vinyl-phosphonate or a 4'-O-phosphonate. In some embodiments, the modified nucleotide is at least one of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2’-amino- modified nucleotide, 2’-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and / or a non-natural base comprising nucleotide.

[0011] In some embodiments, the antisense strand and / or the sense strand comprises at least one internucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoromorpholidate linkage, a phosphoropiperazidate linkage, an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, and a boranophosphate linkage. In some embodiments, the antisense strand and / or the sense strand comprises at least one nucleotide modified linkage. In some embodiments, all the nucleotide linkages in the antisense strand are modified linkages. In some embodiments, the antisense strand and / or the sense strand comprises at least one a phosphorothioate (PS) bond.

[0012] In some embodiments, the dsRNA further comprises a ligand or targeting moiety. In some embodiments, the ligand or targeting moiety is conjugated to the 5’ end, 3’ end or both ends of the dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 3’ end of the sense strand of the dsRNA. In some embodiments, ligand or targeting moiety is at least one N-Acetyl-Galactosamine (GalNAc).

[0013] In some embodiments, a cell comprising a dsRNA of the disclosure is provided. In some embodiments, a vector encoding at least one strand a dsRNA of the disclosure is provided. of the disclosure is provided a cell comprising the vector is provided.

[0014] In some embodiments, a pharmaceutical composition for inhibiting expression of the target gene comprising the dsRNA and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof of the disclosure is provided.

[0015] In some embodiments, a method of inhibiting target gene expression in a cell is provided, the method comprising (a) contacting the cell with the dsRNA of the disclosure or the pharmaceutical composition of the disclosure; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an target gene, thereby inhibiting expression of the target gene in the cell. In some embodiments, the target gene expression is inhibited by at least 30% relative to a control. In some embodiments, the targetAttorney Docket No.: BCR-007WO gene expression is inhibited by at least 30% relative to a negative control dsRNA, optionally wherein target gene expression is measured using a quantitative polymerase chain reaction (PCR) assay.

[0016] In some embodiments, a method of treating a disorder mediated by the target gene is provided, comprising administering to a subject in need of such treatment a therapeutically effective amount of a dsRNA of the disclosure, or a pharmaceutical composition of the disclosure.

[0017] The present disclosure is based, in part, upon the development of double-stranded ribonucleic acid (dsRNA) targeting APOC3 genes, pharmaceutical compositions comprising the dsRNAs targeting APOC3 genes and methods of using the dsRNA to inhibit expression of APOC3 in a cell.

[0018] In some aspects, the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of APOC3 comprising a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence shown in the relevant tables.

[0019] In some aspects, the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of APOC3, wherein the dsRNA is BC-100001, BC-100015, BC- 100019, BC-100020, BC-100021, BC-100022, BC-100024, BC-100026, BC-100027, BC- 100028, BC-100031, or BC-100035.

[0020] In some embodiments, the APOC3 gene is human APOC3. In some embodiments, the APOC3 is human APOC3 comprising the sequence shown in NM_000040.3.

[0021] In some embodiments, the sense strand is 70 - 80% or more identical to the sense strands listed in the relevant tables. In some embodiments, the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence shown in the relevant tables. In some embodiments, the sense strand comprises at least 16, 17, 18, 19, 20, or 21 contiguous nucleotides of a sense strand sequence shown in the relevant tables. In some embodiments, the sense strand comprises 21 contiguous nucleotides of a sense strand sequence shown in the relevant tables. In some embodiments, the antisense strand comprises at least 16, 17, 18, 19, 20, or 21 contiguous nucleotides of an antisense sense strand sequence shown in the relevant tables. In some embodiments, the antisense strand comprises 21 contiguous nucleotides of an antisense sense strand sequence shown in the relevant tables. In some embodiments, the sense strand sequence is selected from a sense strand sequence shown in the relevant tables, and the antisense strand is selected from an antisense strand sequence shown in the relevant tables. In some embodiments, the sense strand sequence is selected from a sense strand sequence shown in theAttorney Docket No.: BCR-007WO relevant tables. In some embodiments, the antisense strand is selected from an antisense strand sequence shown in the relevant tables.

[0022] In some embodiments, the antisense strand has a 3’ end nucleotide overhang compared to the sense strand. In some embodiments, the 3’ end nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand. In some embodiments, the antisense and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. In some embodiments, the antisense strand and the sense strand are at least 80% complementary. In some embodiments, the antisense strand and the sense strand comprise at least one, at least two, at least three, or at least four mismatched nucleotides. In some embodiments, the antisense strand comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a target mRNA corresponding to a fragment of APOC3 mRNA. In some embodiments, the antisense strand of the dsRNA comprises at least 80% complementarity to the fragment of the APOC3 mRNA. In some embodiments, the antisense strand of the dsRNA comprises one, two, three, or four mismatches to the fragment of the APOC3 mRNA.

[0023] In some embodiments, at least one nucleotide of the dsRNA is a modified nucleotide. In some embodiments, the modified nucleotide is at least one of: a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide; an inverted abasic nucleotide, a thymidine-glycol nucleic acid (GNA) S-Isomer; an inosine, and inverted deoxyribonucleotide (3'-3' linked nucleotide), and a thymidine-glycol nucleic acid (GNA) S-Isomer. In some embodiments, the modified nucleotide is at least one of: 5’-vinyl phosphonate nucleotide, a 5’-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2’-MOE (methoxyethyl)nucleotide, and / or a 2’-arabino fluoro (2’-araF) nucleotide. In some embodiments, the antisense strand comprises a phosphate mimic at the 5’ end; optionally wherein the phosphate mimic is a 5'-E-Vinyl-phosphonate or a 4'-O-phosphonate. In some embodiments, the modified nucleotide is at least one of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2’-amino- modified nucleotide, 2’-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and / or a non-natural base comprising nucleotide.

[0024] In some embodiments, the antisense strand and / or the sense strand comprises at least one internucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoromorpholidate linkage, a phosphoropiperazidate linkage, an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, aAttorney Docket No.: BCR-007WO thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, and a boranophosphate linkage. In some embodiments, the antisense strand and / or the sense strand comprises at least one nucleotide modified linkage. In some embodiments, all the nucleotide linkages in the antisense strand are modified linkages. In some embodiments, the antisense strand and / or the sense strand comprises at least one a phosphorothioate (PS) bond.

[0025] In some embodiments, the dsRNA further comprises a ligand or targeting moiety. In some embodiments, the ligand or targeting moiety is conjugated to the 5’ end, 3’ end or both ends of the dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 3’ end of the sense strand of the dsRNA. In some embodiments, ligand or targeting moiety is at least one N-Acetyl-Galactosamine (GalNAc).

[0026] In some embodiments, a cell comprising a dsRNA of the disclosure is provided. In some embodiments, a vector encoding at least one strand a dsRNA of the disclosure is provided. of the disclosure is provided a cell comprising the vector is provided.

[0027] In some embodiments, a pharmaceutical composition for inhibiting expression of APOC3 comprising the dsRNA and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof of the disclosure is provided.

[0028] In some embodiments, a method of inhibiting APOC3 expression in a cell is provided, the method comprising (a) contacting the cell with the dsRNA of the disclosure or the pharmaceutical composition of the disclosure; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an APOC3 gene, thereby inhibiting expression of the APOC3 gene in the cell. In some embodiments, the APOC3 expression is inhibited by at least 30% relative to a control.

[0029] In some embodiments, a method of treating a disorder mediated by APOC3 is provided, comprising administering to a subject in need of such treatment a therapeutically effective amount of a dsRNA of the disclosure, or a pharmaceutical composition of the disclosure. In some embodiments, the disorder is a cardiovascular disorder. In some embodiments, the disorder is cardiovascular disease.

[0030] The present disclosure is based, in part, upon the development of double-stranded ribonucleic acid (dsRNA) targeting ANGPTL3 genes, pharmaceutical compositions comprising the dsRNAs targeting ANGPTL3 genes and methods of using the dsRNA to inhibit expression of ANGPTL3 in a cell.

[0031] In some aspects, the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ANGPTL3 comprising a sense strand and an antisense strand eachAttorney Docket No.: BCR-007WO 15 to 30 nucleotides in length, wherein the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence shown in the relevant tables.

[0032] In some aspects, the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ANGPTL3, wherein the dsRNA is BC-100042, BC-100044, BC- 100047, BC-100054, BC-100062, BC-100067, BC-100069, BC-100085, BC-100109, BC- 100115, BC-100127, or BC-100129.

[0033] In some embodiments, the ANGPTL3 gene is human ANGPTL3. In some embodiments, the ANGPTL3 is human ANGPTL3 comprising the sequence shown in NM_014495.4.

[0034] In some embodiments, the sense strand is 70 - 80% or more identical to the sense strands listed in the relevant tables. In some embodiments, the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence shown in the relevant tables. In some embodiments, the sense strand comprises at least 16, 17, 18, 19, 20, or 21 contiguous nucleotides of a sense strand sequence shown in the relevant tables. In some embodiments, the sense strand comprises 21 contiguous nucleotides of a sense strand sequence shown in the relevant tables. In some embodiments, the antisense strand comprises at least 16, 17, 18, 19, 20, or 21 contiguous nucleotides of an antisense sense strand sequence shown in the relevant tables. In some embodiments, the antisense strand comprises 21 contiguous nucleotides of an antisense sense strand sequence shown in the relevant tables. In some embodiments, the sense strand sequence is selected from a sense strand sequence shown in the relevant tables, and the antisense strand is selected from an antisense strand sequence shown in the relevant tables. In some embodiments, the sense strand sequence is selected from a sense strand sequence shown in the relevant tables. In some embodiments, the antisense strand is selected from an antisense strand sequence shown in the relevant tables.

[0035] In some embodiments, the antisense strand has a 3’ end nucleotide overhang compared to the sense strand. In some embodiments, the 3’ end nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand. In some embodiments, the antisense and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. In some embodiments, the antisense strand and the sense strand are at least 80% complementary. In some embodiments, the antisense strand and the sense strand comprise at least one, at least two, at least three, or at least four mismatched nucleotides. In some embodiments, the antisense strand comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a target mRNA corresponding to a fragment of ANGPTL3 mRNA. In some embodiments, the antisense strand of the dsRNA comprises at least 80% complementarity to the fragment of the ANGPTL3 mRNA. In some embodiments, the antisenseAttorney Docket No.: BCR-007WO strand of the dsRNA comprises one, two, three, or four mismatches to the fragment of the ANGPTL3 mRNA.

[0036] In some embodiments, at least one nucleotide of the dsRNA is a modified nucleotide. In some embodiments, the modified nucleotide is at least one of: a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide; an inverted abasic nucleotide, a thymidine-glycol nucleic acid (GNA) S-Isomer; an inosine, and inverted deoxyribonucleotide (3'-3' linked nucleotide), and a thymidine-glycol nucleic acid (GNA) S-Isomer. In some embodiments, the modified nucleotide is at least one of: 5’-vinyl phosphonate nucleotide, a 5’-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2’-MOE (methoxyethyl)nucleotide, and / or a 2’-arabino fluoro (2’-araF) nucleotide. In some embodiments, the antisense strand comprises a phosphate mimic at the 5’ end; optionally wherein the phosphate mimic is a 5'-E-Vinyl-phosphonate or a 4'-O-phosphonate. In some embodiments, the modified nucleotide is at least one of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2’-amino- modified nucleotide, 2’-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and / or a non-natural base comprising nucleotide.

[0037] In some embodiments, the antisense strand and / or the sense strand comprises at least one internucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoromorpholidate linkage, a phosphoropiperazidate linkage, an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, and a boranophosphate linkage. In some embodiments, the antisense strand and / or the sense strand comprises at least one nucleotide modified linkage. In some embodiments, all the nucleotide linkages in the antisense strand are modified linkages. In some embodiments, the antisense strand and / or the sense strand comprises at least one a phosphorothioate (PS) bond.

[0038] In some embodiments, the dsRNA further comprises a ligand or targeting moiety. In some embodiments, the ligand or targeting moiety is conjugated to the 5’ end, 3’ end or both ends of the dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 3’ end of the sense strand of the dsRNA. In some embodiments, ligand or targeting moiety is at least one N-Acetyl-Galactosamine (GalNAc).Attorney Docket No.: BCR-007WO

[0039] In some embodiments, a cell comprising a dsRNA of the disclosure is provided. In some embodiments, a vector encoding at least one strand a dsRNA of the disclosure is provided. of the disclosure is provided a cell comprising the vector is provided.

[0040] In some embodiments, a pharmaceutical composition for inhibiting expression of ANGPTL3 comprising the dsRNA and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof of the disclosure is provided.

[0041] In some embodiments, a method of inhibiting ANGPTL3 expression in a cell is provided, the method comprising (a) contacting the cell with the dsRNA of the disclosure or the pharmaceutical composition of the disclosure; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell. In some embodiments, the ANGPTL3 expression is inhibited by at least 30% relative to a control.

[0042] In some embodiments, a method of treating a disorder mediated by ANGPTL3 is provided, comprising administering to a subject in need of such treatment a therapeutically effective amount of a dsRNA of the disclosure, or a pharmaceutical composition of the disclosure. In some embodiments, the disorder is a cardiovascular disorder. In some embodiments, the disorder is cardiovascular disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0044] FIGs.1A-1B show dose-response graphs of the percent (%) inhibition of ANGPTL3 mRNA in primary human hepatocyte cells treated with exemplary GalNAc conjugated, modified siRNAs at 100nM, 33nM, 11nM, 3.7nM, 1.2nM, 0.412nM, 0.137nM, and 0.046nM relative to ANGPTL3 mRNA in PBS treated cells. The ANGPTL3 mRNA level was measured by quantitative PCR and normalized to GAPDH.

[0045] FIGs.2A-2D show dose-response graphs of the percent (%) inhibition of APOC3 mRNA in primary human hepatocyte cells treated with GalNAc conjugated, modified siRNA at 100nM, 33nM, 11nM, 3.7nM, 1.2nM, 0.412nM, 0.137nM, and 0.046nM relative to APOC3 mRNA in PBS treated cells. APOC3 mRNA level measured by quantitative PCR and normalized to GAPDH.Attorney Docket No.: BCR-007WO DETAILED DESCRIPTION

[0046] The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description, the drawings, and from the claims.

[0047] The disclosure provides modified double-stranded ribonucleic acid (dsRNA) oligonucleotides and methods of using the modified dsRNA oligonucleotides for inhibiting or silencing the expression of a gene in the genome of a cell or a mammal where the modified dsRNA oligonucleotide targets a gene in the genome of a cell. The disclosure also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of a target gene. The modified dsRNA oligonucleotide directs the sequence- specific degradation of the target gene mRNA.

[0048] The disclosure provides dsRNA oligonucleotides and methods of using the dsRNA oligonucleotides for inhibiting the expression of a Apolipoprotein C3 (APOC3) gene in a cell or a mammal where the dsRNA oligonucleotide targets a APOC3 gene. The disclosure also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of a APOC3 gene, e.g., cardiovascular disease. An APOC3 dsRNA oligonucleotide directs the sequence-specific degradation of APOC3 mRNA.

[0049] The disclosure provides dsRNA oligonucleotides and methods of using the dsRNA oligonucleotides for inhibiting the expression of a Angiopoietin-like 3 (ANGPTL3) gene in a cell or a mammal where the dsRNA oligonucleotide targets a ANGPTL3 gene. The disclosure also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of a ANGPTL3 gene, e.g., cardiovascular disease. An ANGPTL3 dsRNA oligonucleotide directs the sequence-specific degradation of ANGPTL3 mRNA. I. Definitions

[0050] For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

[0051] As used herein, all numerical values or numerical ranges comprise whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, comprises 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%,Attorney Docket No.: BCR-007WO 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000-fold comprises 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-fold, etc., as well as 1.1-, 1.2-, 1.3-, 1.4-, or 1.5-fold, etc., 2.1-, 2.2-, 2.3-, 2.4-, or 2.5-fold, etc., and so forth.

[0052] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

[0053] The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

[0054] The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means±10%. In certain embodiments, about means±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range

[0055] The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 19 nucleotides of a 21 nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

[0056] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range. As used herein, ranges include both the upper and lower limit.

[0057] "G," "C," "A" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware thatAttorney Docket No.: BCR-007WO guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the disclosure by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the disclosure.

[0058] “APOC3” refers to the Apolipoprotein C3 gene. According to the NCBI NLM website, this gene encodes a protein component of triglyceride (TG)-rich lipoproteins (TRLs) including very low density lipoproteins (VLDL), high density lipoproteins (HDL) and chylomicrons.. Mutations in APOC3 are associated with low plasma triglyceride levels and reduced risk of ischemic cardiovascular disease, and hyperalphalipoproteinemia, which is characterized by elevated levels of high density lipoprotein (HDL) and HDL cholesterol in human patients. A human APOC3 mRNA sequence is GenBank accession number NM_000040.3. A rhesus monkey (Macaca mulatta) APOC3 mRNA sequence is GenBank accession number XM_001090312.4; a dog (Canis familiaris) APOC3 mRNA sequence is GenBank accession number NM_001003369.2. A mouse (Mus musculus) mRNA sequence is GenBank accession number NM_001289755.1.

[0059] “ANGPTL3” refers to the Angiopoietin-like 3 gene. According to the NCBI NLM website, this gene encodes a secreted protein that functions in angiogenesis. The encoded protein, which is expressed predominantly in the liver, is further processed into an N-terminal coiled-coil domain-containing chain and a C-terminal fibrinogen chain. The N-terminal chain is important for lipid metabolism, while the C-terminal chain may be involved in angiogenesis. Mutations in this gene cause familial hypobetalipoproteinemia type 2. Diseases associated with ANGPTL3 include Hypobetalipoproteinemia, Familial, 2 and Hypobetalipoproteinemia, Familial, 1. A human ANGPTL3 mRNA sequence is GenBank accession number NM_014495.4,. A rhesus monkey (Macaca mulatta) ANGPTL3 mRNA sequence is GenBank accession number XM_015141187.2; a dog (Canis familiaris) ANGPTL3 mRNA sequence is GenBank accession number XM_038666015.1. A mouse (Mus musculus) mRNA sequence is GenBank accession number NM_013913.4.

[0060] As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcription product.Attorney Docket No.: BCR-007WO

[0061] As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

[0062] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person.

[0063] For example, a first nucleotide sequence can be described as complementary to a second nucleotide sequence when the two sequences hybridize (e.g., anneal) under stringent hybridization conditions. Hybridization conditions include temperature, ionic strength, pH, and organic solvent concentration for the annealing and / or washing steps. The term stringent hybridization conditions refers to conditions under which a first nucleotide sequence will hybridize preferentially to its target sequence, e.g., a second nucleotide sequence, and to a lesser extent to, or not at all to, other sequences. Stringent hybridization conditions are sequence dependent, and are different under different environmental parameters. Generally, stringent hybridization conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the nucleotide sequence at a defined ionic strength and pH. The Tmis the temperature (under defined ionic strength and pH) at which 50% of the first nucleotide sequences hybridize to a perfectly matched target sequence. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-- Hybridization with Nucleic Acid Probes part I, chap.2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier, N.Y. (“Tijssen”).

[0064] Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

[0065] This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequenceAttorney Docket No.: BCR-007WO 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 single stranded 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 described herein.

[0066] “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogsteen base pairing.

[0067] The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

[0068] As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a target gene) including a 5’ UTR, an open reading frame (ORF), or a 3’ UTR. For example, a polynucleotide is complementary to at least a part of a target gene mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding a target gene (e.g. APOC3 or ANGPTL3).

[0069] In one embodiment, the antisense strand of the dsRNA is sufficiently complementary to a target mRNA so as to cause cleavage of the target mRNA.

[0070] The term “double-stranded RNA” or “dsRNA,” 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. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and / or aAttorney Docket No.: BCR-007WO modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, 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 siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.

[0071] The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. 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.” 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 modified dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a modified dsRNA may comprise one or more nucleotide overhangs. The term “siRNA” is also used herein to refer to a modified dsRNA as described above.

[0072] As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a modified dsRNA when a 3'-end of one strand of the modified dsRNA extends beyond the 5'-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the modified dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a modified dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.

[0073] The term “antisense strand” refers to the strand of a modified dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5’ and / or 3’ terminus.

[0074] The term “sense strand,” as used herein, refers to the strand of a modified dsRNA that includes a region that is substantially complementary to a region of the antisense strand.Attorney Docket No.: BCR-007WO

[0075] “Introducing into a cell,” when referring to a modified dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a modified dsRNA may also be "introduced into a cell,” wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, a modified dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein or known in the art.

[0076] The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of” and the like in as far as they refer to a target gene, herein refer to the at least partial suppression of the expression of a target gene (e.g. APOC3 or ANGPTL3), as manifested by a reduction of the amount of mRNA which may be isolated from a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression of a target gene (e.g. APOC3 or ANGPTL3) is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of (mRNAin control cells) - (mRNA in treated cells) •100 % (mRNAin control cells)

[0077] Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to target gene (e.g. APOC3 or ANGPTL3) expression, e.g., the amount of protein encoded by an target gene (e.g. APOC3 or ANGPTL3) which is secreted by a cell, the level of plasma lipid levels or the number of cells displaying a certain phenotype. In principle, target gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given modified dsRNA inhibits the expression of an target gene (e.g. APOC3 or ANGPTL3) by a certain degree and therefore is encompassed by of the disclosure, the assays provided in the Examples below shall serve as such reference.

[0078] For example, in certain instances, expression of a target gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide of the disclosure. In some embodiments, a target gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-strandedAttorney Docket No.: BCR-007WO oligonucleotide of the disclosure. In some embodiments, a target gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded of the disclosure.

[0079] As used herein, in the context of target gene expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by target gene expression. In the context of the present disclosure insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by target gene expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.

[0080] As used herein, the phrases “effective amount” refers to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by target gene expression or an overt symptom of pathological processes mediated by target gene expression. The specific amount that is effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by target gene expression, the patient’s history and age, the stage of pathological processes mediated by target gene expression, and the administration of other anti-pathological processes mediated by target gene expression agents.

[0081] As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a modified dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an modified dsRNA 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 25% 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 25% reduction in that parameter. For example, a therapeutically effective amount of a modified dsRNA targeting the target gene can reduce target gene (e.g. APOC3 or ANGPTL3) levels in a cell or serum by at least 25%.

[0082] The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. 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 andAttorney Docket No.: BCR-007WO calcium phosphate, and lactose, while corn 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. II. Modified Double-stranded Ribonucleic Acids (dsRNA)

[0083] In one aspect of the disclosure, provided herein are modified double-stranded ribonucleic acid (dsRNA) molecules for inhibiting or silencing the expression of a target gene e.g., in a cell within a subject, such as a mammal (for example a human). The use of these modified dsRNA oligonucleotides enables the targeted degradation of mRNAs of the corresponding target gene (for example an ANGPTL3 gene or a APOC3 gene) in mammals.

[0084] In certain embodiments, the dsRNA comprises an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA or an mRNA fragment formed in the expression of a target gene. In some embodiments, the dsRNA comprises at least 70% complementarity to the mRNA or the fragment mRNA of a target mRNA (for example an ANGPTL3 or APOC3 mRNA).

[0085] In some embodiments, the dsRNA is BC-100001, BC-100015, BC-100019, BC-100020, BC-100021, BC-100022, BC-100024, BC-100026, BC-100027, BC-100028, BC-100031, or BC-100035. In some embodiments, the APOC3 is human APOC3.

[0086] In certain embodiments, the dsRNA comprises an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA or an mRNA fragment formed in the expression of a ANGPTL3 gene. In some embodiments, the dsRNA comprises at least 70% complementarity to the mRNA or the fragment mRNA of human ANGPTL3 mRNA.

[0087] In some embodiments, the dsRNA is BC-100042, BC-100044, BC-100047, BC-100054, BC-100062, BC-100067, BC-100069, BC-100085, BC-100109, BC-100115, BC-100127, or BC-100129. In some embodiments, the ANGPTL3 is human ANGPTL3.

[0088] In some embodiments, a modified double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a target gene is provided comprising a sense strand and an antisense strand, wherein each strand is about 15 to about 30 nucleotides in length. In some embodiments, the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end; a 2'-fluoro nucleotide at positions 2, 4, 6, 8, and 14 from the 5’ end; or a 2'-fluoro nucleotide at positions 2, 4, 6, 8, 14, and 16 from the 5’ end. In some embodiments, the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 12, 11, and 9 from the 3’ end; a 2'-fluoroAttorney Docket No.: BCR-007WO nucleotide at positions 15, 13, 11, and 9 from the 3’ end; a 2'-fluoro nucleotide at positions 13, 12, and 11 from the 3’ end; a 2'-fluoro nucleotide at positions 13, 12, 11, and 9 from the 3’ end; or a 2'-fluoro nucleotide at positions 15, 13, 12, and 9 from the 3’ end.

[0089] In some embodiments, the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 12, 11, and 9 from the 3’ end; a 2'-fluoro nucleotide at positions 15, 13, 11, and 9 from the 3’ end; a 2'-fluoro nucleotide at positions 13, 12, and 11 from the 3’ end; a 2'-fluoro nucleotide at positions 13, 12, 11, and 9 from the 3’ end; or a 2'-fluoro nucleotide at positions 15, 13, 12, and 9 from the 3’ end.

[0090] In some embodiments, the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 12, 11, and 9 from the 3’ end; the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 13, 12, 11, and 9 from the 3’ end; antisense strand comprises a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 12, and 9 from the 3’ end; the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 4, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 11, and 9 from the 3’ end; or the antisense strand comprises a 2'- fluoro nucleotide at positions 2, 4, 6, 8, 14, and 16 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 13, 12, and 11 from the 3’ end.

[0091] In some embodiments, the dsRNA has a mismatch to the target mRNA or fragment of the target mRNA. In some embodiments, the dsRNA comprises one or two mismatches to the target mRNA or fragment of the target mRNA. In some embodiments, the dsRNA is more than 70% identical to the target mRNA or fragment of the target mRNA. In some embodiments, the dsRNA is more than 70%, 75%, 80%, 85%, 90%, or 95 % identical to the target mRNA or fragment of the target mRNA. In some embodiments, the antisense strand comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the target mRNA or fragment of the target mRNA. In some embodiments, the antisense strand of the dsRNA comprises at least 80% to the target mRNA or fragment of the target mRNA. In some embodiments, the mismatch is in the sense strand. In some embodiments, the mismatch is in the antisense strand. In some embodiments, the antisense strand of the dsRNA comprises one, two, three, or four mismatches to the target mRNA or fragment of the target mRNA. In some embodiments, the mismatch is located in the middle of the dsRNA. In some embodiments, the mismatch is in the 5’ or 3’ region of the dsRNA. InAttorney Docket No.: BCR-007WO some embodiments, the mismatch is no more than 5 nucleotides from the 5’ or 3’ end of the dsRNA.

[0092] In some embodiments, at least one strand of the dsRNA comprises a 3’ or 5’ overhang of at least 1 nucleotide. In some embodiments, the overhang is at least 2 or a at least 3 nucleotides. In some embodiments, in the dsRNA at least one strand comprises a 3’ overhang. In some embodiments, in the dsRNA at least one strand comprises a 5’ overhang.

[0093] In some embodiments, the antisense strand has a 3’ end nucleotide overhang compared to the sense strand. In some embodiments, the 3’ end nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand. In some embodiments, the antisense and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. In some embodiments, the antisense strand and the sense strand are at least 80% complementary. In some embodiments, the antisense strand and the sense strand comprise at least one, at least two, at least three, or at least four mismatched nucleotides.

[0094] The modified dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. The modified dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) includes a region of complementarity that is complementary to a target gene sequence, derived from the sequence of an mRNA formed during the expression of a target gene, the other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.

[0095] In some embodiments, the duplex structure is between 15 and 30 or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 base pairs in length. In one embodiment the duplex is 19 base pairs in length. In one embodiment the duplex is 20 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different single stranded RNAs (ssRNA) are used in combination, the duplex lengths can be identical or can differ.

[0096] In some embodiments, each strand of the modified dsRNA of the disclosure is between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each is strand is about 25-30 nucleotides in length. In some embodiments, each strand of the duplex is the same length or of different lengths. When two different ssRNAs are used in combination, the lengths of each strand of each ssRNA can be identical or can differ.Attorney Docket No.: BCR-007WO

[0097] In some embodiments, the dsRNA includes dsRNA that is longer than 21-23 nucleotides, e.g., dsRNA that is long enough to be processed by the RNase III enzyme Dicer into 21-23 base pair siRNA which is then incorporated into a RNA-induced silencing complex (RISC). Accordingly, a dsRNA of the disclosure is at least 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or at least 100 base pairs in length.

[0098] Inhibition of the expression of a target gene can be assayed by, for example, a nucleic acid based assay, such as by quantitative PCR, or by a protein-based method, such as by Western blot. Expression of a target gene (for example an ANGPTL3 gene or a APOC3 gene) can be reduced by at least 50% when measured by an assay as described in the Examples below. For example, expression of a ANGPTL3 gene in cell culture, such as in Huh-7 cells, can be assayed by measuring ANGPTL3 mRNA levels, such as by quantitative PCR assay, or by measuring protein levels, such as by ELISA assay.

[0099] In another aspect, the disclosure provides a single-stranded antisense oligonucleotide RNAi. An antisense oligonucleotide is a single-stranded oligonucleotide that is complementary to a sequence within the target mRNA. Antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol. Cancer Ther.1:347-355. Antisense oligonucleotides can also inhibit target protein expression by binding to the mRNA target and promoting mRNA target destruction via RNase-H. The single-stranded antisense RNA molecule can be about 13 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. Modifications

[0100] In certain embodiments, the dsRNA is chemically modified to enhance stability of the dsRNA. The nucleic acids featured in the disclosure may be synthesized and / or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Specific examples of dsRNA compounds useful in this disclosure include dsRNAs containing modified backbones or non-natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.Attorney Docket No.: BCR-007WO

[0101] In some embodiments, a modified dsRNA backbone includes at least one of: a 2'-O- methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide; an inverted abasic nucleotide, a thymidine-glycol nucleic acid (GNA) S- Isomer; an inosine, and inverted deoxyribonucleotide (3'-3' linked nucleotide), and a thymidine- glycol nucleic acid (GNA) S-Isomer.

[0102] In some embodiments, the modification includes one or more phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

[0103] In some embodiments, the modified nucleotide includes at least one of: 5’-vinyl phosphonate nucleotide, a 5’-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2’- MOE (methoxyethyl)nucleotide, and / or a 2’-arabino fluoro (2’-araF) nucleotide. In some embodiments, the modified nucleotide antisense strand comprises a phosphate mimic at the 5’ end; optionally wherein the phosphate mimic is a 5'-E-Vinyl-phosphonate or a 4'-O- phosphonate.

[0104] In some embodiments, the modified nucleotide comprises at least one of: a 2'-deoxy-2'- fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2’-amino-modified nucleotide, 2’-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and / or a non-natural base comprising nucleotide.

[0105] In some embodiments, the antisense strand and / or the sense strand comprises at least one internucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoromorpholidate linkage, a phosphoropiperazidate linkage, an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, and a boranophosphate linkage. In some embodiments, the antisense strand and / or the sense strand comprises at least one nucleotide modified linkage. In someAttorney Docket No.: BCR-007WO embodiments, all the nucleotide linkages in the antisense strand are modified linkages. In some embodiments, the antisense strand and / or the sense strand comprises at least one a phosphorothioate (PS) bond. Conjugates

[0106] Another modification of the dsRNAs of the disclosure involves chemically linking to the dsRNA one or more ligand or targeting moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053- 1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac- glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino- carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0107] In some embodiments, the ligand or targeting moiety is conjugated to the 5’ end, 3’ end or both ends of the modified dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 3’ end of the sense strand of the modified dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 3’ end of the antisense strand of the modified dsRNA. In some embodiments, the ligand or targeting moiety is at least one N-Acetyl- Galactosamine (GalNAc).

[0108] In certain embodiments, the modified dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to modified dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etAttorney Docket No.: BCR-007WO al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison- Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the oligonucleotide sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. The modified dsRNA conjugate can be purified for example by HPLC methods.

[0109] Conjugating a ligand to a modified dsRNA can enhance its cellular absorption as well as targeting to a particular tissue or uptake by specific types of cells such as liver cells. In certain instances, a hydrophobic ligand is conjugated to the modified dsRNA to facilitate direct permeation of the cellular membrane and or uptake across the liver cells. Alternatively, the ligand conjugated to the modified dsRNA is a substrate for receptor-mediated endocytosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides as well as modified dsRNA agents. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are substantially more active compared to their non-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor-mediated endocytosis. modified dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Li and coworkers report that attachment of folic acid to the 3’-terminus of an oligonucleotide resulted in an 8-fold increase in cellular uptake of the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res.1998, 15, 1540. Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents,Attorney Docket No.: BCR-007WO porphyrin conjugates, delivery peptides and lipids such as cholesterol and cholesterylamine. Examples of carbohydrate clusters include Chol-p-(GalNAc)3(N-acetyl galactosamine cholesterol) and LCO(GalNAc)3 (N-acetyl galactosamine – 3’-Lithocholic-oleoyl). Carbohydrate Conjugates

[0110] In some embodiments, a modified dsRNA oligonucleotide of the disclosure further comprises a carbohydrate. The carbohydrate conjugated modified dsRNA is advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0111] In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the disclosure is a monosaccharide. In one embodiment, the monosaccharide is an N- acetylgalactosamine of formula I or formula II, such asFormula I.Attorney Docket No.: BCR-007WO

[0112] In some embodiments, a carbohydrate is conjugated to the 5’ end, 3’ end or both ends of the modified dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 3’ end of the sense strand of the modified dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 3’ end of the antisense strand of the modified dsRNA. In some embodiments, the carbohydrate is at least one N-Acetyl-Galactosamine (GalNAc). III. Pharmaceutical Compositions

[0113] Also disclosed herein are pharmaceutical compositions comprising the modified dsRNAs targeting target genes of the disclosure (for example an ANGPTL3 gene or a APOC3 gene).

[0114] In one embodiment, the disclosure provides pharmaceutical compositions containing a modified dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the modified dsRNA is useful for treating a disease or disorder associated with the expression or activity of a target gene, such as pathological processes mediated by targeting target gene expression. Such pharmaceutical compositions are formulated based on the mode of delivery.

[0115] The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of a target gene (for example an ANGPTL3 gene or a APOC3 gene).

[0116] 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 composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual modified dsRNAs encompassed by theAttorney Docket No.: BCR-007WO disclosure can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

[0117] Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by target gene expression. Such models are used for in vivo testing of dsRNA and modified dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing a human target gene. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses a human target gene (for example an ANGPTL3 gene or a APOC3 gene).

[0118] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the disclosure lies generally 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 any compound used in the methods featured in 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 or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) 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.

[0119] The modified dsRNA featured in the disclosure can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of modified dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein. Liposomal Formulations

[0120] In certain embodiments, pharmaceutical compositions disclosed herein comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.Attorney Docket No.: BCR-007WO

[0121] In some embodiments, the modified dsRNA of the disclosure is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, modified dsRNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue. Excipients

[0122] In some embodiments, the pharmaceutical composition comprises an excipient. In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pre-gelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0123] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0124] Formulations for topical administration of nucleic acids may include sterile and non- sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganicAttorney Docket No.: BCR-007WO excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0125] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. Other Components

[0126] The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0127] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. Administration

[0128] Also disclosed herein are methods of administration for the pharmaceutical compositions and formulations which include the modified dsRNA compositions and pharmaceutical compositions of the disclosure. In some embodiments, the pharmaceutical composition of the disclosure is administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.

[0129] In some embodiments, administration of the pharmaceutical composition is topical (including buccal and sublingual), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. In some embodiments, parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration.Attorney Docket No.: BCR-007WO

[0130] Pharmaceutical compositions containing a modified dsRNA of the disclosure, can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0131] Pharmaceutical formulations, for example, are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[0132] In some embodiments, the modified dsRNA is delivered in a manner to target a particular tissue, for example the liver.

[0133] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound (e.g., modified dsRNA molecule) which produces a therapeutic effect.

[0134] In certain embodiments, a formulation of the present disclosure comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound (e.g., modified dsRNA molecule) of the present disclosure. In certain embodiments, an aforementioned formulation renders orally bioavailable a compound (e.g., modified dsRNA molecule) of the present disclosure.

[0135] Formulations of the disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound (e.g., modified dsRNA molecule) of the present disclosure as an active ingredient. A compound (e.g., modified dsRNA molecule) of the present disclosure may also be administered as a bolus, electuary or paste.

[0136] Liquid dosage forms for oral administration of the compounds (e.g., modified dsRNA molecules) of the disclosure include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.Attorney Docket No.: BCR-007WO IV. Methods for Inhibiting Expression of a Target Gene

[0137] In another aspect, the disclosure provides a method for inhibiting the expression of an target gene (for example an ANGPTL3 gene or a APOC3 gene) in a cell. The method comprises administering a modified dsRNA targeting a target gene to a cell, such that expression of the target gene in the cell is reduced. The disclosure includes methods performed in cells in in vitro or in vivo. In some embodiments, the method is performed in the cell of an animal, e.g., a mouse, a rat, a non-human primate, or a human.

[0138] The present disclosure also provides methods of using a modified dsRNA of the disclosure and / or a composition containing a modified dsRNA of the present disclosure to reduce and / or inhibit target gene expression in a cell. The methods include contacting the cell with a modified dsRNA of the disclosure and maintaining the cell for a time sufficient to obtain degradation of the target mRNA transcript of the target gene, thereby inhibiting expression of the target gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of the target may be determined by determining the mRNA expression level of the target gene using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, by determining the protein level of the target gene using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques, and / or by determining a biological activity of target gene (for example inhibiting an ANGPTL3 gene or a APOC3 gene may effect one or more molecules associated with the cellular blood clotting mechanism).

[0139] In the methods of the disclosure the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

[0140] A cell suitable for treatment using the methods of the disclosure may be any cell that expresses a target gene. A cell suitable for use in the methods of the disclosure may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.

[0141] In some embodiments, the target gene expression is inhibited by at least 30% relative to a control for example as measured by a quantitative polymerase chain reaction (PCR) assay after administration of the modified dsRNA oligonucleotide of the disclosure.Attorney Docket No.: BCR-007WO

[0142] In some embodiments, a method of treating a disorder (e.g., a cardiovascular disorder) mediated by the target gene is provided, comprising administering to a subject in need of such treatment a therapeutically effective amount of a modified dsRNA oligonucleotide or a pharmaceutical composition of the disclosure. EXAMPLES

[0143] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art. Example 1. In vitro dose-response screen with exemplary ANGPTL3 siRNA compounds in primary human hepatocytes

[0144] This example describes a screen of exemplary GalNAc conjugated modified ANGPTL3 siRNA compounds in primary human hepatocytes (PHH) cells in a single dose screen at 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.412 nM, 0.137 nM, and 0.046 nM of the selected siRNA (Table 1).

[0145] “ANGPTL3” refers to the Angiopoietin-like 3 gene. According to the NCBI NLM website, this gene encodes a secreted protein that functions in angiogenesis. A human ANGPTL3 mRNA sequence is GenBank accession number NM_014495.4, included herein as SEQ ID NO:659. A rhesus monkey (Macaca mulatta) ANGPTL3 mRNA sequence is GenBank accession number XM_015141187.2; a dog (Canis familiaris) ANGPTL3 mRNA sequence is GenBank accession number XM_038666015.1. A mouse (Mus musculus) mRNA sequence is GenBank accession number NM_013913.4.

[0146] ANGPTL3 mRNA level was measured by quantitative PCR and normalized to GAPDH relative to mock treated control cells and the average knock down (KD) and SD was determined. The modified sequences for the sense and antisense strands are shown in Table 1 and exemplary results are shown in Table 2 and FIG.1A-1B.

[0147] Table 1 Exemplary 3’ GalNAc conjugated sense strand and anti-sense strand sequences and modifications of ANGPTL3 siRNA compounds.Attorney Docket No.: BCR-007WOAbbreviation: (*) = PS bond; (-) = PO bond; lower case = 2’-OMe; capital = 2'-F; dX = DNA; VP = 5'-E-Vinyl-phosphonate.

[0148] Table 2 Absolute ANGPTL3 mRNA IC50 and Max inhibition (%) for exemplary ANGPTL3 siRNAs.Attorney Docket No.: BCR-007WOExample 2. In vitro dose-response screen with exemplary APOC3 siRNA compounds in primary human hepatocytes

[0149] This example describes a screen of exemplary GalNAc conjugated modified APOC3 siRNA compounds that were evaluated in free uptake assays in primary human hepatocytes (PHH) cells in a single dose screen at 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM, 0.4 nM, 0.1 nM and 0.05 nM of the selected siRNA (Table 3) and % inhibition was measured.

[0150] “APOC3” refers to the Apolipoprotein C3 gene. According to the NCBI NLM website, this gene encodes a protein component of triglyceride (TG)-rich lipoproteins (TRLs) including very low density lipoproteins (VLDL), high density lipoproteins (HDL) and chylomicrons. The encoded protein plays a role in role in the metabolism of these TRLs through multiple modes.

[0151] Each dose of the free uptake assay was performed in triplicate. Absolute IC50of each siRNA listed was generated by GraphPad Prism 9.

[0152] The modified sequences for the sense and antisense strands are shown in Table 3 and exemplary results are shown in Table 4 and FIG.2A-2D.

[0153] Table 3 Exemplary sense and anti-sense sequences and modifications of APOC3 siRNA compounds.Attorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOAbbreviation: (*) = PS bond; (-) = PO bond; lower case = 2’-OMe; capital = 2'-F; rX = RNA; VP = 5'-E-Vinyl-phosphonate Table 4 mRNA IC50 for exemplary APOC3 siRNAs.Attorney Docket No.: BCR-007WO Example 3. In vitro RNA interference (RNAi) screen in primary human hepatocytes

[0154] This example describes a screen for siRNA-based inhibition of the APOC3 gene in primary human hepatocytes. Human hepatocyte cells were transfected with 36 GalNAc conjugated, modified siRNAs (BC-100001- BC-100814, sense strand and antisense strand) at 100nM, 10nM, and 1nM. Sequences of exemplary, unmodified and modified siRNA compounds are shown in Table 1A and Table 2A, respectively. Compounds in Table 2A were 3’ GalNac modified. APOC3 mRNA level was measured by quantitative PCR and relative to APOC3 mRNA levels in PBS mock treated control cells. Table 3A shows the results of single dose screens at 100 nM, 10 nM, and 0.1 nM in Human hepatocyte cells using the selected APOC3 siRNAs. The data are presented as percent inhibition of APOC3 mRNA in the cells transfected with siRNAs relative to APOC3 mRNA level measured by quantitative PCR and normalized to GAPDH.

[0155] Table 1A. APOC3 siRNA unmodified sequencesAttorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WO

[0156] Table 2A. APOC33’-GalNAc conjugated modified sense and antisense sequences conjugated to 3’-GalNAc.Attorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOAbbreviation: (*) = PS bond; (-) = PO bond; lower case = 2’-OMe; capital = 2'-F; rX = RNA; dX = DNA; invAb = inverted abasic; Tgn = thymidine-glycol nucleic acid (GNA) S-Isomer; i = inosine; invdN = inverted deoxyribonucleotide (3'-3' linked nucleotide), Agn =adenosine-glycol nucleic acid (GNA) S-Isomer; Cgn =cytidine-glycol nucleic acid (GNA) S-Isomer; VP = 5'-E- Vinyl-phosphonate.Attorney Docket No.: BCR-007WO

[0157] Table 3A Results of single dose screens at 100nM, 10nM and 0.1nM in human hepatocyte cells using the selected modified APOC3 siRNAs in % inhibition (KD).Example 4. In vitro RNAi screen in primary cynomolgus hepatocytes

[0158] This example describes a screen of exemplary APOC3 siRNA compounds in cryopreserved primary cynomolgus hepatocytes (PCH) in a single dose screen at 100nM, 10nM,Attorney Docket No.: BCR-007WO and 1nM of the selected siRNA. The data are presented as percent inhibition of APOC3 mRNA in the cells transfected with siRNAs relative to APOC3 mRNA level measured by quantitative PCR and normalized to GAPDH and in % inhibition and SD was determined. Table 4A and shows the results of single dose screens at 100nM, 10nM, and 1nM.

[0159] Table 4A. Mean percent inhibition and SD for selected compounds.Example 5. Evaluation of knockdown of human APOC3 in humanized transgenic miceAttorney Docket No.: BCR-007WO

[0160] This example describes the evaluation of in vivo activity of APOC3 siRNAs that do not cross-react with the rodent APOC3 gene. Briefly, a transgenic mouse model in which human APOC3 gene was knocked randomly into the mouse genome was used for in vivo screening of siRNAs that were identified based on their potency in vitro. All siRNAs were administrated subcutaneously at 3 mg / kg, with PBS as a negative control. The expression of human APOC3 protein in the mice were measured in serum collected at 7, 14, 21, and 28 days by enzyme- linked immunosorbent assay (ELISA). Data is shown as a ratio to the baseline (Day -1, before siRNA treatment). The results are shown in Table 5A.

[0161] Table 5A.7, 14, 21, and 28 days ratio to the baseline inhibition and SD for exemplary APOC3 siRNAs.Example 6. In vitro RNA interference (RNAi) screen in Huh7 cell line

[0162] This example describes a screen for siRNA-based inhibition of the ANGPTL3 gene in a hepatocyte derived cellular carcinoma cell model (Huh-7). Huh-7 cells were transfected with 96 3’-GalNAc conjugated, modified siRNAs (BC-100039 - BC-100134, sense strand and antisense strand) at 10 nM and 0.1 nM. Sequences of exemplary, unmodified and modified siRNA compounds are shown in Table 1B and Table 2B, respectively. Compounds in Table 2 were 3’ GalNac modified. ANGPTL3 mRNA level was measured by quantitative PCR and normalized to GAPDH relative to mock treated control cells. Table 3B shows the results of single dose screens at 10 nM and 0.1 nM in Huh7 cells using the selected ANGPTL3 siRNAs. The data are presented as percent inhibition of ANGPLT3 mRNA in the cells transfected with siRNAs relative to ANGPTL3 mRNA in the mock treated control cells. Additional exemplary 3’ GalNac modified sense and antisense strands of ANGPTL3 siRNA are listed in Table 2-1B.Attorney Docket No.: BCR-007WO

[0163] Table 1B. ANGPTL3 siRNA unmodified sequencesAttorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WO

[0164] Table 2B. ANGPTL3 GalNac modified sense strand and antisense sequences conjugated to 3’-GalNAc.Attorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOdX = DNA; invAb = inverted abasic; Tgn = thymidine-glycol nucleic acid (GNA) S-Isomer; i = inosine; invdN = inverted deoxyribonucleotide (3'-3' linked nucleotide) Agn =adenosine-glycol nucleic acid (GNA) S-Isomer; Cgn =cytidine-glycol nucleic acid (GNA) S-Isomer; VP = 5'-E-Vinyl-phosphonate.

[0165] Table 2-1B Exemplary 3’-GalNac modified sense strands and antisense strands of ANGPTL3 siRNA.Attorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOAbbreviation: (*) = PS bond; (-) = PO bond; lower case = 2’-OMe; capital = 2'-F; rX = RNA; dX = DNA; invAb = inverted abasic; Tgn = thymidine-glycol nucleic acid (GNA) S-Isomer; i = inosine; invdN = inverted deoxyribonucleotide (3'-3' linked nucleotide) Agn =adenosine-glycol nucleic acid (GNA) S-Isomer; Cgn =cytidine-glycol nucleic acid (GNA) S-Isomer; VP = 5'- E-Vinyl-phosphonate.

[0166] Table 3B Results of single dose screens at 10 nM and 0.1 nM in Huh7 cells using the selected modified ANGPTL3 siRNAsAttorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOExample 7. In vitro RNAi screen in primary human hepatocytes

[0167] Exemplary siRNA compounds that showed ANGPTL3 knockdown were selected and tested for knockdown of ANGPTL3 at 100 nM, 10 nM and 1 nM in cryopreserved primary human hepatocyte (PHH) cells. The ANGPTL3 mRNA level was measured by quantitative PCR and normalized to GAPDH relative to mock treated control cells and the Mean KD and SD was determined. Table 4B shows the results of single dose ANGPTL3 siRNA screens at 100 nM, 10 nM, and 1 nM in cryopreserved primary human hepatocyte (PHH) cells.

[0168] Table 4B. Mean % KD and SD for selected compoundsAttorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WO

[0169] Selected ANGPTL3 siRNA compounds BC-100044, BC-100047, BC-100115, BC- 100067, BC-100127, and BC-100042 were further modified and tested in primary human hepatocyte cells treated with GalNAc conjugated, modified siRNA at 100 nM, 10 nM, and 1 nM relative to ANGPTL3 mRNA in PBS treated control cells. ANGPTL3 mRNA level was measured by quantitative PCR and normalized to GAPDH. The modified sequences for the sense and antisense strands are shown in Table 5B and exemplary results are shown in Table 6B.

[0170] Table 5B Exemplary 3’-GalNAc conjugated sense and anti-sense sequences and modifications of ANGPTL3 siRNA compounds.Attorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOAbbreviation: (*) = PS bond; (-) = PO bond; lower case = 2’-OMe; capital = 2'-F; rX = RNA; dX = DNA; invAb = inverted abasic; Tgn = thymidine-glycol nucleic acid (GNA) S-Isomer; i = inosine; invdN = inverted deoxyribonucleotide (3'-3' linked nucleotide) Agn =adenosine-glycol nucleic acid (GNA) S-Isomer; Cgn =cytidine-glycol nucleic acid (GNA) S-Isomer; VP = 5'- E-Vinyl-phosphonate.Attorney Docket No.: BCR-007WO

[0171] Table 6B. Mean % KD and SD for selected ANGPTL3 siRNA compounds.Attorney Docket No.: BCR-007WOExample 8. In vitro RNAi screen in primary cynomolgus hepatocytes

[0172] This example describes a screen of exemplary ANGPTL3 siRNA compounds in cryopreserved primary cynomolgus hepatocytes (PCH) in a single dose screen at 100 nM, 10 nM, and 1 nM of the selected siRNA. ANGPTL3 mRNA level was measured by quantitative PCR and normalized to GAPDH relative to mock treated control cells and the mean knock down (KD) and SD was determined. Table 7B shows the results of single dose screens at 100 nM, 10 nM, and 1 nM.

[0173] Table 7B Mean % KD and SD for selected ANGPTL3 siRNA compounds.Attorney Docket No.: BCR-007WOExample 9. Evaluation of knockdown of mouse ANGPTL3 in wildtype mice

[0174] This example describes the evaluation of exemplary ANGPTL3 siRNAs of mouse ANGPTL3 in vivo. siRNAs that crossed to rodent ANGPTL3 were tested in wildtype mice for their ability to inhibit expression of mouse ANGPTL3. All siRNAs were administrated subcutaneously at 3 mg / kg, with PBS administrated as a negative control. The expression of mouse ANGPTL3 protein was measured in serum collected at 3, 7, and 14 days post-injection by enzyme-linked immunosorbent assay (ELISA). Table 8B shows the results as a ratio to the baseline (Day -1, before siRNA treatment).

[0175] Table 8B 3 day, 7 day, and 14 day mean knockdown ratios for exemplary ANGPTL3 siRNAs.Example 10. Time course evaluation of knockdown of mouse ANGPTL3 in wildtype mice

[0176] This example describes a time course evaluation of knockdown of mouse ANGPTL3 mRNA with exemplary ANGPTL3 siRNAs. Exemplary siRNAs that cross to rodent ANGPTL3 mRNA were tested in wildtype mice for their ability to inhibit expression of mouse ANGPTL3. All siRNAs were administrated subcutaneously at 3 mg / kg, with PBS administrated as a negative control. The expression of mouse ANGPTL3 protein was measured in serum at 7, 14, 21, and 28 days by enzyme-linked immunosorbent assay (ELISA). Data is shown as a ratio to the baseline (Day -1, before siRNA treatment). Exemplary results are shown in Table 9B.Attorney Docket No.: BCR-007WO

[0177] Table 9B 7 day, 14 day, and 21 day mean knockdown ratios for exemplary ANGPTL3 siRNAs.Attorney Docket No.: BCR-007WOExample 11. Evaluation of knockdown of human ANGPTL3 in humanized transgenic mice

[0178] This example describes the evaluation of in vivo activity of ANGPTL3 siRNAs that do not cross-react with the rodent ANGPTL3 gene. Briefly, a transgenic mouse model in which human ANGPTL3 gene was knocked into the mouse ANGPTL3 locus that also inactivates mouse ANGPTL3 expression, was used for in vivo screening of siRNAs that were identified based on their potency in vitro. All siRNAs were administrated subcutaneously at 3 mg / kg, with PBS as a negative control. The expression of human ANGPTL3 protein in the mice were measured in serum collected at 7 and 14 days by enzyme-linked immunosorbent assay (ELISA). Data is shown as a ratio to the baseline (Day -1, before siRNA treatment). The results are shown in Table 10B.

[0179] Table 10B 7 day and 14 day mean and SD for exemplary ANGPTL3 siRNAs.Attorney Docket No.: BCR-007WO Example 12. Time course evaluation of knockdown of human ANGPTL3 in humanized transgenic mice

[0180] This example describes a time course evaluation of knockdown of human ANGPTL3 protein in vivo. To evaluate in vivo activity of siRNAs that do not cross-react with the rodent ANGPTL3 gene or mRNA, a transgenic mouse model in which human ANGPTL3 gene was knocked into the mouse ANGPTL3 locus and also inactivates mouse ANGPTL3 protein expression, was used for in vivo screening of siRNAs that were identified based on their potency in vitro. All siRNAs were administrated subcutaneously at 3mg / kg, with PBS administered as a negative control. The expression of human ANGPTL3 were measured in serum collected at 7, 14, and 21 days by enzyme-linked immunosorbent assay (ELISA). Data is shown as a ratio to the baseline (Day -1). The results are shown in Table 11B.

[0181] The results show, that BC-100042, BC-100067, BC-100115, and BC-100127 siRNA and their modified derivative siRNAs reduce ANGPTL3 protein expression in vivo by >50 % over 21 days after administration compared with the PBS control.

[0182] Table 11B 7 day, 14 day, and 21 day mean human ANGPTL3 expression and SD for exemplary ANGPTL3 siRNAs.Attorney Docket No.: BCR-007WO

[0183] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. INFORMAL SEQUENCE LISTING FOR EXAMPLES 1-2Attorney Docket No.: BCR-007WOAttorney Docket No.: BCR-007WOINCORPORATION BY REFERENCE

[0184] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. EQUIVALENTS

[0185] The disclosure can be embodied in other specific forms without departing from the essential characteristics thereof. The foregoing embodiments therefore are to be considered illustrative rather than limiting on the disclosure described herein. The scope of the disclosure is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

Attorney Docket No.: BCR-007WO WHAT IS CLAIMED IS:

1. A modified double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a target gene, wherein the modified dsRNA comprises a sense strand and an antisense strand, wherein each strand is about 15 to about 30 nucleotides in length, and wherein the antisense strand comprises a) a 2'-fluoro nucleotide at positions 2, 4, 6, 8, 14, and 16 from the 5’ end; b) a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end; or c) a 2'-fluoro nucleotide at positions 2, 4, 6, 8, and 14 from the 5’ end.

2. A modified double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a target gene, wherein the modified dsRNA comprises a sense strand and an antisense strand, wherein each strand is about 15 to about 30 nucleotides in length, and wherein the sense strand comprises a) a 2'-fluoro nucleotide at positions 15, 13, 12, 11, and 9 from the 3’ end; b) a 2'-fluoro nucleotide at positions 13, 12, and 11 from the 3’ end; c) a 2'-fluoro nucleotide at positions 15, 13, 11, and 9 from the 3’ end; d) a 2'-fluoro nucleotide at positions 13, 12, 11, and 9 from the 3’ end; or e) a 2'-fluoro nucleotide at positions 15, 13, 12, and 9 from the 3’ end.

3. The modified dsRNA of claim 1, wherein the sense strand comprises a) a 2'-fluoro nucleotide at positions 15, 13, 12, 11, and 9 from the 3’ end; b) a 2'-fluoro nucleotide at positions 15, 13, 11, and 9 from the 3’ end; c) a 2'-fluoro nucleotide at positions 13, 12, and 11 from the 3’ end; d) a 2'-fluoro nucleotide at positions 13, 12, 11, and 9 from the 3’ end; or e) a 2'-fluoro nucleotide at positions 15, 13, 12, and 9 from the 3’ end.

4. The modified dsRNA of any one of claims 1 to 3, wherein a) the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 12, 11, and 9 from the 3’ end; b) the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 13, 12, 11, and 9 from the 3’ end; c) antisense strand comprises a 2'-fluoro nucleotide at positions 2, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 12, and 9 from the 3’ end;Attorney Docket No.: BCR-007WO d) the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 4, 6, 8, and 14 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 15, 13, 11, and 9 from the 3’ end; or e) the antisense strand comprises a 2'-fluoro nucleotide at positions 2, 4, 6, 8, 14, and 16 from the 5’ end and the sense strand comprises a 2'-fluoro nucleotide at positions 13, 12, and 11 from the 3’ end.

5. The modified dsRNA of any one of claims 1 to 4, wherein the antisense strand has a 3’ end nucleotide overhang compared to the sense strand.

6. The modified dsRNA of claim 5, wherein the 3’ end nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand.

7. The modified dsRNA of any one of claims 1 to 6, wherein the antisense and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary.

8. The modified dsRNA of any one of claims 1 to 6, wherein the antisense strand and the sense strand are at least 80% complementary.

9. The modified dsRNA of any one of claims 1 to 6, wherein the antisense strand and the sense strand comprise at least one, at least two, at least three, or at least four mismatched nucleotides.

10. The modified dsRNA of any one of claims 1 to 9, wherein the antisense strand comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a target mRNA corresponding to the target gene.

11. The modified dsRNA of any one of claims 1 to 9, wherein the antisense strand of the modified dsRNA comprises at least 80% complementarity to a target mRNA corresponding to the target gene.

12. The modified dsRNA of any one of claims 1 to 9, wherein the antisense strand of the modified dsRNA comprises one, two, three, or four mismatches to a target mRNA corresponding to the target gene.

13. The modified dsRNA of any one of claims 1 to 12, wherein at least one additional nucleotide is a modified nucleotide.

14. The modified dsRNA of claim 13, wherein the modified nucleotide is at least one of: a 2'-O- methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide; an inverted abasic nucleotide, a thymidine-glycol nucleic acid (GNA) S-Isomer; an inosine, an inverted deoxyribonucleotide (3'-3' linked nucleotide), or a thymidine-glycol nucleic acid (GNA) S-Isomer.Attorney Docket No.: BCR-007WO 15. The modified sRNA of claim 13, wherein the modified nucleotide is at least one of: 5’-vinyl phosphonate nucleotide, a 5’-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2’-MOE (methoxyethyl)nucleotide, and / or a 2’-arabino fluoro (2’-araF) nucleotide.

16. The modified dsRNA of claim 13, wherein the antisense strand comprises a phosphate mimic at the 5’ end; optionally wherein the phosphate mimic is a 5'-E-Vinyl-phosphonate or a 4'-O-phosphonate.

17. The modified dsRNA of claim 13, wherein the modified a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2’- amino-modified nucleotide, a 2’-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and / or a non-natural base comprising nucleotide 18. The modified dsRNA of claim 13, wherein the antisense strand and / or the sense strand comprises at least one internucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoromorpholidate linkage, a phosphoropiperazidate linkage, an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, and a boranophosphate linkage.

19. The modified dsRNA of claim 18, wherein the antisense strand and / or the sense strand comprises at least one nucleotide modified linkage.

20. The modified dsRNA of claim 18, wherein all the nucleotide linkages in the antisense strand are modified linkages.

21. The modified dsRNA of claim 18, wherein the antisense strand and / or the sense strand comprises at least one nucleotide with a phosphorothioate (PS) bond.

22. The modified dsRNA of any one of claims 1 or 3-21, further comprising a ligand or targeting moiety.

23. The modified dsRNA of claim 22, wherein the ligand or targeting moiety is conjugated to the 5’ end, 3’ end or both ends of the modified dsRNA.

24. The modified dsRNA of claim 22, wherein the ligand or targeting moiety is conjugated to the 3’ end of the sense strand of the modified dsRNA.

25. The modified dsRNA of any one of claims 22 to 24, wherein ligand or targeting moiety is at least one N-Acetyl-Galactosamine (GalNAc).

26. A cell comprising the modified dsRNA of any one of claims 1-25.Attorney Docket No.: BCR-007WO 27. A pharmaceutical composition for inhibiting expression of a target gene comprising the modified dsRNA of any one of claims 1-25 and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

28. A method of inhibiting expression of a target gene in a cell, comprising: (a) contacting the cell with the modified dsRNA of any one of claims 1-25 or the pharmaceutical composition of claim 27; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the target mRNA transcript of the target gene, thereby inhibiting expression of the target gene in the cell.

29. The method of claim 28, wherein the target gene expression is inhibited by at least 30% relative to a negative control dsRNA, optionally wherein target gene expression is measured using a quantitative polymerase chain reaction (PCR) assay.

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