Modified double-stranded RNA agents

Abstract

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

Description

[0001] Cross-referencing related applications This application claims the benefit and priority of 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 herein by reference in its entirety.

[0002] sequence list This application contains an electronically submitted sequence list XML, which is hereby incorporated in its entirety by reference. The copy of the XML was created on [Date], named [Name], and has a size of [Number] bytes. Technical Field

[0003] This disclosure relates to nucleotide modifications in double-stranded ribonucleic acid (dsRNA) targeting genes in the genome, and methods for using modified dsRNA to suppress gene expression in the genome. Summary of the Invention

[0004] This disclosure is in part based on the development of modified double-stranded RNA (dsRNA) of mRNA transcripts of target genes in the genome, pharmaceutical compositions comprising the dsRNA of said target genes, and methods of using said dsRNA to inhibit the expression of target genes in cells.

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

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

[0007] In some embodiments, the sense strand comprises 2'-fluoronucleotides at positions 15, 13, 12, 11, and 9 from the 3' end; 2'-fluoronucleotides at positions 15, 13, 11, and 9 from the 3' end; 2'-fluoronucleotides at positions 13, 12, and 11 from the 3' end; 2'-fluoronucleotides at positions 13, 12, 11, and 9 from the 3' end; or 2'-fluoronucleotides at positions 15, 13, 12, and 9 from the 3' end.

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

[0009] In some embodiments, the antisense strand has a 3' nucleotide overhang compared to the sense strand. In some embodiments, the 3' nucleotide overhang comprises one, two, or three nucleotides compared to the sense strand. In some embodiments, the antisense strand 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 at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical nucleotide sequences to the target mRNA corresponding to the target gene. In some embodiments, the antisense strand of the dsRNA comprises at least 80% complementarity to the target mRNA corresponding to the target gene. In some embodiments, the antisense strand of the dsRNA comprises one, two, three, or four mismatches to the 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 the following: a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-thiophosphate group, a 2'-fluorine modified nucleotide; a reverse abase-free nucleotide, a thymine-glycol nucleic acid (GNA) S-isomer; inosine and a reverse deoxyribonucleotide (3'-3' linked nucleotide), and a thymine-glycol nucleic acid (GNA) S-isomer. In some embodiments, the modified nucleotide is at least one of the following: a 5'-vinylphosphonate nucleotide, a 5'-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2'-MOE (methoxyethyl) nucleotide, and / or a 2'-arabinoflus (2'-araF) nucleotide. In some embodiments, the antisense strand contains a phosphate mimic at the 5' end; optionally, the phosphate mimic is a 5'-E-vinylphosphonate or a 4'-O-phosphonate. In some embodiments, the modified nucleotide is at least one of the following: 2'-deoxy-2'-fluorine modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, baseless nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, phosphoramide ester and / or non-natural bases containing nucleotides.

[0011] In some embodiments, the antisense strand and / or the sense strand comprises at least one internucleotide bond selected from the group consisting of: phosphorothioate linkage, dithiothioate linkage, phosphotriester linkage, alkylphosphonate linkage, aminoalkylphosphotriester linkage, alkylenephosphotriester linkage, hypophosphite linkage, phosphoramidite linkage, morpholinophosphotriester linkage, piperazine phosphate linkage, aminoalkylphosphamidite linkage, thiophosphamidite linkage, thiocarbonylalkylphosphotriester linkage, thiophosphate linkage, selenophosphate linkage, and boron phosphate linkage. In some embodiments, the antisense strand and / or the sense strand comprises at least one nucleotide-modified bond. In some embodiments, all nucleotide bonds in the antisense strand are modified bonds. In some embodiments, the antisense strand and / or the sense strand comprises at least one phosphothioate (PS) bond.

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

[0013] In some embodiments, a cell is provided that contains the dsRNA of this disclosure. In some embodiments, a vector is provided that encodes at least one strand of dsRNA of this disclosure. This disclosure provides a cell containing said vector.

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

[0015] In some embodiments, a method for inhibiting the expression of a target gene in cells is provided, the method comprising (a) contacting the cells with the dsRNA or pharmaceutical composition disclosed herein; and (b) maintaining the cells produced in step (a) for a time sufficient to degrade the mRNA transcript of the target gene, thereby inhibiting the expression of the target gene in the cells. In some embodiments, the target gene expression is inhibited by at least 30% relative to a control. In some embodiments, the target gene expression is inhibited by at least 30% relative to a negative control dsRNA, optionally wherein the target gene expression is measured using a quantitative polymerase chain reaction (PCR) assay.

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

[0017] This disclosure is partly based on targeting. APOC3 Development of double-stranded ribonucleic acid (dsRNA) for genes, including the targeted APOC3 Pharmaceutical compositions of dsRNA of genes and the use of said dsRNA to inhibit the growth of cellular organisms. APOC3 Methods of expression.

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

[0019] In some aspects, this disclosure provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the 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 containing 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 table. In some embodiments, the sense strand comprises at least 15 consecutive nucleotides of the sense strand sequence shown in the relevant table. In some embodiments, the sense strand comprises at least 16, 17, 18, 19, 20, or 21 consecutive nucleotides of the sense strand sequence shown in the relevant table. In some embodiments, the sense strand comprises 21 consecutive nucleotides of the sense strand sequence shown in the relevant table. In some embodiments, the antisense strand comprises at least 16, 17, 18, 19, 20, or 21 consecutive nucleotides of the antisense strand sequence shown in the relevant table. In some embodiments, the antisense strand comprises 21 consecutive nucleotides of the antisense strand sequence shown in the relevant table. In some embodiments, the sense strand sequence is selected from the sense strand sequences shown in the relevant table, and the antisense strand is selected from the antisense strand sequences shown in the relevant table. In some embodiments, the sense strand sequence is selected from the sense strand sequences shown in the relevant table. In some embodiments, the antisense strand is selected from the antisense strand sequences shown in the relevant table.

[0022] In some embodiments, the antisense strand has a 3' nucleotide overhang compared to the sense strand. In some embodiments, the 3' nucleotide overhang comprises one, two, or three nucleotides compared to the sense strand. In some embodiments, the antisense strand 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 contain at least one, at least two, at least three, or at least four mismatched nucleotides. In some embodiments, the antisense strand contains at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical nucleotide sequences to the target mRNA corresponding to the fragment of APOC3 mRNA. In some embodiments, the antisense strand of the dsRNA contains at least 80% complementarity to the fragment of the APOC3 mRNA. In some embodiments, the antisense strand of the dsRNA contains one, two, three, or four mismatches with 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 the following: a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-thiophosphate group, a 2'-fluorine modified nucleotide; a reverse abase-free nucleotide, a thymine-glycol nucleic acid (GNA) S-isomer; inosine and a reverse deoxyribonucleotide (3'-3' linked nucleotide), and a thymine-glycol nucleic acid (GNA) S-isomer. In some embodiments, the modified nucleotide is at least one of the following: a 5'-vinylphosphonate nucleotide, a 5'-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2'-MOE (methoxyethyl) nucleotide, and / or a 2'-arabinoflus (2'-araF) nucleotide. In some embodiments, the antisense strand contains a phosphate mimic at the 5' end; optionally, the phosphate mimic is a 5'-E-vinylphosphonate or a 4'-O-phosphonate. In some embodiments, the modified nucleotide is at least one of the following: 2'-deoxy-2'-fluorine modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, baseless nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, phosphoramide ester and / or non-natural bases containing nucleotides.

[0024] In some embodiments, the antisense strand and / or the sense strand comprises at least one internucleotide bond selected from the group consisting of: thiophosphate bond, dithiophosphate bond, phosphate trimer bond, alkylphosphonate bond, aminoalkylphosphate trimer bond, alkylenephosphonate bond, hypophosphonate bond, phosphoramidite bond, morpholinophosphate bond, piperazine phosphate bond, aminoalkylphosphatite bond, thiophosphatite bond, thiocarbonylalkylphosphonate bond, thiocarbonylalkylphosphate trimer bond, thiophosphate bond, selenophosphate bond, and boron phosphate bond. In some embodiments, the antisense strand and / or the sense strand comprises at least one nucleotide-modified bond. In some embodiments, all nucleotide bonds in the antisense strand are modified bonds. In some embodiments, the antisense strand and / or the sense strand comprises at least one thiophosphate (PS) bond.

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

[0026] In some embodiments, a cell is provided that contains the dsRNA of this disclosure. In some embodiments, a vector is provided that encodes at least one strand of dsRNA of this disclosure. This disclosure provides a cell containing said vector.

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

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

[0029] In some embodiments, a method of treating an APOC3-mediated condition is provided, the method comprising administering a therapeutically effective amount of the disclosed dsRNA or the disclosed pharmaceutical composition to a subject requiring such treatment. In some embodiments, the condition is a cardiovascular condition. In some embodiments, the condition is a cardiovascular disease.

[0030] This disclosure is partly based on targeting. ANGPTL3 Development of double-stranded ribonucleic acid (dsRNA) for genes, including the targeted ANGPTL3 Pharmaceutical compositions of dsRNA of genes and the use of said dsRNA to inhibit the growth of cellular organisms. ANGPTL3 Methods of expression.

[0031] In some aspects, this disclosure provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of ANGPTL3, said dsRNA comprising a sense strand and an antisense strand each having a length of 15 to 30 nucleotides, wherein said antisense strand comprises at least 15 consecutive nucleotides of the antisense strand sequence shown in the relevant table.

[0032] In some aspects, this disclosure provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the 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 containing the sequence shown in NM_014495.4.

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

[0035] In some embodiments, the antisense strand has a 3' nucleotide overhang compared to the sense strand. In some embodiments, the 3' nucleotide overhang comprises one, two, or three nucleotides compared to the sense strand. In some embodiments, the antisense strand 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 at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical nucleotide sequences to the target mRNA corresponding to the ANGPTL3 mRNA fragment. In some embodiments, the antisense strand of the dsRNA comprises at least 80% complementarity to the ANGPTL3 mRNA fragment. In some embodiments, the antisense strand of the dsRNA contains one, two, three, or four mismatches with 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 the following: a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-thiophosphate group, a 2'-fluorine modified nucleotide; a reverse abase-free nucleotide, a thymine-glycol nucleic acid (GNA) S-isomer; inosine and a reverse deoxyribonucleotide (3'-3' linked nucleotide), and a thymine-glycol nucleic acid (GNA) S-isomer. In some embodiments, the modified nucleotide is at least one of the following: a 5'-vinylphosphonate nucleotide, a 5'-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2'-MOE (methoxyethyl) nucleotide, and / or a 2'-arabinoflus (2'-araF) nucleotide. In some embodiments, the antisense strand contains a phosphate mimic at the 5' end; optionally, the phosphate mimic is a 5'-E-vinylphosphonate or a 4'-O-phosphonate. In some embodiments, the modified nucleotide is at least one of the following: 2'-deoxy-2'-fluorine modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, baseless nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, phosphoramide ester and / or non-natural bases containing nucleotides.

[0037] In some embodiments, the antisense strand and / or the sense strand comprises at least one internucleotide bond selected from the group consisting of: thiophosphate bond, dithiophosphate bond, phosphate trimer bond, alkylphosphonate bond, aminoalkylphosphate trimer bond, alkylenephosphonate bond, hypophosphonate bond, phosphoramidite bond, morpholinophosphate bond, piperazine phosphate bond, aminoalkylphosphatite bond, thiophosphatite bond, thiocarbonylalkylphosphonate bond, thiocarbonylalkylphosphate trimer bond, thiophosphate bond, selenophosphate bond, and boron phosphate bond. In some embodiments, the antisense strand and / or the sense strand comprises at least one nucleotide-modified bond. In some embodiments, all nucleotide bonds in the antisense strand are modified bonds. In some embodiments, the antisense strand and / or the sense strand comprises at least one thiophosphate (PS) bond.

[0038] In some embodiments, the dsRNA further comprises a ligand or a targeting portion. In some embodiments, the ligand or the targeting portion is conjugated to the 5' end, the 3' end, or both ends of the dsRNA. In some embodiments, the ligand or the targeting portion is conjugated to the 3' end of the sense strand of the dsRNA. In some embodiments, the ligand or the targeting portion is at least one N-acetylgalactosamine (GalNAc).

[0039] In some embodiments, a cell is provided that contains the dsRNA of this disclosure. In some embodiments, a vector is provided that encodes at least one strand of dsRNA of this disclosure. This disclosure provides a cell containing said vector.

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

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

[0042] In some embodiments, a method of treating an ANGPTL3-mediated condition is provided, the method comprising administering a therapeutically effective amount of the disclosed dsRNA or the disclosed pharmaceutical composition to a subject requiring such treatment. In some embodiments, the condition is a cardiovascular condition. In some embodiments, the condition is a cardiovascular disease. Attached Figure Description

[0043] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments, which utilize the principles of the invention, and are illustrated in the accompanying drawings: Figures 1A-1B A dose-response plot shows the percentage (%) of ANGPTL3 mRNA inhibition in primary human hepatocytes treated with exemplary GalNAc-conjugated modified siRNAs at levels of 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.412 nM, 0.137 nM, and 0.046 nM, relative to PBS-treated cells. ANGPTL3 mRNA levels were measured by quantitative PCR and normalized relative to GAPDH.

[0044] Figures 2A-2D The dose-response plot shows the percentage (%) of APOC3 mRNA inhibition in primary human hepatocytes treated with modified siRNA conjugated with GalNAc at concentrations of 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.412 nM, 0.137 nM, and 0.046 nM, relative to PBS-treated cells. APOC3 mRNA levels were measured by quantitative PCR and normalized relative to GAPDH. Detailed Implementation

[0045] Details of one or more embodiments of the invention are set forth in the following description. Other features, objects, and advantages of the invention will become apparent from the specification, the accompanying drawings, and the claims.

[0046] This disclosure provides modified double-stranded RNA (dsRNA) oligonucleotides and methods for using modified dsRNA oligonucleotides to inhibit or silence gene expression in the genome of cells or mammals, wherein the modified dsRNA oligonucleotides target genes in the genome of cells. This disclosure also provides compositions and methods for treating pathological symptoms and diseases in mammals caused by the expression of target genes. The modified dsRNA oligonucleotides direct sequence-specific degradation of target gene mRNA.

[0047] This disclosure provides dsRNA oligonucleotides and the use of dsRNA oligonucleotides to inhibit apolipoprotein C3 in cells or mammals. APOC3 Methods for gene expression, including dsRNA oligonucleotide targeting APOC3 Genes. This disclosure also provides for the treatment of mammals by... APOC3 Compositions and methods for addressing pathological symptoms and diseases caused by gene expression, such as cardiovascular diseases. APOC3 dsRNA oligonucleotide guidance. APOC3 Sequence-specific degradation of mRNA.

[0048] This disclosure provides dsRNA oligonucleotides and the use of dsRNA oligonucleotides to inhibit angiopoietin-like 3 in cells or mammals. ANGPTL3 Methods for gene expression, including dsRNA oligonucleotide targeting ANGPTL3 Genes. This disclosure also provides for the treatment of mammals by... ANGPTL3 Compositions and methods for addressing pathological symptoms and diseases caused by gene expression, such as cardiovascular diseases. ANGPTL3 dsRNA oligonucleotide guidance. ANGPTL3 Sequence-specific degradation of mRNA.

[0049] I. Definition For convenience, the meanings of certain terms and phrases used in this specification, examples, and appended claims are provided below. If there is a significant difference between the terminology used in other parts of this specification and the definitions provided in this section, the definitions in this section shall prevail.

[0050] As used herein, unless the context explicitly indicates otherwise, all numerical values ​​or ranges are inclusive of or encompass whole integers within or encompassing such ranges, as well as fractions of values ​​or integers within or encompassing such ranges. Thus, for example, references to the range 90-100% include 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., and 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so on. In another instance, references to the range of 1-5,000 times include 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x, as well as 1.1x, 1.2x, 1.3x, 1.4x, or 1.5x, 2.1x, 2.2x, 2.3x, 2.4x, or 2.5x, and so on.

[0051] The articles “a” and “an” are used in this document to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “an element” means one element or more elements, such as multiple elements.

[0052] The term “including” is used herein to mean the phrase “including but not limited to” and may be used interchangeably with the phrase.

[0053] The term “about” is used herein to mean a typical tolerance range in the field. For example, “about” can be understood as approximately 2 standard deviations from the mean. In some embodiments, it is approximately ±10% of the mean. In some embodiments, it is approximately ±5% of the mean. When “about” appears before a series of numbers or ranges, it should be understood that “about” may modify each of the numbers in the series or range.

[0054] The term "at least" preceding a number or series of numbers is understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included, as is clearly apparent from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For instance, "at least 19 nucleotides in a nucleic acid molecule having 21 nucleotides" means that 19, 20, or 21 nucleotides have the indicated property. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in the series or range.

[0055] As used herein, “no more than” or “less than” is understood to be the value and logical upper or lower limit or integer adjacent to the phrase, which is logical in context and is zero. For example, a double strand with “no more than 2 nucleotides” overhangs has 2, 1, or 0 nucleotide overhangs. When “no more than” appears before a series of numbers or ranges, it should be understood that “no more than” can modify each number in the series or range. As used herein, a range includes both the upper and lower limits.

[0056] “G,” “C,” “A,” and “U” typically represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. “T” and “dT” are used interchangeably herein and refer to deoxyribonucleotides in which the nucleobase is thymine, such as deoxyriboglycosamine. However, it is understood that the terms “ribonucleotide,” “nucleotide,” or “deoxyribonucleotide” can also refer to modified nucleotides, as further detailed below, or alternative substitutions. Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil can be substituted with other portions without substantially altering the base-pairing properties of the oligonucleotide containing the nucleotide carrying such substitutions. For example, but not limited to, nucleotides containing inosine as a base can pair with nucleotides containing adenine, cytosine, or uracil. Therefore, in the nucleotide sequences of this disclosure, nucleotides containing uracil, guanine, or adenine can be substituted with nucleotides containing, for example, inosine. Sequences containing such substitutions are embodiments of this disclosure.

[0057] “ APOC3 "This refers to the apolipoprotein C3 gene. According to the NCBI NLM website, this gene encodes protein components of triglyceride (TG)-rich lipoproteins (TRL), including very low-density lipoprotein (VLDL), high-density lipoprotein (HDL), and chylomicrons." APOC3 The mutations in this protein are associated with low plasma triglyceride levels, a reduced risk of ischemic cardiovascular disease, and hyperalpha-lipoproteinemia, characterized by elevated levels of high-density lipoprotein (HDL) and HDL cholesterol in human patients. APOC3 The mRNA sequence is GenBank accession number NM_000040.3. Rhesus monkey (macaque) Macaca mulatta )) APOC3 The mRNA sequence is GenBank accession number XM_001090312.4; dog (domestic dog) Canis familiaris APOC3 The mRNA sequence is GenBank accession number NM_001003369.2. Mouse (domestic mouse) Mus musculus The mRNA sequence is GenBank accession number NM_001289755.1.

[0058] “ ANGPTL3"ANGPTL3" refers to the angiopoietin-like 3 gene. According to the NCBI NLM website, this gene encodes a secreted protein that plays a role in angiogenesis. The encoded protein, primarily expressed in the liver, is further processed into an N-terminal chain containing a coiled-coil domain 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 familial hypobetalipoproteinemia type 2 and familial hypobetalipoproteinemia type 1. ANGPTL3 The mRNA sequence is GenBank accession number NM_014495.4. Rhesus monkey (Macaque). ANGPTL3 The mRNA sequence is GenBank accession number XM_015141187.2; dog ( Canis familiaris ANGPTL3 The mRNA sequence is GenBank accession number XM_038666015.1. The mouse (domestic rat) mRNA sequence is GenBank accession number NM_013913.4.

[0059] As used herein, “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA products of RNA processing as primary transcription products.

[0060] As used herein, the term "chain containing a sequence" refers to an oligonucleotide chain containing a nucleotide chain described by using a sequence description indicated by standard nucleotide nomenclature.

[0061] As used herein, and unless otherwise specified, the term “complementary” when used to describe a first nucleotide sequence relative to a second nucleotide sequence means the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize under certain conditions with an oligonucleotide or polynucleotide containing the second nucleotide sequence and to form a double-stranded structure, as will be understood by those skilled in the art.

[0062] For example, when two sequences hybridize under stringent hybridization conditions (e.g., annealing), the first nucleotide sequence can be described as complementary to the second nucleotide sequence. Hybridization conditions include the temperature, ionic strength, pH, and organic solvent concentration used for annealing and / or washing steps. The term stringent hybridization conditions refer to conditions under which the first nucleotide sequence will preferentially hybridize with its target sequence, such as the second nucleotide sequence, and hybridize to a lesser extent with other sequences or not with any other sequences at all. Stringent hybridization conditions are sequence-dependent and vary under different environmental parameters. Typically, stringent hybridization conditions are chosen to be at a specified ionic strength and pH higher than the thermal melting point (T0) of the nucleotide sequence. m Approximately 5°C lower. mThis is the temperature at which 50% of the first nucleotide sequence hybridizes with a perfectly matching target sequence (at defined ionic strength and pH). Extensive guidance on nucleic acid hybridization has been found, for example, in Tijssen (1993), *Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes*, Part I, Chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, NY (“Tijssen”).

[0063] Other conditions can be applied, such as physiologically relevant conditions that may be encountered within the organism. Technicians will be able to determine the most suitable set of conditions for testing the complementarity of the two sequences based on the final application of the hybrid nucleotides.

[0064] This includes base pairing of an oligonucleotide or polynucleotide containing the first nucleotide sequence with an oligonucleotide or polynucleotide containing the second nucleotide sequence over the entire length of the first and second nucleotide sequences. Such sequences may be referred to herein as “perfectly complementary” to each other. However, when the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may be perfectly complementary, or they may form one or more, but generally no more than four, three, or two mismatched base pairs after hybridization, while retaining the ability to hybridize under the conditions most relevant to their final application. However, in cases where the two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered definitively mismatched relative to complementarity. For example, a dsRNA containing one 21-nucleotide oligonucleotide and another 23-nucleotide oligonucleotide, where the longer oligonucleotide contains a 21-nucleotide sequence perfectly complementary to the shorter oligonucleotide, may still be referred to as “perfectly complementary” for the purposes described herein.

[0065] As used herein, “complementary” sequences may also include non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, or formed entirely from them, provided that the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogsteen base pairing.

[0066] The terms “complementary,” “fully complementary,” and “substantially complementary” used in this article may be used relative to base matching between the sense and antisense strands of the dsRNA or between the antisense strand of the dsRNA and the target sequence, as understood in the context in which they are used.

[0067] As used herein, a polynucleotide that is at least partially “substantially complementary” to messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding a target gene), including a 5' UTR, an open reading frame (ORF), or a 3' UTR. For example, if the sequence is substantially complementary to a sequence encoding a target gene (e.g., APOC3 or ANGPTL3 If the uninterrupted portions of the mRNA of a target gene are substantially complementary, then the polynucleotide is complementary to at least a portion of the target gene's mRNA.

[0068] In one embodiment, the antisense strand of the dsRNA is fully complementary to the target mRNA in order to induce cleavage of the target mRNA.

[0069] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands as defined above. Generally, the majority of the nucleotides in each strand are ribonucleotides, but as described in detail herein, each or both strands may also include at least one non-ribonucleotide, such as a deoxyribonucleotide, and / or a modified nucleotide. Additionally, as used herein, "dsRNA" can include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides, and includes all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modifications are covered by "dsRNA" as used in siRNA-type molecules.

[0070] The two strands forming a double-stranded structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of a larger molecule and are therefore linked by an unbroken strand of nucleotides between the 3' end of one strand forming the double-stranded structure and the 5' end of the corresponding other strand, the linking RNA strand is called a "hairpin loop." When the two strands are covalently linked in a manner other than an unbroken strand of nucleotides between the 3' end of one strand forming the double-stranded structure and the 5' end of the corresponding other strand, the linking structure is called a "connector." RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the modified dsRNA minus any overhangs present in the double-stranded structure. In addition to the double-stranded structure, modified dsRNA may contain one or more nucleotide overhangs. The term "siRNA" is also used herein to refer to modified dsRNA as described above.

[0071] As used herein, a "nucleotide overhang" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of a modified dsRNA when the 3' end of one strand extends beyond the 5' end of the other strand, and vice versa. "Dull" or "blunt" means that no unpaired nucleotides are present at the said end of the modified dsRNA; that is, no nucleotide overhang is present. A "dull-ended" dsRNA is a modified dsRNA that is double-stranded throughout its entire length; that is, there are no nucleotide overhangs at either end of the molecule.

[0072] The term “antisense strand” refers to a strand of modified dsRNA that includes regions substantially complementary to the target sequence. As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to a sequence as defined herein (e.g., the target sequence). In cases where the complementary region is not perfectly complementary to the target sequence, mismatches are most tolerant in terminal regions and, if present, are typically within one or more terminal regions, for example, within 6, 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0073] As used herein, the term "sense strand" refers to a strand of modified dsRNA that comprises regions substantially complementary to the regions of the antisense strand.

[0074] When referring to modified dsRNA, "introduced into cells" means to facilitate uptake or absorption into cells, as understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unassisted diffusion or active cellular processes, or through aids or devices. This term is not limited to in vitro cells; modified dsRNA can also be "introduced into cells," where cells are part of a living organism. In this case, introduction into cells will include delivery to the organism. For example, for in vivo delivery, modified dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipid transfection. Other methods are described below or are known in the art.

[0075] The terms "silencing," "suppressing expression," "downregulating expression," and "repressing expression," while referring to the target gene in these contexts, in this article refer to the target gene (e.g., APOC3 or ANGPTL3 At least partial repression of the expression of a target gene, as manifested by a reduction in the amount of mRNA, can be achieved from which, compared with a second cell or cell group substantially identical to the first cell or cell group but already or not so treated (control cells), the target gene can be transcribed and treated such that the target gene (e.g.) is transcribed. APOC3 or ANGPTL3 The expression of ) was inhibited in the first cell or cell group isolated. The degree of inhibition is usually expressed as follows: Alternatively, the degree of repression can be based on the functional relationship with the target gene (e.g., APOC3 or ANGPTL3 The decrease in expression-related parameters, such as those of target genes secreted by cells (e.g., APOC3 or ANGPTL3 The amount of protein encoded, plasma lipid levels, or the number of cells exhibiting a particular phenotype can be given. In principle, target gene silencing can be determined in any cells expressing the target, whether constitutively or through genome engineering, and by any appropriate assay. However, when reference is needed to determine whether a given modified dsRNA represses a target gene to some extent (e.g., APOC3 or ANGPTL3 When the expression of ) is and is therefore covered by this disclosure, the determinations provided in the following examples shall serve as such references.

[0076] For example, in some cases, by applying the double-stranded oligonucleotides of this disclosure, the expression of a target gene is repressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, by applying the double-stranded oligonucleotides of this disclosure, the target gene is repressed by at least about 60%, 70%, or 80%. In some embodiments, by applying the double strands of this disclosure, the target gene is repressed by at least about 85%, 90%, or 95%.

[0077] As used herein, in the context of target gene expression, the terms “treat”, “treatment”, etc., refer to the relief or reduction of a pathological process mediated by target gene expression. In the context of this disclosure, in relation to any other condition described below (other than a pathological process mediated by target gene expression), the terms “treat”, “treatment”, etc., mean the relief or reduction of at least one symptom associated with the progression of such condition, or the slowing or reversal of the progression of such condition.

[0078] As used herein, the phrase "effective dose" refers to the amount that provides therapeutic benefit in treating, preventing, or managing a pathological process mediated by target gene expression or in managing the obvious symptoms of a pathological process mediated by target gene expression. The specific effective dose can be readily determined by a general practitioner and can vary based on factors known in the art, such as the type of pathological process mediated by target gene expression, the patient's medical history and age, the stage of the pathological process mediated by target gene expression, and the administration of other antipathological processes mediated by drugs targeting target gene expression.

[0079] As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of modified dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeuticly effective amount,” or simply “effective amount” refers to the amount of modified dsRNA that effectively produces the intended pharmacological, therapeutic, or preventative outcome. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition is reduced by at least 25%, then a therapeutically effective amount of a drug for treating said disease or condition is the amount necessary to reduce said parameter by at least 25%. For example, a therapeutically effective amount of modified dsRNA targeting a target gene can reduce the target gene (e.g., ...) in cells or serum. APOC3 or ANGPTL3 The level decreased by at least 25%.

[0080] The term "pharmaceutically acceptable carrier" refers to a carrier used for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextran, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginate are suitable disintegrants. Binders may include starch and gelatin, while lubricants (if present) are typically magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

[0081] II. Modified double-stranded ribonucleic acid (dsRNA) In one aspect of this disclosure, modified double-stranded RNA (dsRNA) molecules are provided for inhibiting or silencing the expression of target genes, for example, in cells of a subject, such as a mammal (e.g., a human). The use of these modified dsRNA oligonucleotides enables the targeting of corresponding target genes (e.g., in mammals) to be expressed. APOC3 Gene or ANGPTL3 Targeted degradation of mRNA (genes).

[0082] In some embodiments, the dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA or mRNA fragment formed during the expression of the target gene. In some embodiments, the dsRNA comprises a target mRNA (e.g., ANGPTL3 or APOC3 The mRNA or fragment mRNA has at least 70% complementarity.

[0083] 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, ANGPTL3 It is a person APOC3 .

[0084] In some embodiments, the dsRNA comprises an antisense strand with a complementary region, the complementary region being... APOC3 At least a portion of the mRNA or mRNA fragment formed during gene expression is complementary. In some embodiments, the dsRNA contains components similar to those in human... APOC3The mRNA or fragment mRNA has at least 70% complementarity.

[0085] 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, ANGPTL3 It is a person ANGPTL3 .

[0086] In some embodiments, a modified double-stranded RNA (dsRNA) for repressing the expression of a target gene is provided, the modified dsRNA 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 2'-fluoronucleotides at positions 2, 6, 8, and 14 from the 5' end; 2'-fluoronucleotides at positions 2, 4, 6, 8, and 14 from the 5' end; or 2'-fluoronucleotides at positions 2, 4, 6, 8, 14, and 16 from the 5' end. In some embodiments, the sense strand comprises 2'-fluoronucleotides at positions 15, 13, 12, 11, and 9 from the 3' end; 2'-fluoronucleotides at positions 15, 13, 11, and 9 from the 3' end; 2'-fluoronucleotides at positions 13, 12, and 11 from the 3' end; 2'-fluoronucleotides at positions 13, 12, 11, and 9 from the 3' end; or 2'-fluoronucleotides at positions 15, 13, 12, and 9 from the 3' end.

[0087] In some embodiments, the sense strand comprises 2'-fluoronucleotides at positions 15, 13, 12, 11, and 9 from the 3' end; 2'-fluoronucleotides at positions 15, 13, 11, and 9 from the 3' end; 2'-fluoronucleotides at positions 13, 12, and 11 from the 3' end; 2'-fluoronucleotides at positions 13, 12, 11, and 9 from the 3' end; or 2'-fluoronucleotides at positions 15, 13, 12, and 9 from the 3' end.

[0088] In some embodiments, the antisense strand comprises 2'-fluoronucleotides at positions 2, 6, 8, and 14 from the 5' end, and the sense strand comprises 2'-fluoronucleotides at positions 15, 13, 12, 11, and 9 from the 3' end; the antisense strand comprises 2'-fluoronucleotides at positions 2, 6, 8, and 14 from the 5' end, and the sense strand comprises 2'-fluoronucleotides at positions 13, 12, 11, and 9 from the 3' end; the antisense strand comprises 2'-fluoronucleotides at positions 2, 6, 8, and 14 from the 5' end. The sense strand comprises 2'-fluoronucleotides at positions 15, 13, 12, and 9 from the 3' end; the antisense strand comprises 2'-fluoronucleotides at positions 2, 4, 6, 8, and 14 from the 5' end, and the sense strand comprises 2'-fluoronucleotides at positions 15, 13, 11, and 9 from the 3' end; or the antisense strand comprises 2'-fluoronucleotides at positions 2, 4, 6, 8, 14, and 16 from the 5' end, and the sense strand comprises 2'-fluoronucleotides at positions 13, 12, and 11 from the 3' end.

[0089] In some embodiments, the dsRNA has a mismatch with the target mRNA or a fragment of the target mRNA. In some embodiments, the dsRNA contains one or two mismatches with the target mRNA or a fragment of the target mRNA. In some embodiments, the dsRNA is more than 70% identical to the target mRNA or a 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 a fragment of the target mRNA. In some embodiments, the antisense strand contains at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical nucleotide sequences to the target mRNA or a fragment of the target mRNA. In some embodiments, the antisense strand of the dsRNA contains at least 80% of the target mRNA or a 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 contains one, two, three, or four mismatches with the target mRNA or a fragment of the target mRNA. In some embodiments, the mismatch is located in the middle of the dsRNA. In some embodiments, the mismatch occurs in the 5' or 3' region of the dsRNA. In some embodiments, the mismatch is no more than 5 nucleotides from the 5' or 3' end of the dsRNA.

[0090] In some embodiments, at least one strand of the dsRNA includes a 3' or 5' overhang of at least one nucleotide. In some embodiments, the overhang is at least two or at least three nucleotides. In some embodiments, at least one strand of the dsRNA includes a 3' overhang. In some embodiments, at least one strand of the dsRNA includes a 5' overhang.

[0091] In some embodiments, the antisense strand has a 3' nucleotide overhang compared to the sense strand. In some embodiments, the 3' nucleotide overhang comprises one, two, or three nucleotides compared to the sense strand. In some embodiments, the antisense strand 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.

[0092] Modified dsRNA can be synthesized using standard methods known in the art, as discussed further below, for example, by using an automated DNA synthesizer, commercially available from companies such as Biosearch and Applied Biosystems, Inc. The modified dsRNA comprises two RNA strands sufficiently complementary to hybridize and form a double-stranded structure. One strand of the dsRNA (the antisense strand) includes a complementary region to the target gene sequence derived from the sequence of mRNA formed during the expression of the target gene, and the other strand (the sense strand) includes a region complementary to the antisense strand, such that when combined under suitable conditions, the two strands hybridize and form a double-stranded structure.

[0093] In some embodiments, the length of the double-stranded structure is 15 to 30, 25 to 30, 18 to 25, 19 to 24, 19 to 21, or 19, 20, or 21 base pairs. In one embodiment, the length of the double-stranded structure is 19 base pairs. In one embodiment, the length of the double-stranded structure is 20 base pairs. In another embodiment, the length of the double-stranded structure is 21 base pairs. When two different single-stranded RNAs (ssRNAs) are used in combination, the length of the double-stranded structure may be the same or different.

[0094] In some embodiments, each strand of the modified dsRNA of this disclosure is 15 to 30, or 18 to 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each strand is about 25-30 nucleotides in length. In some embodiments, each strand of the duplex has the same or different lengths. When two different ssRNAs are used in combination, each strand of each ssRNA may be the same or different in length.

[0095] In some embodiments, the dsRNA comprises dsRNA longer than 21-23 nucleotides, for example, dsRNA long enough to be processed by the RNase III enzyme Dicer into siRNA having 21-23 base pairs, and then incorporated into an RNA-induced silencing complex (RISC). Therefore, the length of the dsRNA disclosed herein is at least 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or at least 100 base pairs.

[0096] Inhibition of target gene expression can be determined, for example, by nucleic acid-based assays such as quantitative PCR, or by protein-based methods such as Western blotting. When measured by assays as described in the following examples, the expression of the target gene (e.g.) ANGPTL3 Gene or ANGPTL3 The expression of genes can be reduced by at least 50%. For example, ANGPTL3 Gene expression in cell cultures, such as in Huh-7 cells, can be measured. APOC3 mRNA levels can be determined, for example, by quantitative PCR, or by measuring protein levels, for example, by ELISA.

[0097] On the other hand, this disclosure provides single-stranded antisense oligonucleotide RNAi. Antisense oligonucleotides are single-stranded oligonucleotides complementary to a sequence within the target mRNA. Antisense oligonucleotides can stoichiometrically inhibit translation by base pairing with mRNA and physically block translation mechanisms; see Dias, N. et al., (2002) Molecular Cancer Therapy (Molecular Cancer Therapy). ANGPTL3 ANGPTL3 (1:347-355). Antisense oligonucleotides can also inhibit target protein expression by binding to mRNA targets and promoting mRNA target destruction via RNase-H. Single-stranded antisense RNA molecules can be from about 13 to about 30 nucleotides in length and have a sequence complementary to the target sequence.

[0098] Mol. In some embodiments, the dsRNA is chemically modified to enhance its stability. The nucleic acids characterized in this disclosure can be synthesized and / or modified using methods recognized in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which are hereby incorporated by reference. Specific examples of dsRNA compounds that can be used in this disclosure include dsRNAs containing a modified backbone or non-natural nucleoside internucleotide bonds. As defined in this specification, dsRNAs having a modified backbone include dsRNAs that retain a phosphorus atom in the backbone and dsRNAs that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes mentioned in the art, modified dsRNAs that do not have a phosphorus atom in their internucleotide backbone may also be considered oligonucleotides.

[0099] In some embodiments, the modified dsRNA backbone includes at least one of the following: a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-thiophosphate group, a 2'-fluorine modified nucleotide; a reverse abase-free nucleotide, a thymine-glycol nucleic acid (GNA) S-isomer; inosine and a reverse deoxyribonucleotide (3'-3' linked nucleotide), and a thymine-glycol nucleic acid (GNA) S-isomer.

[0100] In some embodiments, the modification comprises one or more thiophosphates, chiral thiophosphates, dithiophosphates, triphosphates, aminoalkyl phosphates, methyl and other alkylphosphonates, including 3'-alkylene phosphonates and chiral phosphonates, hypophosphonates, phosphatidyl esters including 3'-aminophosphatidyl esters and aminoalkylphosphatidyl esters, thiophosphatidyl esters, thioalkylphosphonates, thioalkyl phosphates, and borate phosphates having a normal 3'-5' bond, 2'-5' linked analogs of these having opposite polarity, wherein adjacent pairs of nucleoside units are 3'-5' to 5'-3' or 2'-5' to 5'-2' linked. Various salts, mixed salts, and free acid forms are also included.

[0101] In some embodiments, the modified nucleotide comprises at least one of the following: 5'-vinylphosphonate nucleotide, 5'-phosphate or phosphate mimicry, locked nucleic acid (LNA), 2'-MOE (methoxyethyl) nucleotide, and / or 2'-arabinoflus (2'-araF) nucleotide. In some embodiments, the modified nucleotide antisense strand comprises a phosphate mimicry at the 5' end; optionally, said phosphate mimicry is 5'-E-vinylphosphonate or 4'-O-phosphonate.

[0102] In some embodiments, the modified nucleotide comprises at least one of the following: 2'-deoxy-2'-fluorine modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, baseless nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, phosphoramide ester and / or non-natural bases containing nucleotides.

[0103] In some embodiments, the antisense strand and / or the sense strand comprises at least one internucleotide bond selected from the group consisting of: thiophosphate bond, dithiophosphate bond, phosphate trimer bond, alkylphosphonate bond, aminoalkylphosphate trimer bond, alkylenephosphonate bond, hypophosphonate bond, phosphoramidite bond, morpholinophosphate bond, piperazine phosphate bond, aminoalkylphosphatite bond, thiophosphatite bond, thiocarbonylalkylphosphonate bond, thiocarbonylalkylphosphate trimer bond, thiophosphate bond, selenophosphate bond, and boron phosphate bond. In some embodiments, the antisense strand and / or the sense strand comprises at least one nucleotide-modified bond. In some embodiments, all nucleotide bonds in the antisense strand are modified bonds. In some embodiments, the antisense strand and / or the sense strand comprises at least one thiophosphate (PS) bond.

[0104] Cancer Ther Another modification of the dsRNA disclosed herein involves chemically linking one or more ligands or targeting moieties or conjugates to the dsRNA, which enhances the activity, cellular distribution, or cellular uptake of the dsRNA.Such fractions include, but are not limited to, lipid fractions, such as cholesterol fractions (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), bile acids (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, for example, beryllium-S-triphenylmethanethiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), and sulfur cholesterol (Oberhauser et al., Nucleic Acids Res., 1992, ). 20:533-538), aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., *Journal of the European Society for Molecular Biology (EMBO J)*, 1991, 10:1111-1118; Kabanov et al., *Letter from the Federation of European Biochemical Societies (FEBS Lett)*, 1990, 259:327-330; Svinarchuk et al., *Biochemistry*, 1993, 75:49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Mvinarchuk et al., *Tetrahedron Letters*, 1995, 36:3651-3654; Shea et al., *Nucleic Acid Research*, 1990, 18:3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973) or adamantaneacetic acid (Manoharan et al., Tetrahedral Letters, 1995, 36:3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237) or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0105] In some embodiments, the ligand or the targeting portion is conjugated to the 5' end, the 3' end, or both ends of the modified dsRNA. In some embodiments, the ligand or the targeting portion is conjugated to the 3' end of the sense strand of the modified dsRNA. In some embodiments, the ligand or the targeting portion is conjugated to the 3' end of the antisense strand of the modified dsRNA. In some embodiments, the ligand or the targeting portion is at least one N-acetylgalactosamine (GalNAc).

[0106] In some embodiments, the modified dsRNA may be modified with non-ligand groups. Many non-ligand molecules have been conjugated to the modified dsRNA to enhance its activity, cellular distribution, or cellular uptake, and procedures for such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Letsinger et al., Proceedings of the National Academy of Sciences, 1989, 86:6553), bile acids (Manoharan et al., Letters of Bioorganic and Medicinal Chemistry, 1994, 4:1053), thioethers, such as hexyl-S-triphenylmethanethiol (Manoharan et al., Annals of the New York Academy of Sciences, 1992, 660:306; Manoharan et al., Letters of Bioorganic and Medicinal Chemistry, 1993, 3:2765), sulfur cholesterol (Oberhauser et al., Nucleic Acid Research, 1992, 20:533), and fatty acid chains, such as dodecyl glycol or undecyl residues (Saison-Behmoaras et al., Journal of the European Society for Molecular Biology, 1991, 10:111; Kabanov et al., Letters of the Federation of European Biochemical Societies, 1990). 259:327; Svinarchuk et al., *Biochemistry*, 1993, 75:49), phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate (Manoharan et al., *Tetrahedral Letters*, 1995, 36:3651; Shea et al., *Nucleic Acid Research*, 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., *Nucleosides and Nucleotides*, 1995, 14:969) or adamantaneacetic acid (Manoharan et al., *Tetrahedral Letters*, 1995, 36:3651), palmitic moieties (Mishra et al., *Chinese Journal of Biochemistry and Biophysics*, 1995, (1264:229) or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., *Journal of Pharmacology and Experimental Therapeutics*, 1996, 277:923). Typical conjugation schemes involve synthesizing dsRNA carrying an amino linker at one or more positions in the oligonucleotide sequence. The amino group is then reacted with the conjugated molecule using a suitable coupling or activating agent. The conjugation reaction can be carried out in solution while the dsRNA is still bound to a solid-phase support or after dsRNA cleavage. Modified dsRNA conjugates can be purified, for example, by HPLC.

[0107] Conjugating ligands to modified dsRNA can enhance their cellular uptake and targeting to specific tissues or specific cell types, such as hepatocytes. In some cases, hydrophobic ligands are conjugated to modified dsRNA to facilitate direct cell membrane penetration and / or transhepatocyte uptake. Alternatively, the ligand conjugated to modified dsRNA may be a substrate for receptor-mediated endocytosis. These approaches have been used to enhance the cellular penetration of antisense oligonucleotides and modified dsRNA agents. For example, cholesterol has been conjugated to various antisense oligonucleotides to produce compounds with significantly higher activity compared to their unconjugated analogs. See M. Manoharan, "Antisense and Nucleic Acid Drug Development" (…). modification conjugate )》 2002, 12 , 103. Other lipophilic compounds conjugated to oligonucleotides include 1-pyrenebutyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. An example of a ligand used for receptor-mediated endocytosis is folic acid. Folic acid enters cells via folate receptor-mediated endocytosis. Modified dsRNA compounds carrying folic acid are efficiently transported into cells via folate receptor-mediated endocytosis. Li and colleagues reported that attaching folic acid to the 3'-terminus of an oligonucleotide increased cellular uptake of the oligonucleotide by 8-fold. Li, S.; Deshmukh, HM; Huang, L. *Drug Research* (… Antisense & Nucleic Acid Drug )》 1998, 15 , 1540. Other ligands conjugated with oligonucleotides include polyethylene glycol, carbohydrate clusters, cross-linking agents, porphyrin conjugates, delivery peptides, and lipids such as cholesterol and cholesterol amines. Examples of carbohydrate clusters include Chol-p-(GalNAc)3 (N-acetylgalactosamine cholesterol) and LCO(GalNAc)3 (N-acetylgalactosamine-3'-lithocholic acid-oleoyl).

[0108] Development In some embodiments, the modified dsRNA oligonucleotides of this disclosure further comprise carbohydrates. Carbohydrate-conjugated modified dsRNAs facilitate in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound that is itself composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), wherein an oxygen, nitrogen, or sulfur atom is bonded to each carbon atom; or a compound having a carbohydrate moiety as part thereof, said carbohydrate moiety being composed of one or more monosaccharide units, each of said one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), wherein an oxygen, nitrogen, or sulfur atom is bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Certain monosaccharides include sugars with 5 or more saccharides (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0109] In one embodiment, the carbohydrate conjugate used in the compositions and methods of this disclosure is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine of Formula I or II, such as... Formula I. Formula II.

[0110] In some embodiments, the carbohydrate is conjugated to the 5' end, the 3' end, or both ends of the modified dsRNA. In some embodiments, the ligand or the targeting portion is conjugated to the 3' end of the sense strand of the modified dsRNA. In some embodiments, the ligand or the targeting portion is conjugated to the 3' end of the antisense strand of the modified dsRNA. In some embodiments, the carbohydrate is at least one N-acetylgalactosamine (GalNAc).

[0111] III. Pharmaceutical Composition This document also discloses pharmaceutical compositions comprising targeting genes disclosed herein (e.g., Pharm. Res. Gene or carbohydrate conjugate Modified dsRNA of genes.

[0112] In one embodiment, this disclosure provides a pharmaceutical composition comprising a modified dsRNA as described herein, and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising modified dsRNA can be used to treat diseases or conditions associated with the expression or activity of a target gene, such as pathological processes mediated by targeting the expression of the target gene. Such pharmaceutical compositions are formulated based on a delivery modality.

[0113] The pharmaceutical compositions characterized in this article are sufficient to inhibit target genes (e.g., ANGPTL3 Gene or APOC3 Dosage administration of gene expression.

[0114] Those skilled in the art will understand that certain factors may influence the dosage and timing required for effective treatment of a subject, including but not limited to the severity of the disease or condition, prior treatment, the subject's overall health and / or age, and any other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of the composition may include a single treatment or a series of treatments. As described elsewhere herein, the effective dose and in vivo half-life of the single modified dsRNA covered by this disclosure can be estimated using conventional methods or based on in vivo testing using appropriate animal models.

[0115] Advances in mouse genetics have led to the development of numerous mouse models for studying various human diseases, such as pathological processes mediated by target gene expression. These models are used for in vivo testing of dsRNA and modified dsRNA, as well as for determining effective therapeutic doses. Suitable mouse models are, for example, mice containing plasmids expressing human target genes. Another suitable mouse model is one carrying a plasmid expressing a human target gene (e.g., ...). ANGPTL3 Gene or APOC3 Transgenic mice containing genetically modified genes.

[0116] Data obtained from cell culture assays and animal studies can be used to formulate dosage ranges for human use. The dosages of the compositions characterized in this disclosure are generally within a circulating concentration range including the ED50, with little or no toxicity. Dosages can vary within this range depending on the dosage form and route of administration used. For any compound used in the methods characterized in this disclosure, the therapeutically effective dose can be initially estimated based on cell culture assays. Dosages can be formulated in animal models to achieve circulating plasma concentration ranges of the compound, or, where appropriate, to achieve circulating plasma concentration ranges of peptide products of target sequences (e.g., to achieve reduced concentrations of the peptide), said target sequences including the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine the useful dose in humans more accurately. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0117] The modified dsRNA characterized in this disclosure can be administered in combination with other known agents that are effective in treating pathological processes mediated by target gene expression. In any case, the administering physician can adjust the amount and timing of the modified dsRNA administration based on results observed using standard measures of efficacy known in the art or described herein.

[0118] ANGPTL3 In some embodiments, the pharmaceutical compositions disclosed herein comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems can be used to prepare pharmaceutical compositions comprising hydrophobic compounds. In some embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0119] In some embodiments, the modified dsRNA of this disclosure is introduced into pre-formed liposomes or liposome complexes made of a mixture of cationic and neutral lipids. In some methods, the modified dsRNA complex is formed with monocationic or polycationic lipids in the absence of neutral lipids. In some embodiments, the lipid portion is selected to increase the distribution of the agent to specific cells or tissues. In some embodiments, the lipid portion is selected to increase the distribution of the agent to adipose tissue. In some embodiments, the lipid portion is selected to increase the distribution of the agent to muscle tissue.

[0120] APOC3 In some embodiments, the pharmaceutical composition comprises an excipient. Compared to a carrier compound, a “drug carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert medium used to deliver one or more nucleic acids to animals. Excipients may be liquid or solid and are selected taking into account the planned administration method to provide the desired volume, consistency, etc., when combined with nucleic acids and other components of a given pharmaceutical composition. Typical drug carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, or dicalcium phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium carboxyacetic acid starch, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0121] Pharmaceutically acceptable organic or inorganic excipients that are suitable for non-parenteral application and do not react harmfully with nucleic acids may also be used to formulate the compositions disclosed herein. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0122] Formulations for topical application of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents (such as alcohol), or solutions of nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral application and not causing harmful reactions with nucleic acids may be used.

[0123] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0124] liposomal formulation The compositions disclosed herein may additionally contain other auxiliary components routinely found in pharmaceutical compositions, used at levels determined in the art. Thus, for example, the compositions may contain additional compatible pharmaceutically active materials, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials suitable for the physical formulation of various dosage forms of the compositions disclosed herein, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials, when added, should not unduly interfere with the bioactivity of the components of the compositions disclosed herein. The formulation may be sterilized and, if desired, may be mixed with adjuvants that do not harmfully interact with the nucleic acids of said formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavoring agents, and / or aromatic substances.

[0125] Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. Suspensions may also contain stabilizers.

[0126] excipient This document also discloses methods of administering pharmaceutical compositions and formulations, including the modified dsRNA compositions and pharmaceutical compositions of this disclosure. In some embodiments, the pharmaceutical compositions of this disclosure are administered in a variety of ways, depending on whether local or systemic treatment is desired, and depending on the area to be treated.

[0127] In some embodiments, the pharmaceutical composition is administered topically (including via the buccal and sublingual route), pulmonaryly (e.g., by inhalation or blowing in of a powder or aerosol, including via a nebulizer); intratracheally, intranasally, epidermally, and percutaneously, orally or parenterally. In some embodiments, parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, such as intracranial, intrathecal, or intraventricular administration.

[0128] Pharmaceutical compositions containing the modified dsRNA of this disclosure may be presented in dose-unit form and may be prepared by any suitable method. The pharmaceutical composition shall be formulated to be compatible with its intended route of administration. Useful formulations may be prepared by methods well known in the pharmaceutical field. See, for example, Remington's Pharmaceutical Sciences, 18th edition (Mack Publishing Company, 1990).

[0129] The pharmaceutical formulation is, for example, sterile. Sterilization can be achieved, for example, by filtration through a sterile filter membrane. In the case of a lyophilized composition, filter sterilization can be performed before or after lyophilization and reconstitution.

[0130] In some embodiments, the modified dsRNA is delivered in a manner that targets a specific tissue, such as the liver.

[0131] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific administration method. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is typically the amount of the compound that produces the therapeutic effect (e.g., a modified dsRNA molecule).

[0132] In some embodiments, the formulations of this disclosure comprise excipients selected from the group consisting of cyclodextrins, cellulose, liposomes, micelle forming agents such as bile acids, and polymer carriers such as polyesters and polyanhydrides; and compounds of this disclosure (e.g., modified dsRNA molecules). In some embodiments, the foregoing formulations make the compounds of this disclosure (e.g., modified dsRNA molecules) orally bioavailable.

[0133] Formulations of this disclosure suitable for oral administration may be in the form of capsules, flat capsules, pills, tablets, lozenges (using a flavoring matrix, typically sucrose and gum arabic or tragacanth), powders, granules; or as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-oil liquid emulsions; or as elixirs or syrups; or as soft lozenges (using an inert matrix, such as gelatin and glycerin or sucrose and gum arabic); and / or as mouthwashes, etc., each containing a predetermined amount of a compound of this disclosure (e.g., a modified dsRNA molecule) as an active ingredient. Compounds of this disclosure (e.g., modified dsRNA molecules) may also be administered as boluses, ointments, or pastes.

[0134] Liquid dosage forms for oral administration of the compounds disclosed herein (e.g., modified dsRNA molecules) include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.

[0135] IV. Methods for suppressing target gene expression On the other hand, this disclosure provides a method for inhibiting target genes in cells (e.g. other components Gene or administration Methods for expressing a target gene. The methods include administering a modified dsRNA targeting a target gene to a cell, thereby reducing the expression of the target gene in the cell. This disclosure includes methods performed in vitro or in vivo in cells. In some embodiments, the methods are performed in cells of an animal, such as a mouse, rat, non-human primate, or human.

[0136] This disclosure also provides methods for reducing and / or inhibiting the expression of a target gene in cells using the modified dsRNA of this disclosure and / or compositions containing the modified dsRNA of this disclosure. The methods include contacting cells with the modified dsRNA of this disclosure and maintaining the cells for a time sufficient to allow degradation of the target mRNA transcript of the target gene, thereby inhibiting the expression of the target gene in the cells. The reduction in gene expression can be assessed by any method known in the art. For example, a reduction in target expression can be determined by: determining the mRNA expression level of the target gene using methods conventional to those skilled in the art (e.g., Northern blotting, qRT-PCR); determining the protein level of the target gene using methods conventional to those skilled in the art (e.g., Western blotting, immunoassay); and / or by determining the biological activity of the target gene (e.g., inhibitory effect). ANGPTL3 Gene or APOC3 Genes can influence one or more molecules associated with cellular coagulation mechanisms.

[0137] In the methods disclosed herein, cells can be contacted in vitro or in vivo, i.e., cells can be inside a subject.

[0138] The cells suitable for treatment using the methods of this disclosure can be any cells expressing the target gene. Cells suitable for use in the methods of this disclosure can be mammalian cells, such as primate cells (e.g., human cells or non-human primate cells, e.g., monkey cells or chimpanzee cells), non-primate cells (e.g., bovine cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster cells, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), bird cells (e.g., duck cells or goose cells), or whale cells. In one embodiment, the cell is a human cell, e.g., a human liver cell.

[0139] In some embodiments, target gene expression is suppressed by at least 30% relative to a control, for example, as measured by quantitative polymerase chain reaction (PCR) after administration of the modified dsRNA oligonucleotide of this disclosure.

[0140] In some embodiments, a method is provided for treating a condition mediated by a target gene (e.g., cardiovascular disease), the method comprising administering a therapeutically effective amount of the modified dsRNA oligonucleotide or pharmaceutical composition of this disclosure to a subject requiring such treatment.

[0141] Example The following examples are given for the purpose of illustrating various embodiments of this disclosure and are not intended to limit this disclosure in any way. These examples and the methods described herein represent exemplary preferred embodiments and are not intended to limit the scope of this disclosure. Variations therein and other uses covered within the spirit of this disclosure as defined by the claims will be apparent to those skilled in the art.

[0142] Example 1. Using an exemplary method in primary human hepatocytes ANGPTL3 In vitro dose-response screening of siRNA compounds This example describes the exemplary GalNAc-conjugated modified siRNA in primary human hepatocyte (PHH) cells during single-dose screening with selected siRNAs at concentrations of 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.412 nM, 0.137 nM, and 0.046 nM. APOC3 Screening of siRNA compounds (Table 1).

[0143] “ ANGPTL3 "" refers to the angiopoietin-like 3 gene. According to the NCBI NLM website, this gene encodes a secreted protein that plays a role in angiogenesis. ANGPTL3The mRNA sequence is GenBank accession number NM_014495.4, included in this article as SEQ ID NO: 659. Rhesus monkey (macaque). ANGPTL3 The mRNA sequence is GenBank accession number XM_015141187.2; dog ( ANGPTL3 The mRNA sequence is GenBank accession number XM_038666015.1. The mouse (domestic rat) mRNA sequence is GenBank accession number NM_013913.4.

[0144] Measurement by quantitative PCR ANGPTL3 mRNA levels, and relative to control cells treated with the simulated treatment Canis familiaris Normalization was performed, and the average knockdown (KD) and SD were determined. Modified sequences of sense and antisense strands are shown in Table 1, and exemplary results are shown in Table 2 and... ANGPTL3 middle.

[0145] Table 1. Exemplary 3' GalNAc conjugated sense and antisense sequences and modifications of ANGPTL3 siRNA compounds. Abbreviations: (*) = PS key; (-) = PO key; lowercase = 2'-OMe; uppercase = 2'-F; dX = DNA; VP = 5'-E-vinylphosphonate.

[0146] Table 2. Absolute values ​​of exemplary ANGPTL3 siRNAs GAPDH mRNA IC 50 And maximum inhibition (%). Example 2. Using an exemplary method in primary human hepatocytes Figures 1A-1B In vitro dose-response screening of siRNA compounds This example describes an exemplary GalNAc-conjugated modified siRNA evaluated in a free uptake assay in primary human hepatocytes (PHH) cells during single-dose screening with selected siRNAs (Table 3) at doses of 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM, 0.4 nM, 0.1 nM, and 0.05 nM. ANGPTL3 Screening of siRNA compounds and measurement of inhibition.

[0147] “APOC3” refers to the apolipoprotein C3 gene. According to the NCBI NLM website, this gene encodes protein components of triglyceride (TG)-rich lipoproteins (TRLs), including very low-density lipoprotein (VLDL), high-density lipoprotein (HDL), and chylomicrons. The encoded protein functions in the metabolism of these TRLs through multiple pathways.

[0148] Free access assays were performed in triplicate for each dose. The absolute IC50 values ​​for each listed siRNA are... 50 Generated by GraphPadPrism 9.

[0149] Modified sequences with and without sense strands are shown in Table 3, and exemplary results are shown in Table 4 and... APOC3 middle.

[0150] Table 3 APOC3 Exemplary sense and antisense sequences of siRNA compounds and modifications thereof. Abbreviations: (*) = PS bond; (-) = PO bond; lowercase = 2'-OMe; uppercase = 2'-F; rX = RNA; VP = 5'-E-vinylphosphonate Table 4 Examples Figures 2A-2D siRNA mRNA IC 50 . Example 3. In vitro RNA interference (RNAi) screening in primary human hepatocytes This example describes the application of siRNA-based methods in primary human hepatocytes. APOC3 Screening for gene repression. Human hepatocytes were transfected with 36 GalNAc-conjugated modified siRNAs (BC-100001-BC-100814, sense and antisense strands) at 100 nM, 10 nM, and 1 nM. The sequences of exemplary, unmodified, and modified siRNA compounds are shown in Tables 1A and 2A, respectively. The compounds in Table 2A are 3' GalNac modified. APOC3 mRNA levels were measured by quantitative PCR and relative to PBS-simulated control cells. APOC3 Table 3A shows the mRNA levels using the selected... APOC3 Results of single-dose screening of siRNA in human hepatocytes at 100 nM, 10 nM, and 0.1 nM. Data are presented as APOC3 mRNA levels in cells transfected with siRNA relative to APOC3 mRNA levels measured by quantitative PCR and normalized relative to GAPDH. APOC3 Percentage of mRNA inhibition.

[0151] Table 1A. APOC3 unmodified siRNA sequence Table 2A. Combinations with 3'-GalNAc APOC3 The semantic and antisemantic sequences modified by the 3'-GalNAc conjugation. Abbreviations: (*) = PS bond; (-) = PO bond; lowercase = 2'-OMe; uppercase = 2'-F; rX = RNA; dX = DNA; invAb = reverse baseless; Tgn = thymine-glycol nucleic acid (GNA) S-isomer; i = inosine; invdN = reverse 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.

[0152] Table 3A uses the selected modified... APOC3 Results of single-dose screening of siRNA in human hepatocytes at 100 nM, 10 nM, and 0.1 nM are expressed as inhibition % (KD). Example 4. In vitro RNAi screening in primary cynomolgus monkey hepatocytes This example describes an exemplary study of cryopreserved primary cynomolgus monkey hepatocytes (PCH) in a single-dose screening with selected siRNAs at 100 nM, 10 nM, and 1 nM. APOC3 Screening of siRNA compounds. Data are presented as APOC3 mRNA levels in cells transfected with siRNA, relative to APOC3 mRNA levels measured by quantitative PCR and normalized relative to GAPDH. APOC3 The percentage of mRNA inhibition was expressed as a percentage of inhibition, and the SD was determined. Table 4A shows the results of single-dose screening at 100 nM, 10 nM, and 1 nM.

[0153] Table 4A. Average inhibition percentage and SD of the selected compounds. Example 5. Evaluation of human APOC3 knockdown in humanized transgenic mice This example describes the evaluation of the in vivo activity of APOC3 siRNAs that do not cross-react with the rodent APOC3 gene. Briefly, in vivo screening of siRNAs was performed using a transgenic mouse model in which the human APOC3 gene is randomly knocked into the mouse genome, the screening being based on their in vitro potency. All siRNAs were administered subcutaneously at 3 mg / kg, with PBS used as a negative control. Human APOC3 protein expression in mice was measured in serum collected at days 7, 14, 21, and 28 by enzyme-linked immunosorbent assay (ELISA). Data are presented as ratios to baseline (day -1, before siRNA treatment). Results are shown in Table 5A.

[0154] Table 5A. Ratios of inhibition at 7, 14, 21, and 28 days to baseline and SD of exemplary APOC3 siRNA. Example 6. In vitro RNA interference (RNAi) screening in the Huh7 cell line This example describes siRNA-based [technology / method] in a hepatocyte-derived cancer cell model (Huh-7). APOC3 Screening for gene repression. Huh-7 cells were transfected with 96 modified siRNAs (BC-100039-BC-100134, sense and antisense strands) conjugated with 10 nM and 0.1 nM respectively. The sequences of exemplary, unmodified, and modified siRNA compounds are shown in Tables 1B and 2B, respectively. The compounds in Table 2 are 3' GalNac modified. Measurements were performed by quantitative PCR. APOC3 mRNA levels, and relative to control cells treated with the simulated treatment ANGPTL3 Normalization. Table 3B shows the normalization used with the selected... ANGPTL3 Results of single-dose screening of siRNA in Huh7 cells at 10 nM and 0.1 nM. Data are presented as a percentage of control cells treated with a simulated effect. GAPDH The percentage of ANGPLT3 mRNA inhibition in cells transfected with siRNA is listed in Table 2-1B. ANGPTL3 Other exemplary siRNAs include 3' GalNac modified sense and antisense strands.

[0155] Table 1B. ANGPTL3 unmodified siRNA sequence Table 2B. Combinations with 3'-GalNAc ANGPTL3 GalNac-modified sense chains and antisense sequences. Abbreviations: (*) = PS bond; (-) = PO bond; lowercase = 2'-OMe; uppercase = 2'-F; rX = RNA; dX = DNA; invAb = inverse baseless; Tgn = thymine-glycol nucleic acid (GNA) S-isomer; i = inosine; invdN = inverse 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.

[0156] Table 2-1B ANGPTL3 Exemplary 3'-GalNac modified sense and antisense strands of siRNA. Abbreviations: (*) = PS bond; (-) = PO bond; lowercase = 2'-OMe; uppercase = 2'-F; rX = RNA; dX = DNA; invAb = inverse baseless; Tgn = thymine-glycol nucleic acid (GNA) S-isomer; i = inosine; invdN = inverse 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.

[0157] Table 3B uses the selected modified... ANGPTL3 Results of single-dose screening of siRNA in Huh7 cells at 10 nM and 0.1 nM. Example 7. In vitro RNAi screening in primary human hepatocytes Select display ANGPTL3Exemplary siRNA compounds were knocked down, and knockdown was tested in cryopreserved primary human hepatocytes (PHH) at 100 nM, 10 nM, and 1 nM. ANGPTL3 Quantitative PCR measurement ANGPTL3 mRNA levels, and relative to control cells treated with the simulated treatment ANGPTL3 Normalization was performed, and the mean KD and SD were determined. Table 4B shows the single-dose administration at 100 nM, 10 nM, and 1 nM in cryopreserved primary human hepatocytes (PHH) cells. ANGPTL3 Results of siRNA screening.

[0158] Table 4B. Average KD and SD% of the selected compounds Compared to control cells treated with PBS GAPDH The mRNA was further modified in primary human hepatocytes treated with modified siRNA conjugated with 100 nM, 10 nM, and 1 nM GalNAc, and the selected mRNA was then tested. ANGPTL3 siRNA compounds BC-100044, BC-100047, BC-100115, BC-100067, BC-100127, and BC-100042. Measurement was performed by quantitative PCR. ANGPTL3 mRNA levels and relative to ANGPTL3 Normalization. Modified sequences of sense and antisense strands are shown in Table 5B, and exemplary results are shown in Table 6B.

[0159] Table 5B ANGPTL3 Exemplary 3'-GalNAc conjugated sense and antisense sequences of siRNA compounds, and modifications thereof. Abbreviations: (*) = PS bond; (-) = PO bond; lowercase = 2'-OMe; uppercase = 2'-F; rX = RNA; dX = DNA; invAb = inverse baseless; Tgn = thymine-glycol nucleic acid (GNA) S-isomer; i = inosine; invdN = inverse 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.

[0160] Table 6B. Selected GAPDH Average KD and SD of siRNA compounds. Example 8. In vitro RNAi screening in primary cynomolgus monkey hepatocytes This example describes an exemplary study of cryopreserved primary cynomolgus monkey hepatocytes (PCH) in a single-dose screening with selected siRNAs at 100 nM, 10 nM, and 1 nM. ANGPTL3 Screening of siRNA compounds. Measurement by quantitative PCR. ANGPTL3 mRNA levels, and relative to control cells treated with the simulated treatment ANGPTL3 Normalization was performed and mean knockdown (KD) and SD were determined. Table 7B shows the results of single-dose screening at 100 nM, 10 nM, and 1 nM.

[0161] Selected from Table 7B ANGPTL3 Average KD and SD of siRNA compounds. Example 9. Evaluation of mice in wild-type mice GAPDH knocking down This example describes an in vivo mouse. ANGPTL3 Exemplary ANGPTL3 Evaluation of siRNA. Testing in wild-type mice and rodents. ANGPTL3 Hybrid siRNA inhibits mice ANGPTL3 Expression capacity. All siRNAs were administered subcutaneously at 3 mg / kg, with PBS administered as a negative control. ANGPTL3 protein expression in mice 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 baseline (day -1, before siRNA treatment).

[0162] Table 8B Examples ANGPTL3 Average knockdown ratio of siRNA at 3 days, 7 days and 14 days. Example 10. Mice in wild-type mice ANGPTL3 The timeline for the knockdown is low. This example describes the use of an exemplary model. ANGPTL3 siRNA knockdown mice ANGPTL3 Time-course evaluation of mRNA. Tested in wild-type mice and rodents. ANGPTL3 Exemplary siRNA-inhibited mice with mRNA hybridization ANGPTL3 ANGPTL3 ANGPTL3Expression capacity. All siRNAs were administered subcutaneously at 3 mg / kg, with PBS administered as a negative control. Mouse ANGPTL3 protein expression was measured in serum at days 7, 14, 21, and 28 by enzyme-linked immunosorbent assay (ELISA). Data are presented as ratios to baseline (day -1, before siRNA treatment). Exemplary results are shown in Table 9B.

[0163] Table 9B shows the average knockdown ratios of exemplary ANGPTL3 siRNAs at 7, 14, and 21 days. Example 11. Evaluation of human ANGPTL3 knockdown in humanized transgenic mice This example describes the evaluation of the in vivo activity of ANGPTL3 siRNAs that do not cross-react with the rodent ANGPTL3 gene. Briefly, in vivo screening of siRNAs was performed using a transgenic mouse model in which knocking the human ANGPTL3 gene into the mouse ANGPTL3 locus also inactivated ANGPTL3 expression in mice, based on their in vitro potency. All siRNAs were administered subcutaneously at 3 mg / kg, with PBS used as a negative control. Human ANGPTL3 protein expression in mice was measured in serum collected at days 7 and 14 by enzyme-linked immunosorbent assay (ELISA). Data are presented as ratios to baseline (day -1, before siRNA treatment). Results are shown in Table 10B.

[0164] Table 10B shows the 7-day and 14-day mean and SD of exemplary ANGPTL3 siRNAs. Example 12. Time-series evaluation of human ANGPTL3 knockdown in humanized transgenic mice This example describes the time-series evaluation of in vivo knockdown of human ANGPTL3 protein. To evaluate the in vivo activity of siRNAs that do not cross-react with rodent ANGPTL3 gene or mRNA, in vivo screening of siRNAs was performed using a transgenic mouse model in which the human ANGPTL3 gene was knocked into the mouse ANGPTL3 locus and mouse ANGPTL3 protein expression was also inactivated. This in vivo screening was based on their in vitro potency assessment. All siRNAs were administered subcutaneously at 3 mg / kg, with PBS administered as a negative control. Human ANGPTL3 protein expression was measured in serum collected at days 7, 14, and 21 by enzyme-linked immunosorbent assay (ELISA). Data are presented as ratios to baseline (day -1). Results are shown in Table 11B.

[0165] The results showed that, compared with the PBS control, BC-100042, BC-100067, BC-100115 and BC-100127 siRNAs and their modified derivative siRNAs reduced the in vivo expression of ANGPTL3 protein by >50% within 21 days after administration.

[0166] Table 11B shows the 7-day, 14-day, and 21-day average human ANGPTL3 expression and SD of exemplary ANGPTL3 siRNAs. While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, 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. The appended claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0167] Informal sequence lists of Example 1-2 By incorporating via reference The full disclosure of each of the patents and scientific documents mentioned in this article is incorporated herein by reference for all purposes.

[0168] Equivalent form This disclosure may be embodied in other specific forms without departing from its essential characteristics. Therefore, the foregoing embodiments should be considered illustrative and not limiting of the disclosure described herein. The scope of this disclosure is indicated by the appended claims rather than the foregoing description, and all variations within the meaning and equivalence of the claims are intended to be included therein.

Claims

1. A modified double-stranded RNA (dsRNA) for inhibiting the 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) 2'-fluoronucleotides at positions 2, 4, 6, 8, 14, and 16 from the 5' end; b) 2'-fluoronucleotides at positions 2, 6, 8, and 14 from the 5' end; or c) 2'-fluoronucleotides at positions 2, 4, 6, 8, and 14 from the 5' end.

2. A modified double-stranded ribonucleic acid (dsRNA) for inhibiting the 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) 2'-fluoronucleotides at positions 15, 13, 12, 11 and 9 from the 3' end; b) 2'-fluoronucleotides at positions 13, 12, and 11 from the 3' end; c) 2'-fluoronucleotides located at positions 15, 13, 11, and 9 from the 3' end; d) 2'-fluoronucleotides located at positions 13, 12, 11, and 9 from the 3' end; or e) 2'-fluoronucleotides at positions 15, 13, 12 and 9 from the 3' end.

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

4. The modified dsRNA according to any one of claims 1 to 3, wherein a) The antisense strand comprises 2'-fluoronucleotides at positions 2, 6, 8 and 14 from the 5' end, and the sense strand comprises 2'-fluoronucleotides at positions 15, 13, 12, 11 and 9 from the 3' end; b) The antisense strand comprises 2'-fluoronucleotides at positions 2, 6, 8 and 14 from the 5' end, and the sense strand comprises 2'-fluoronucleotides at positions 13, 12, 11 and 9 from the 3' end; c) The antisense strand comprises 2'-fluoronucleotides at positions 2, 6, 8 and 14 from the 5' end, and the sense strand comprises 2'-fluoronucleotides at positions 15, 13, 12 and 9 from the 3' end; d) The antisense strand comprises 2'-fluoronucleotides at positions 2, 4, 6, 8, and 14 from the 5' end, and the sense strand comprises 2'-fluoronucleotides at positions 15, 13, 11, and 9 from the 3' end; or e) The antisense strand comprises 2'-fluoronucleotides at positions 2, 4, 6, 8, 14 and 16 from the 5' end, and the sense strand comprises 2'-fluoronucleotides at positions 13, 12 and 11 from the 3' end.

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

6. The modified dsRNA according to claim 5, wherein the 3' end nucleotide overhang comprises one, two, or three nucleotides compared to the sense strand.

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

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

9. The modified dsRNA according to 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 according to 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 the target mRNA corresponding to the target gene.

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

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

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

14. The modified dsRNA according to claim 13, wherein the modified nucleotide is at least one of the following: a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-thiophosphate group, a 2'-fluorine modified nucleotide; a reverse abase-free nucleotide, a thymine-glycol nucleic acid (GNA) S-isomer; inosine, a reverse deoxyribonucleotide (3'-3' linked nucleotide), or a thymine-glycol nucleic acid (GNA) S-isomer.

15. The modified sRNA of claim 13, wherein the modified nucleotide is at least one of the following: 5'-vinylphosphonate nucleotide, 5'-phosphate ester or phosphate ester mimic, locked nucleic acid (LNA), 2'-MOE (methoxyethyl) nucleotide and / or 2'-arabinoflus (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-vinylphosphonate or a 4'-O-phosphonate.

17. The modified dsRNA of claim 13, wherein the modified 2'-deoxy-2'-fluoro nucleotide, 2'-deoxy nucleotide, locked nucleotide, baseless nucleotide, 2'-amino nucleotide, 2'-alkyl nucleotide, morpholino nucleotide, phosphoramide ester and / or non-natural bases containing nucleotides.

18. The modified dsRNA of claim 13, wherein the antisense strand and / or the sense strand comprises at least one nucleoside internucleotide bond selected from: phosphorothioate linkage, dithiothioate linkage, phosphotriester linkage, alkylphosphonate linkage, aminoalkylphosphotriester linkage, alkylenephosphotriester linkage, hypophosphite linkage, phosphoramidite linkage, morpholinophosphotriester linkage, piperazine phosphate linkage, aminoalkylphosphamidite linkage, thiophosphamidite linkage, thiocarbonylalkylphosphotriester linkage, thiophosphate linkage, selenophosphate linkage, and boron phosphate linkage.

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

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

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

22. The modified dsRNA according to any one of claims 1 to 21, further comprising a ligand or a targeting portion.

23. The modified dsRNA of claim 22, wherein the ligand or the targeting portion 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 the targeting portion is conjugated to the 3' end of the sense strand of the modified dsRNA.

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

26. A cell comprising the modified dsRNA according to any one of claims 1 to 25.

27. A pharmaceutical composition for inhibiting the expression of a target gene, said pharmaceutical composition comprising a modified dsRNA according to any one of claims 1 to 25, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

28. A method for inhibiting the expression of a target gene in a cell, the method comprising: (a) Contacting the cells with the modified dsRNA according to any one of claims 1 to 25 or the pharmaceutical composition according to claim 27; and (b) The cells produced in step (a) are maintained for a time sufficient to allow the target mRNA transcript of the target gene to be degraded, thereby inhibiting the expression of the target gene in the cells.

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

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