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WO2025228441A2 - Petit arn interférant ciblant inhbe et utilisations associées - Google Patents

Petit arn interférant ciblant inhbe et utilisations associées

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Publication number
WO2025228441A2
WO2025228441A2 PCT/CN2025/092633 CN2025092633W WO2025228441A2 WO 2025228441 A2 WO2025228441 A2 WO 2025228441A2 CN 2025092633 W CN2025092633 W CN 2025092633W WO 2025228441 A2 WO2025228441 A2 WO 2025228441A2
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WO
WIPO (PCT)
Prior art keywords
isolated oligonucleotide
inhbe
present disclosure
antisense strand
sense strand
Prior art date
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Pending
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PCT/CN2025/092633
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English (en)
Other versions
WO2025228441A3 (fr
Inventor
Elisabeth WONDIMU
Peng Zhang
Chunxue ZHOU
Xiaochen Xu
Xiaowei Sun
Shiyu WANG
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Sanegene Bio USA Inc
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Sanegene Bio USA Inc
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Publication of WO2025228441A2 publication Critical patent/WO2025228441A2/fr
Publication of WO2025228441A3 publication Critical patent/WO2025228441A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • INHBE Inhibin Subunit Beta E encodes a circulating growth factor of the activin family and is highly and specifically expressed in hepatocytes. INHBE has been implicated in regulating numerous cellular processes including cell proliferation, apoptosis, immune response and hormone secretion. This gene may be upregulated under conditions of endoplasmic reticulum stress, and this protein may inhibit cellular proliferation and growth in pancreas and liver.
  • INHBE levels Analysis of INHBE levels in multiple studies have implicated high INHBE levels as an independent risk factor for metabolic dysfunction and disease, such as obesity, diabetes, and cardiometabolic disease.
  • exome-sequencing analysis has also implicated carriers of INHBE loss of function gene variants have a more favorable metabolic profiled and lower odds of coronary heart disease and type II diabetes compared to non-carriers. Accordingly, there is a need for therapies for subject having diseases, disorders and symptoms associated with elevated INHBE expression levels.
  • the present disclosure provides compositions targeting INHBE and methods of reducing INHBE expression for treatment of subjects having a disease, disorder or symptom associated with INHBE expression level.
  • the expression level is elevated.
  • the expression level of INHBE may not be elevated but suppression of its expression is of benefit.
  • compositions targeting INHBE and methods of reducing INHBE expression for treatment of subjects having a disease, disorder, or symptom associated with INHBE expression level are provided.
  • the expression level is elevated.
  • the expression level of INHBE may not be elevated but suppression of its expression is of benefit.
  • the present disclosure provides an isolated oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is substantially identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 26-59; b) 122-142; c) 171-191; d) 289-309; e) 355-385; f) 400-426; g) 474-522; h) 519-550; i) 632-656; j) 705-725; k) 739-759; l) 775-805; m) 823-843; n) 873-909; o) 985-1005; p) 1037-1057; q) 1095-1134; r) 1124-1153; s) 1202-1259; t) 1269-1289; u) 1295-1325; v) 1384-1417; w) 1418-1486;
  • the sense strand comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 26-59; b) 122-142; c) 171-191; d) 289-309; e) 355-385; f) 400-426; g) 474-522; h) 519-550; i) 632-656; j) 705-725; k) 739-759; l) 775-805; m) 823-843; n) 873-909; o) 985-1005; p) 1037-1057; q) 1095-1134; r) 1124-1153; s) 1202-1259; t) 1269-1289; u) 1295-1325; v) 1384
  • the sense strand comprises a nucleotide sequence that is identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 26-59; b) 122-142; c) 171-191; d) 289-309; e) 355-385; f) 400-426; g) 474-522; h) 519-550; i) 632-656; j) 705-725; k) 739-759; l) 775-805; m) 823-843; n) 873-909; o) 985-1005; p) 1037-1057; q) 1095-1134; r) 1124-1153; s) 1202-1259; t) 1269-1289; u) 1295-1325; v) 1384-1417; w) 1418-1486; x) 1586-1606; y) 1610
  • the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 289-309; b) 494-514, c) 1610-1631, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the sense strand comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region between any one of the nucleotide positions selected from: a) 289-309; b) 494-514, c) 1610-1631, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 289-309; b) 494-514, c) 1610-1631, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the sense strand comprises a nucleotide sequence that is substantially identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 32-55; b) 122-142; c) 171-191; d) 363-383; e) 400-421; f) 405-426; g) 474-494; h) 495-522; i) 632-656; j) 705-725; k) 739-759; l) 780-805; m) 823-843; n) 875-895; o) 878-899; p) 985-1005; q) 1037-1057; r) 1095-1115; s) 1128-1153; t) 1202-1230; u) 1239-1259; v) 1269-1289; w) 1295-1315; x) 1304-1325; y) 1384-1414;
  • the sense strand comprises a sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 32-55; b) 122-142; c) 171-191; d) 363-383; e) 400-421; f) 405-426; g) 474-494; h) 495-522; i) 632-656; j) 705-725; k) 739-759; l) 780-805; m) 823-843; n) 875-895; o) 878-899; p) 985-1005; q) 1037-1057; r) 1095-1115; s) 1128-1153; t) 1202-1230; u) 1239-1259; v) 1269-1289; w) 1295-1315
  • the sense strand comprises a sequence that is identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 32-55; b) 122-142; c) 171-191; d) 363-383; e) 400-421; f) 405-426; g) 474-494; h) 495-522; i) 632-656; j) 705-725; k) 739-759; l) 780-805; m) 823-843; n) 875-895; o) 878-899; p) 985-1005; q) 1037-1057; r) 1095-1115; s) 1128-1153; t) 1202-1230; u) 1239-1259; v) 1269-1289; w) 1295-1315; x) 1305-1325; y) 1384-1414; z) 1397-1417
  • the sense strand comprises a sequence that is substantially identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 26-49; b) 39-59; c) 403-423; d) 874-894; e) 876-896; f) 889-909; g) 1218-1238; h) 1302-1322; i) 1395-1415; j) 1458-1478; k) 1785-1805; and l) 1855-1875, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the sense strand comprises a sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 26-49; b) 39-59; c) 403-423; d) 874-894; e) 876-896; f) 889-909; g) 1218-1238; h) 1302-1322; i) 1395-1415; j) 1458-1478; k) 1785-1805; and l) 1855 -1875, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the sense strand comprises a sequence that is identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 26-49; b) 39-59; c) 403-423; d) 874-894; e) 876-896; f) 889-909; g) 1218-1238; h) 1302-1322; i) 1395-1415; j) 1458-1478; k) 1785-1805; and l) 1855 -1875, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the isolated oligonucleotide is capable of inducing degradation of the human INHBE mRNA.
  • the sense strand is a single stranded RNA molecule
  • the antisense strand is a single stranded molecule
  • both the sense strand and the antisense strand are single stranded RNA molecules.
  • the antisense strand comprises a 3’ overhang.
  • the 3’ overhang comprises at least one nucleotide.
  • the 3’ overhang comprises two nucleotides.
  • the 3’ overhang comprises any one of thymidine-thymidine (dTdT) , Adenine-Adenine (AA) , Cysteine-Cysteine (CC) , Guanine-Guanine (GG) or Uracil-Uracil (UU) .
  • the sense strand comprises an RNA sequence of at least 20 nucleotides in length. In some embodiments of the isolated oligonucleotide, the sense strand comprises an RNA sequence of 20 nucleotides in length.
  • the antisense strand comprises an RNA sequence of at least 22 nucleotides in length. In some embodiments of the isolated oligonucleotide, the antisense strand comprises an RNA sequence of 22 nucleotides in length.
  • the double stranded region is between 19 and 21 nucleotides in length. In some embodiments of the isolated oligonucleotide, the double stranded region is 20 nucleotides in length.
  • the present disclosure provides an isolated oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand is substantially complementary to its corresponding sense strand such that the sense strand and the antisense strand form a double stranded region.
  • the double stranded region comprises an antisense strand selected from SEQ ID NOs: 2-150 and its corresponding sense strand selected from SEQ ID NOs: 151-299, as set forth in Table 2.
  • the double stranded region comprises an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5’ UCUGUGCUUGACCCUCACAGCU 3’) , and a corresponding sense strand of nucleic acid sequence according to SEQ ID NO: 151 (5’ CUGUGAGGGUCAAGCACAGA 3’) , as set forth in Table 2.
  • SEQ ID NO: 2 and corresponding SEQ ID NO: 151 are identified as duplex number 1 ( “Duplex No. 1” ) , as set forth in Table 2.
  • the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 2-150. In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 151-299.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA by at least 50% (e.g., 50%to 55%, 55%to 60%, 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%or 95%to 100%) at a dose of 10 nM, wherein the antisense strand and its corresponding sense strand are selected from Table 3.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA by 20%to 50% (e.g., 20%to 25%, 25%to 30%, 30%to 35%, 35%to 40%, 40%to 45%or 45%to 50%) , at a dose of 10 nM, wherein the antisense strand and its corresponding sense strand are selected from Table 4.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA by at least 50% (e.g., 50%to 55%, 55%to 60%, 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%or 95%to 100%) at a dose of 0.4 nM, wherein the antisense strand and its corresponding sense strand are selected from Table 5.
  • 50%to 55%, 55%to 60%, 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%or 95%to 100% at a dose of 0.4 nM, wherein the antisense strand and its corresponding sense strand are selected from Table 5.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA by 20%to 50% (e.g., 20%to 25%, 25%to 30%, 30%to 35%, 35%to 40%, 40%to 45%or 45%to 50%) , at a dose of 0.4 nM, wherein the antisense strand and its corresponding sense strand are selected from Table 6.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA in vivo by at least 50% (e.g., 50%to 55%, 55%to 60%, 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100%) at a dose of 0.5 mg/kg, wherein the antisense strand and its corresponding sense strand are selected from Table 7.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA in vivo by at least 60% (e.g., 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100%) at a dose of 0.5 mg/kg, wherein the antisense strand and its corresponding sense strand are selected from Table 8.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA in vivo by at least 70% (e.g., 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100%) at a dose of 1.0 mg/kg, wherein the antisense strand and its corresponding sense strand are selected from Table 8.
  • 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100% at a dose of 1.0 mg/kg, wherein the antisense strand and its corresponding sense strand are selected from Table 8.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA in vivo by at least 70% (e.g., 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100%) at a dose level of 2.0 mg/kg, wherein the antisense strand and its corresponding sense strand are selected from Table 8.
  • 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100% at a dose level of 2.0 mg/kg, wherein the antisense strand and its corresponding sense strand are selected from Table 8.
  • the present disclosure also provides an isolated oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand or the antisense strand or both comprise one or more modified nucleotide (s) .
  • the antisense strand comprises a modified sequence selected from SEQ ID NOs: 300-448 and its corresponding sense strand selected from SEQ ID NOs: 449-597, as set forth in Table 9.
  • FIGs. 1A-1F are graphs showing the efficacy of siRNA compound duplexes 254 (FIG. 1A) , 279 (FIG. 1B) , 286 (FIG. 1C) , 296 (FIG. 1D) , 297 (FIG. 1E) , and 298 (FIG. 1F) in silencing INHBE mRNA of primary human hepatocytes.
  • the siRNA compounds were assessed at concentrations of 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM, 0.0001 nM and 0.00001 nM. Data is presented as %of INHBE mRNA remaining relative to mock control when normalized to GAPDH mRNA levels (Mean, +/-SD) .
  • FIGs. 2A-2C are graphs showing the efficacy of siRNA compounds 254 (FIG. 2A) , 296 (FIG. 2B) , and 297 (FIG. 2C) in silencing INHBE mRNA of primary cynomolgus hepatocytes.
  • the siRNA compounds were assessed at concentrations of 1000 nM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM and 0.0001 nM.
  • Data is presented as %of Macaca fascicularis INHBE mRNA remaining relative to mock control when normalized to Macaca fascicularis GAPDH mRNA levels (Mean, +/-SD) .
  • FIG. 3 is a graph showing the in vivo efficacy of siRNA compound duplex 148 in silencing INHBE mRNA of human INHBE knock-in mice. 8-9 weeks old male mice were administered subcutaneously with a single dose of 0.1 mg/kg, 1 mg/kg, or 10 mg/kg of selected siRNA compounds followed by collection of liver samples at days 6, 20 and 34 post dose. Liver mRNA was extracted and analyzed by RT-QPCR. Data is presented as %of INHBE mRNA remaining relative to vehicle-treated control group when normalized to Gapdh mRNA levels (Mean, +/-SD) .
  • FIG. 4 is a graph showing the in vivo efficacy of siRNA compound duplexes 247, 252, 254, 294, and 295 in silencing INHBE mRNA of cynomolgus monkeys.
  • Obese cynomolgus monkeys were administered subcutaneously with a single dose of 3 mg/kg of selected siRNA compounds followed by collection of liver samples at day 8 before dose, on the dosing date (before dose) , and days 14, 28, 56, 84, and 112 after dose. Liver mRNA was extracted and analyzed by RT-QPCR. Data is presented as %of INHBE mRNA remaining relative to vehicle-treated control group when normalized to Gapdh mRNA levels (Mean, +/-SD) .
  • FIG. 5 is a graph showing the in vivo efficacy of siRNA compound duplexes 296, 297, and 298 in silencing INHBE mRNA of cynomolgus monkeys.
  • Obese cynomolgus monkeys were administered subcutaneously with a single dose of 3 mg/kg of selected siRNA compounds followed by collection of liver samples on day 7 before dose, on the dosing date (before dose) , and day 14, 28, 56 and 84 after dose. Data is presented as %of INHBE mRNA remaining relative to vehicle-treated control group when normalized to Gapdh mRNA levels (Mean, +/-SD) .
  • FIG. 6 is a graph showing the in vivo efficacy of siRNA compound duplex 297 in silencing INHBE mRNA of cynomolgus monkeys.
  • Obese cynomolgus monkeys were administered subcutaneously with a single dose of 3 mg/kg of siRNA compound duplex 297 followed by collection of liver samples on day 7 before dose, on the dosing date (before dose) , and day 14, 28, 42, 56 and 84 after dose. Blood was collected to harvest plasma on day 7 before dose, the dosing date (before dose) , and days 7, 14, 28, 56 and 84 after dose.
  • Plasma INHBE protein was analyzed by Western blot. Data is presented as %of protein INHBE remaining relative to individual money’s predose level (Mean, +/-SD) .
  • oligonucleotide that form a double stranded region, preferably small interfering RNAs (siRNAs) , that can decrease INHBE mRNA expression, in turn leading to a decrease in the degree of INHBE protein (e.g., Activin E) expression in target cells.
  • siRNAs small interfering RNAs
  • the oligonucleotides disclosed herein can have therapeutic application in regulating the expression of INHBE for treatment of a disease or disorder involving INHBE such as, but not limited to a metabolic disease or disorder, metabolic syndrome and related diseases such as diabetes, obesity, lipodystrophy, lipohypertrophy, hyperlipidemia, polycystic ovary syndrome, stroke, muscle wasting or atrophy hypertension, and cardiovascular or cardiometabolic disease, liver diseases including hepatic steatosis, inflammatory and fibrotic liver diseases, such as nonalcoholic steatohepatitis (NASH) , nonalcoholic fatty liver disease (NAFLD) , cirrhosis of the liver, alcoholic liver disease (ALD) , drug induced liver injury, and hepatocellular necrosis.
  • a disease or disorder involving INHBE such as, but not limited to a metabolic disease or disorder, metabolic syndrome and related diseases such as diabetes, obesity, lipodystrophy, lipohypertrophy, hyperlipidemia, polyc
  • the present invention provides compositions and methods of treating a subject having a disorder that would benefit from the reduction in INHBE expression.
  • the methods disclosed herein prevent at least one symptom in a subject having a disease or disorder that would benefit from reduction in INHBE expression.
  • the present disclosure has identified specific regions within the INHBE mRNA, that provide targets for binding double stranded oligonucleotides, e.g., siRNA, leading to reduction in level of expression of the INHBE mRNA.
  • double stranded oligonucleotides e.g., siRNA
  • the INHBE mRNA sequence described herein is an mRNA sequence encoded by an INHBE gene according to GenBank Accession No. NM_031479.5:
  • the present disclosure provides an isolated oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is substantially identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 26-59; b) 122-142; c) 171-191; d) 289-309; e) 355-385; f) 400-426; g) 474-522; h) 519-550; i) 632-656; j) 705-725; k) 739-759; l) 775-805; m) 823-843; n) 873-909; o) 985-1005; p) 1037-1057; q) 1095-1134; r) 1124-1153; s) 1202-1259; t) 1269-1289; u) 1295-1325; v) 1384-1417; w) 1418-1486;
  • the sense strand comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 26-59; b) 122-142; c) 171-191; d) 289-309; e) 355-385; f) 400-426; g) 474-522; h) 519-550; i) 632-656; j) 705-725; k) 739-759; l) 775-805; m) 823-843; n) 873-909; o) 985-1005; p) 1037-1057; q) 1095-1134; r) 1124-1153; s) 1202-1259; t) 1269-1289; u) 1295-1325; v) 1384
  • the sense strand comprises a nucleotide sequence that is identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 26-59; b) 122-142; c) 171-191; d) 289-309; e) 355-385; f) 400-426; g) 474-522; h) 519-550; i) 632-656; j) 705-725; k) 739-759; l) 775-805; m) 823-843; n) 873-909; o) 985-1005; p) 1037-1057; q) 1095-1134; r) 1124-1153; s) 1202-1259; t) 1269-1289; u) 1295-1325; v) 1384-1417; w) 1418-1486; x) 1586-1606; y) 1610
  • the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 289-309; b) 494-514, c) 1610-1631, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the sense strand comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region between any one of the nucleotide positions selected from: a) 289-309; b) 494-514, c) 1610-1631, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 289-309; b) 494-514, c) 1610-1631, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the sense strand comprises a nucleotide sequence that is substantially identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 32-55; b) 122-142; c) 171-191; d) 363-383; e) 400-421; f) 405-426; g) 474-494; h) 495-522; i) 632-656; j) 705-725; k) 739-759; l) 780-805; m) 823-843; n) 875-895; o) 878-899; p) 985-1005; q) 1037-1057; r) 1095-1115; s) 1128-1153; t) 1202-1230; u) 1239-1259; v) 1269-1289; w) 1295-1315; x) 1305-1325; y) 1384-1414;
  • the sense strand comprises a sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 32-55; b) 122-142; c) 171-191; d) 363-383; e) 400-421; f) 405-426; g) 474-494; h) 495-522; i) 632-656; j) 705-725; k) 739-759; l) 780-805; m) 823-843; n) 875-895; o) 878-899; p) 985-1005; q) 1037-1057; r) 1095-1115; s) 1128-1153; t) 1202-1230; u) 1239-1259; v) 1269-1289; w) 1295-1315
  • the sense strand comprises a sequence that is identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 32-55; b) 122-142; c) 171-191; d) 363-383; e) 400-421; f) 405-426; g) 474-494; h) 495-522; i) 632-656; j) 705-725; k) 739-759; l) 780-805; m) 823-843; n) 875-895; o) 878-899; p) 985-1005; q) 1037-1057; r) 1095-1115; s) 1128-1153; t) 1202-1230; u) 1239-1259; v) 1269-1289; w) 1295-1315; x) 1305-1325; y) 1384-1414; z) 1397-1417
  • the sense strand comprises a sequence that is substantially identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 26-49; b) 39-59; c) 403-423; d) 874-894; e) 876-896; f) 889-909; g) 1218-1238; h) 1302-1322; i) 1395-1415; j) 1458-1478; k) 1785-1805; and l) 1855-1875, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the sense strand comprises a sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 26-49; b) 39-59; c) 403-423; d) 874-894; e) 876-896; f) 889-909; g) 1218-1238; h) 1302-1322; i) 1395-1415; j) 1458-1478; k) 1785-1805; and l) 1855 -1875, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the sense strand comprises a sequence that is identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 26-49; b) 39-59; c) 403-423; d) 874-894; e) 876-896; f) 889-909; g) 1218-1238; h) 1302-1322; i) 1395-1415; j) 1458-1478; k) 1785-1805; and l) 1855 -1875, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the present disclosure provides an isolated oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand is substantially complementary to its corresponding sense strand such that the sense strand and the antisense strand form a double stranded region.
  • the double stranded region comprises an antisense strand selected from SEQ ID NOs: 2-150 and its corresponding sense strand selected from SEQ ID NOs: 151-299, as set forth in Table 2.
  • the double stranded region comprises an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5’ UCUGUGCUUGACCCUCACAGCU 3’) , and a corresponding sense strand of nucleic acid sequence according to SEQ ID NO: 151 (5’ CUGUGAGGGUCAAGCACAGA 3’) , as set forth in Table 2.
  • SEQ ID NO: 2 and corresponding SEQ ID NO: 151 are identified as duplex number 1 ( “Duplex No. 1” ) , as set forth in Table 2.
  • the present disclosure also provides an isolated oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand or the antisense strand or both comprise one or more modified nucleotide (s) .
  • the antisense strand comprises a modified sequence selected from SEQ ID NOs: 300-448 and its corresponding sense strand selected from SEQ ID NOs: 449-597, as set forth in Table 9.
  • the INHBE mRNA sequence according to SEQ ID NO: 1, as described herein, is any heterologous mRNA sequence with sufficient identity to an mRNA sequence encoded by an INHBE gene according to Accession No. NM_031479.5, as described herein, that allows binding to the antisense strand of the oligonucleotides of the present disclosure.
  • the isolated oligonucleotide is capable of inducing degradation of the INHBE mRNA.
  • the sense strand is a single stranded RNA molecule.
  • the antisense strand is a single stranded RNA molecule.
  • both the sense strand and the antisense strand are single stranded RNA molecules.
  • the isolated oligonucleotide of the present disclosure is a small interfering RNA (siRNA) .
  • siRNA small interfering RNA
  • the disclosure provides siRNAs, wherein the siRNA comprises a sense region and antisense region complementary to the sense region that together form an RNA duplex, and wherein the sense region comprises a sequence at least 70%to 100%identical to an INHBE mRNA sequence. Definitions
  • RNAi refers to the process of sequence-specific post-transcriptional gene silencing, mediated by double-stranded RNA (dsRNA) .
  • Duplex RNA siRNA small interfering RNA
  • miRNA miRNA
  • miRNA miRNA
  • shRNA shRNA
  • ddRNA DNA-directed RNA
  • piRNA piRNA
  • rasiRNA rasiRNA
  • modified forms thereof are all capable of mediating RNA interference.
  • dsRNA molecules may be commercially available or may be designed and prepared based on known sequence information, etc.
  • the antisense strand of these molecules can include RNA, DNA, peptide nucleic acid (PNA) , or a combination thereof.
  • DNA/RNA chimera polynucleotide includes, but is not limited to, a double-strand polynucleotide composed of DNA and RNA that inhibits the expression of a target gene.
  • dsRNA molecules can also include one or more modified nucleotides, as described herein, which can be incorporated on either strand.
  • dsRNA comprising a first (antisense) strand that is complementary to a portion of a target gene and a second (sense) strand that is fully or partially complementary to the first antisense strand is introduced into an organism.
  • the target gene-specific dsRNA is processed into relatively small fragments (siRNAs) and can subsequently become distributed throughout the organism, decrease messenger RNA of target gene, leading to a phenotype that may come to closely resemble the phenotype arising from a complete or partial deletion of the target gene.
  • RNAi also involves an endonuclease complex known as the RNA induced silencing complex (RISC) .
  • RISC RNA induced silencing complex
  • siRNAs enter the RISC complex and direct cleavage of a single stranded RNA target having a sequence complementary to the antisense strand of the siRNA duplex. The other strand of the siRNA is the passenger strand. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.
  • siRNAs can thus down regulate or knock down gene expression by mediating RNA interference in a sequence-specific manner.
  • target gene or “target sequence” refers to a gene or gene sequence whose corresponding RNA is targeted for degradation through the RNAi pathway using dsRNAs or siRNAs as described herein.
  • the siRNA comprises an antisense region complementary to, or substantially complementary to, at least a portion of the target gene or sequence, and sense strand complementary to the antisense strand.
  • the siRNA directs the RISC complex to cleave an RNA comprising a target sequence, thereby degrading the RNA.
  • oligonucleotide As used herein, “oligonucleotide” , “nucleic acid, ” “nucleotide sequence, ” and “polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • the term polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain.
  • the nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand.
  • the nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides) . Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • the present disclosure further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide of this disclosure.
  • dsRNA When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. Other modifications, such as modification to the phosphodiester backbone, or the 2’-fluoro, the 2′-hydroxy or 2’O-methyl in the ribose sugar group of the RNA can also be made.
  • isolated can refer to a nucleic acid, nucleotide sequence or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques) , or chemical precursors or other chemicals (when chemically synthesized) .
  • an “isolated fragment” is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state. “Isolated” does not mean that the preparation is technically pure (homogeneous) , but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.
  • region or “fragment” is used interchangeably and as applied to an oligonucleotide.
  • the INHBE mRNA sequence as described herein, will be understood to mean a full length INHBE mRNA nucleotide sequence, unless indicated otherwise.
  • the INHBE mRNA sequence can be a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence of the INHBE mRNA sequence comprising, consisting essentially of, and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 60%, 70%, 80%, 90%, 92%, 95%, 98%or 99%identical) to the reference nucleic acid or nucleotide sequence.
  • nucleic acid fragment according to the disclosure may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive nucleotides of a nucleic acid or nucleotide sequence according to the disclosure.
  • complementary polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G: C) and adenine paired with either thymine (A: T) in the case of DNA, or adenine paired with uracil (A: U) in the case of RNA. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A. ” It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.
  • the term “substantially complementary” is at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98 or 99%) complementary to the sense strand that is substantially identical to the nucleotide sequence within the defined regions in SEQ ID NO: 1.
  • the term “substantially complementary” means that two nucleic acid sequences are complementary at least at about 90%, 95%or 99%of their nucleotides.
  • the two nucleic acid sequences can be complementary at least at 90%, 95%, 96%, 97%, 98%, 99%or more of their nucleotides. In some embodiments, the two nucleic acid sequences can be between 90%to 95%complementary, between 70%to 100%complementary, between 95%and 96%complementary, between 90%and 100%complementary, between 96%to 97%complementary, between 60%to 80%complementary, between 97%and 98%complementary, between 70%and 90%complementary, between 98%and 99%complementary, between 80%and 100%complementary, or between 99%and 100%complementary.
  • substantially complementary can also mean that two nucleic acid sequences, sense strand and antisense strand have sufficient complementarity that allows binding between the sense strand and antisense strand to form a double stranded region comprising of between 19-25 nucleotides in length.
  • substantially complementary can also mean that two nucleic acid sequences can hybridize under high stringency conditions, and such conditions are well known in the art.
  • the term "substantially identical” or “sufficient identity” used interchangeably herein, is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% (e.g., between 70%to 805, 8-%to 90%or 90%to 95%or 95%to 99%or 99%to 100%) identical to the nucleotide sequence within the defined regions in SEQ ID NO: 1.
  • the term “identity” means that sequences are compared with one another as follows. In order to determine the percentage identity of two nucleic acid sequences, the sequences can first be aligned with respect to one another in order subsequently to make a comparison of these sequences possible. For this e.g., gaps can be inserted into the sequence of the first nucleic acid sequence and the nucleotides can be compared with the corresponding position of the second nucleic acid sequence. If a position in the first nucleic acid sequence is occupied by the same nucleotide as is the case at a position in the second sequence, the two sequences are identical at this position.
  • the percentage identity between two sequences is a function of the number of identical positions divided by the number of all the positions compared in the sequences investigated.
  • a “percent identity” or “%identity” as used interchangeably herein, for aligned segments of a test sequence and a reference sequence is the percent of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.
  • nucleotide sequence and “nucleic acid sequence” are used interchangeably herein, unless indicated otherwise.
  • the percentage identity of two sequences can be determined with the aid of a mathematical algorithm.
  • a preferred, but not limiting, example of a mathematical algorithm which can be used for comparison of two sequences is the algorithm of Karlin et al. (1993) , PNAS USA, 90: 5873-5877. Such an algorithm is integrated in the NBLAST program, with which sequences which have a desired identity to the sequences of the present disclosure can be identified.
  • the “Gapped BLAST” program can be used, as is described in Altschul et al. (1997) , Nucleic Acids Res, 25: 3389-3402. If BLAST and Gapped BLAST programs are used, the preset parameters of the particular program (e.g.
  • NBLAST NBLAST
  • the sequences can be aligned further using version 9 of GAP (global alignment program) of the “Genetic Computing Group” using the preset (BLOSUM62) matrix (values -4 to +11) with a gap open penalty of -12 (for the first zero of a gap) and a gap extension penalty of -4 (for each additional successive zero in the gap) .
  • GAP global alignment program
  • BLOSUM62 preset
  • the percentage identity is calculated by expressing the number of agreements as a percentage content of the nucleic acids in the sequence claimed.
  • the methods described for determination of the percentage identity of two nucleic acid sequences can also be used correspondingly, if necessary, on the coded amino acid sequences.
  • BLAST Basic Local Alignment Search Tool
  • Percent identity can be 70%identity or greater, e.g., at least 70%identity, at least 75%identity, at least 80%identity, at least 85%identity, at least 90%identity, at least 95%identity, at least 98%identity, at least 99%identity or 100%identity.
  • heterologous refers to a nucleic acid sequence that either originates from another species or is from the same species or organism but is modified from either its original form or the form primarily expressed in the cell.
  • a nucleotide sequence derived from an organism or species different from that of the cell into which the nucleotide sequence is introduced is heterologous with respect to that cell and the cell's descendants.
  • a heterologous nucleotide sequence includes a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., a different copy number, and/or under the control of different regulatory sequences than that found in nature.
  • Double Stranded RNAs Targeting Human Inhibin Subunit Beta E (INHBE)
  • the disclosure provides isolated oligonucleotides comprising a double stranded RNAs (dsRNAs) duplex region which target an INHBE mRNA sequence for degradation.
  • the double stranded RNA molecule of the disclosure may be in the form of any type of RNA interference molecule known in the art.
  • the double stranded RNA molecule is a small interfering RNA (siRNA) .
  • the double stranded RNA molecule is a short hairpin RNA (shRNA) molecule.
  • the double stranded RNA molecule is a Dicer substrate that is processed in a cell to produce an siRNA.
  • the double stranded RNA molecule is part of a microRNA precursor molecule.
  • the dsRNA is a small interfering RNA (siRNA) which targets an INHBE mRNA sequence for degradation.
  • siRNA targeting INHBE is packaged in a delivery system described herein (e.g., nanoparticle) .
  • the isolated oligonucleotides of the present disclosure targeting INHBE for degradation can comprise a sense strand at least 70%identical to any fragment of an INHBE mRNA, for example the INHBE mRNA of SEQ ID NO: 1.
  • the sense strand comprises or consists essentially of a sequence at least 70%, at least 80%, at least 90%, at least 95%or is 100%identical to any fragment of SEQ ID NO: 1.
  • the siRNAs targeting INHBE for degradation can comprise an antisense strand at least 70%identical to a sequence complementary to any fragment of an INHBE mRNA, for example the INHBE mRNA of SEQ ID NO: 1.
  • the antisense strand comprises or consists essentially of a sequence at least 70%, at least 80%, at least 90%, at least 95%or is 100%identical to a sequence complementary to any fragment of SEQ ID NO: 1.
  • the sense region and antisense regions are complementary, and base pair to form an RNA duplex structure.
  • the fragment of the INHBE mRNA that has percent identity to the sense region of the siRNA, and which is complementary to the antisense region of the siRNA can be protein coding sequence of the mRNA, an untranslated region (UTR) of the mRNA (5’ UTR or 3’ UTR) , or both.
  • the isolated oligonucleotides of the present disclosure comprises a sense region and antisense region complementary to the sense region that together form an RNA duplex, and the sense region comprises a sequence at least 70%identical to an INHBE mRNA sequence. In some embodiments, the sense region is identical to an INHBE mRNA sequence.
  • the term “sense strand” or “sense region” refers to a nucleotide sequence of an siRNA molecule that is partially or fully complementary to at least a portion of a corresponding antisense strand or antisense region of the siRNA molecule.
  • the sense strand of an isolated oligonucleotides of the present disclosure molecule can include a nucleic acid sequence having some percentage identity with a target nucleic acid sequence such as an INHBE mRNA sequence.
  • the sense region may have 100%identity, i.e., complete identity or homology, to the target nucleic acid sequence.
  • there may be one or more mismatches between the sense region and the target nucleic acid sequence there may be 1, 2, 3, 4, 5, 6, or 7 mismatches between the sense region and the target nucleic acid sequence.
  • antisense strand or “antisense region” refers to a nucleotide sequence of the isolated oligonucleotides of the present disclosure, that is partially or fully complementary to at least a portion of a target nucleic acid sequence.
  • the antisense strand of an isolated oligonucleotides of the present disclosure molecule can include a nucleic acid sequence that is complementary to at least a portion of a corresponding sense strand of the isolated oligonucleotides.
  • the sense region comprises a sequence that is at least 70%identical, at least 75%identical, at least 80%identical, at least 85%identical, at least 90%identical, at least 95%identical, at least 97%identical, at least 99%identical or 100%identical to a sequence of SEQ ID NO: 1 or a region of SEQ ID NO: 1, as disclosed herein.
  • the sense region consists essentially of a sequence that is at least 70%identical, at least 75%identical, at least 80%identical, at least 85%identical, at least 90%identical, at least 95%identical, at least 97%identical, at least 99%identical or 100%identical to a sequence of SEQ ID NO: 1 or a region of SEQ ID NO: 1, as disclosed herein.
  • the sense region comprises a sequence that is identical to a sequence of SEQ ID NO: 1 or a region of SEQ ID NO: 1, as disclosed herein.
  • the sense region consists essentially of a sequence that is identical to a sequence of SEQ ID NO: 1 or a region of SEQ ID NO: 1, as disclosed herein.
  • the sense region of the isolated oligonucleotides of the present disclosure targeting INHBE has one or more mismatches between the sequence of the isolated oligonucleotides and the INHBE sequence.
  • the sequence of the sense region may have 1, 2, 3, 4 or 5 mismatches between the sequence of the sense region of the isolated oligonucleotides and the INHBE sequence.
  • the INHBE sequence is an INHBE 3’ untranslated region sequence (3’ UTR) . Without wishing to be bound by theory, it is thought that siRNAs targeting the 3’ UTR have elevated mismatch tolerance when compared to mismatches in the isolated oligonucleotides targeting coding regions of a gene.
  • the isolated oligonucleotides RNAs may be tolerant of mismatches outside the seed region.
  • the “seed region” of the isolated oligonucleotides refers to base pairs 2-8 of the antisense region of the isolated oligonucleotides, i.e., the strand of the isolated oligonucleotides that is complementary to and hybridizes to the target mRNA.
  • the antisense region comprises a sequence that is at least 70%identical, at least 75%identical, at least 80%identical, at least 85%identical, at least 90%identical, at least 95%identical, at least 97%identical, at least 99%identical or 100%identical to a sequence complementary to a sequence of SEQ ID NO: 1 or a region of SEQ ID NO: 1, as disclosed herein.
  • the antisense region consists essentially of a sequence that is at least 70%identical, at least 75%identical, at least 80%identical, at least 85%identical, at least 90%identical, at least 95%identical, at least 97%identical, at least 99%or 100%identical to a sequence complementary to a sequence of SEQ ID NO: 1 or a region of SEQ ID NO: 1.
  • the antisense region comprises a sequence that is identical to a sequence complementary to a sequence of SEQ ID NO: 1 or a region of SEQ ID NO: 1.
  • the sense region consists essentially of a sequence that is complementary to a sequence of SEQ ID NO: 1 or a region of SEQ ID NO: 1.
  • the antisense region of the INHBE targeting isolated oligonucleotide of the present disclosure is complementary to the sense region.
  • the sense region and the antisense region are fully complementary (no mismatches) .
  • the antisense region is partially complementary to the sense region, i.e., there are 1, 2, 3, 4 or 5 mismatches between the sense region and the antisense region.
  • isolated oligonucleotide of the present disclosure comprise an RNA duplex that is about 16 to about 25 nucleotides in length. In some embodiments, the RNA duplex is between about 17 and about 24 nucleotides in length, between about 18 and about 23 nucleotides in length, or between about 19 and about 22 nucleotides in length. In some embodiments, the RNA duplex is 19 nucleotides in length. In some embodiments, the RNA duplex is 20 nucleotides in length.
  • the sense strand is a single stranded RNA molecule.
  • the antisense strand is a single stranded RNA molecule.
  • both the sense strand and the antisense strand are single stranded RNA molecules.
  • the isolated oligonucleotide of the present disclosure is an siRNA targeting INHBE that comprises two different single stranded RNAs, the first comprising the sense region and the second comprising the antisense region, which hybridize to form an RNA duplex.
  • the isolated oligonucleotide of the present disclosure can have one or more overhangs from the duplex region.
  • the overhangs which are non-base-paired, single strand regions, can be from one to eight nucleotides in length, or longer.
  • the overhang can be a 3’ overhang, wherein the 3’-end of a strand has a single strand region of from one to eight nucleotides.
  • the overhang can be a 5’ overhang, wherein the 5 '-end of a strand has a single strand region of from one to eight nucleotides.
  • the overhangs of the isolated oligonucleotide are the same length. In some embodiments, the overhangs of the isolated oligonucleotide are different lengths.
  • the single stranded RNA molecule of the sense strand comprises a 3’ overhang. In some embodiments, the 3’ overhang of the single stranded RNA molecule of the sense strand comprises at least one nucleotide. In some embodiments, the 3’ overhang of the single stranded RNA molecule of the sense strand comprises two nucleotides.
  • the single stranded RNA molecule of the antisense strand comprises a 3’ overhang. In some embodiments, the 3’ overhang of the single stranded RNA molecule of the antisense strand comprises at least one nucleotide. In some embodiments, the 3’ overhang of the single stranded RNA molecule of the antisense strand comprises two nucleotides.
  • both ends of isolated oligonucleotide have an overhang, for example, a 3’ dinucleotide overhang on each end.
  • the overhangs at the 5'-and 3 '-ends are of different lengths. In some embodiments, the overhangs at the 5'-and 3 '-ends are of the same length.
  • the overhang can contain one or more deoxyribonucleotides, one or more ribonucleotides, or a combination of deoxyribonucleotides and ribonucleotides.
  • one, or both, of the overhang nucleotides of an siRNA may be 2'-deoxyribonucleotides.
  • the first single stranded RNA molecule comprises a first 3’ overhang.
  • the second single stranded RNA molecule comprises a second 3’ overhang.
  • the first and second 3’ overhangs comprise a dinucleotide.
  • the 3’ overhang comprises any one of thymidine-thymidine (dTdT) , Adenine-Adenine (AA) , Cysteine-Cysteine (CC) , Guanine-Guanine (GG) or Uracil-Uracil (UU) .
  • the isolated oligonucleotide of the present disclosure the 3’ overhang comprises a thymidine-thymidine (dTdT) or a Uracil-Uracil (UU) overhang.
  • the 3’ overhang comprises a Uracil-Uracil (UU) overhang.
  • 3’ overhangs such as dinucleotide overhangs, enhance siRNA mediated mRNA degradation by enhancing siRNA-RISC complex formation, and/or rate of cleavage of the target mRNA by the siRNA-RISC complex.
  • the isolated oligonucleotide of the present disclosure can have one or more blunt ends, in which the duplex region ends with no overhang, and the strands are base paired to the end of the duplex region.
  • the isolated oligonucleotide of the present disclosure can have one or more blunt ends, or can have one or more overhangs, or can have a combination of a blunt end and an overhang end.
  • the 5’ end of the siRNA can be blunt and the 3’ end of the same isolated oligonucleotide comprise an overhang, or vice versa.
  • both ends of the isolated oligonucleotide of the present disclosure are blunt ends.
  • the double stranded region comprises an antisense strand and a sense strand, according to any one of the pairs of antisense strand and sense strand sequences in Table 2 and/or Table 9, as described below.
  • the isolated oligonucleotides disclosed in the present disclosure are useful in treating or preventing a disease or disorder associated with aberrant or increased expression or activity of INHBE or a disease or disorder where INHBE plays a role.
  • Exemplary isolated oligonucleotides of the present disclosure are described in Table 2.
  • the sense strand comprises a sequence selected from any one of the group of sense strand/passenger strand sequences listed in Table 2.
  • the antisense strand comprises a sequence selected from any one of the group of antisense strand/guide strand sequences listed in Table 2.
  • the sense and antisense regions comprise complementary sequences selected from the group listed in Table 2.
  • the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 2-150.
  • the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 151-299.
  • the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 2-150; and the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 151-299, wherein the antisense strand and the sense strand sequences have sufficient complementarity to allow formation of a double stranded region between the antisense and the sense strand.
  • the present disclosure provides an isolated oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand is substantially complementary to the sense strand such that the sense strand and the antisense strand form a double stranded region, wherein the double stranded region comprises an antisense strand selected from SEQ ID NOs: 2-150 and its corresponding sense strand selected from SEQ ID NOs: 151-299, as set forth in Table 2.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA by at least 50% (e.g., 50%to 55%, 55%to 60%, 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%or 95%to 100%) at a dose of 10 nM, wherein the antisense strand and its corresponding sense strand are selected from Table 3.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA by 20%to 50% (e.g., 20%to 25%, 25%to 30%, 30%to 35%, 35%to 40%, 40%to 45%or 45%to 50%) , at a dose of 10 nM, wherein the antisense strand and its corresponding sense strand are selected from Table 4.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA by at least 50% (e.g., 50%to 55%, 55%to 60%, 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%or 95%to 100%) at a dose of 0.4 nM, wherein the antisense strand and its corresponding sense strand are selected from Table 5.
  • 50%to 55%, 55%to 60%, 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%or 95%to 100% at a dose of 0.4 nM, wherein the antisense strand and its corresponding sense strand are selected from Table 5.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA by 20%to 50% (e.g., 20%to 25%, 25%to 30%, 30%to 35%, 35%to 40%, 40%to 45%or 45%to 50%) , at a dose of 0.4 nM, wherein the antisense strand and its corresponding sense strand are selected from Table 6.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA in vivo by at least 50% (e.g., 50%to 55%, 55%to 60%, 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100%) at a dose of 0.5 mg/kg, wherein the antisense strand and its corresponding sense strand are selected from Table 7.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA in vivo by at least 60% (e.g., 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100%) at a dose of 0.5 mg/kg, wherein the antisense strand and its corresponding sense strand are selected from Table 8.
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA in vivo by at least 70% (e.g., 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100%) at a dose of 1.0 mg/kg, wherein the antisense strand and its corresponding sense strand are selected from Table 8.
  • 70% e.g., 60%to 65%, 65%to 70%, 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100%
  • the isolated oligonucleotide attenuates expression of the INHBE mRNA in vivo by at least 70% (e.g., 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100%) at a dose of 2.0 mg/kg, wherein the antisense strand and its corresponding sense strand are selected from Table 8.
  • 70%to 75%, 75%to 80%, 80%to 85%, 85%to 90%, 90%to 95%, or 95%to 100% at a dose of 2.0 mg/kg, wherein the antisense strand and its corresponding sense strand are selected from Table 8.
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 26-59; b) 122-142; c) 171-191; d) 289-309; e) 355-385; f) 400-426; g) 474-522; h) 519-550; i) 632-656; j) 705-725; k) 739-759; l) 775-805; m) 823-843; n) 873-909; o) 985-1005; p) 1037-1057; q) 1095-1134; r) 1124-1153; s) 1202-1259; t) 1269-1289; u) 1295-1325; v) 1384-1417; w) 1418-1486; x) 1586-1606; y)
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 26-59; b) 122-142; c) 171-191; d) 289-309; e) 355-385; f) 400-426; g) 474-522; h) 519-550; i) 632-656; j) 705-725; k) 739-759; l) 775-805; m) 823-843; n) 873-909; o) 985-1005; p) 1037-1057; q) 1095-1134; r) 1124-1153; s) 1202-1259; t) 1269-1289; u)
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 26-59; b) 122-142; c) 171-191; d) 289-309; e) 355-385; f) 400-426; g) 474-522; h) 519-550; i) 632-656; j) 705-725; k) 739-759; l) 775-805; m) 823-843; n) 873-909; o) 985-1005; p) 1037-1057; q) 1095-1134; r) 1124-1153; s) 1202-1259; t) 1269-1289; u) 1295-1325; v) 1384-1417; w) 1418-1486; x)
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 289-309; b) 494-514, c) 1610-1631, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region between any one of the nucleotide positions selected from: a) 289-309; b) 494-514, c) 1610-1631, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 289-309; b) 494-514, c) 1610-1631, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a sequence that is substantially identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 32-55; b) 122-142; c) 171-191; d) 363-383; e) 400-421; f) 405-426; g) 474-494; h) 495-522; i) 632-656; j) 705-725; k) 739-759; l) 780-805; m) 823-843; n) 875-895; o) 878-899; p) 985-1005; q) 1037-1057; r) 1095-1115; s) 1128-1153; t) 1202-1230; u) 1239-1259; v) 1269-1289; w) 1295-1315; x) 1305-1325; y) 13
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 32-55; b) 122-142; c) 171-191; d) 363-383; e) 400-421; f) 405-426; g) 474-494; h) 495-522; i) 632-656; j) 705-725; k) 739-759; l) 780-805; m) 823-843; n) 875-895; o) 878-899; p) 985-1005; q) 1037-1057; r) 1095-1115; s) 1128-1153; t) 1202-1230; u) 1239-1259; v) 1269
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 32-55; b) 122-142; c) 171-191; d) 363-383; e) 400-421; f) 405-426; g) 474-494; h) 495-522; i) 632-656; j) 705-725; k) 739-759; l) 780-805; m) 823-843; n) 875-895; o) 878-899; p) 985-1005; q) 1037-1057; r) 1095-1115; s) 1128-1153; t) 1202-1230; u) 1239-1259; v) 1269-1289; w) 1295-1315; x) 1305-1325;
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein sense strand comprises a sequence that is substantially identical to a region comprising the sequence between nucleotide positions selected from: a) 26-49; b) 39-59; c) 403-423; d) 874-894; e) 876-896; f) 889-909; g) 1218-1238; h) 1302-1322; i) 1395-1415; j) 1458-1478; k) 1785-1805; and l) 1855-1875, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein sense strand comprises a sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%identical to a region comprising the sequence between nucleotide positions selected from: a) 26-49; b) 39-59; c) 403-423; d) 874-894; e) 876-896; f) 889-909; g) 1218-1238; h) 1302-1322; i) 1395-1415; j) 1458-1478; k) 1785-1805; and l) 1855-1875, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is identical to a region comprising the sequence between nucleotide positions selected from: a) 26-49; b) 39-59; c) 403-423; d) 874-894; e) 876-896; f) 889-909; g) 1218-1238; h) 1302-1322; i) 1395-1415; j) 1458-1478; k) 1785-1805; and l) 1855-1875, from the 5’ end of a human INHBE mRNA sequence according to SEQ ID NO: 1.
  • the isolated oligonucleotide of the present disclosure can comprise a linker, sometimes referred to as a loop.
  • siRNAs comprising a linker or loop are sometimes referred to as short hairpin RNAs (shRNAs) .
  • shRNAs short hairpin RNAs
  • both the sense and the antisense regions of the siRNA are encoded by one single-stranded RNA.
  • the antisense region and the sense region hybridize to form a duplex region.
  • the sense and antisense regions are joined by a linker sequence, forming a “hairpin” or “stem-loop” structure.
  • the siRNA can have complementary sense and antisense regions at opposing ends of a single stranded molecule, so that the molecule can form a duplex region with the complementary sequence portions, and the strands are linked at one end of the duplex region by a linker.
  • the linker can be either a nucleotide or non-nucleotide linker or a combination thereof.
  • the linker can interact with the first, and optionally, second strands through covalent bonds or non-covalent interactions.
  • An siRNA of this disclosure may include a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the nucleic acid to the antisense region of the nucleic acid.
  • a nucleotide linker can be a linker of ⁇ 2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleotides in length.
  • non-nucleotide linker examples include an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric agents, for example polyethylene glycols such as those having from 2 to 100 ethylene glycol units.
  • nucleotide linker sequences include, but are not limited to, AUG, CCC, UUCG, CCACC, AAGCAA, CCACACC and UUCAAGAGA.
  • the isolated oligonucleotide of the present disclosure is an siRNA that can be a dsRNA of a length suitable as a Dicer substrate, which can be processed to produce a RISC active siRNA molecule. See, e.g., Rossi et al., US2005/0244858.
  • a Dicer substrate double stranded RNA can be of a length sufficient that it is processed by Dicer to produce an active siRNA, and may further include one or more of the following properties: (i) the Dicer substrate dsRNA can be asymmetric, for example, having a 3' overhang on the antisense strand, (ii) the Dicer substrate dsRNA can have a modified 3' end on the sense strand to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA, for example the incorporation of one or more DNA nucleotides, and (iii) the first and second strands of the Dicer substrate ds RNA can from 19-30 bp in length.
  • the isolated oligonucleotide of the present disclosure comprises at least one modified nucleotide.
  • the sense strand or the antisense strand or both comprise one or more modified nucleotide (s) .
  • only the sense strand comprises one or more modified nucleotide (s) .
  • only the antisense strand comprises one or more modified nucleotide (s) .
  • both the sense strand and antisense strand comprise one or more modified nucleotide (s) .
  • the isolated oligonucleotide is partially chemically modified. In some embodiments, the isolated oligonucleotide is fully chemically modified.
  • the isolated oligonucleotide comprises at least two modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least three modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least four modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least five modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least six modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least seven modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least eight modified nucleotides.
  • the isolated oligonucleotide comprises at least nine modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least ten modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least eleven modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least twelve modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least thirteen modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least fourteen modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least fifteen modified nucleotides.
  • the isolated oligonucleotide comprises at least sixteen modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least seventeen modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least eighteen modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least nineteen modified nucleotides. In some embodiments, the isolated oligonucleotide comprises at least twenty modified nucleotides. In some embodiments, the isolated oligonucleotide comprises more than twenty modified nucleotides. In some embodiments, the isolated oligonucleotide comprises between twenty and thirty modified nucleotides. In some embodiments, the isolated oligonucleotide comprises between thirty and forty modified nucleotides. In some embodiments, the isolated oligonucleotide comprises between forty and fifty modified nucleotides.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least one modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least two modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least three modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least four modified nucleotides.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least five modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least six modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least seven modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least eight modified nucleotides.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least nine modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least ten modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least eleven modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least twelve modified nucleotides.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least thirteen modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least fourteen modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least fifteen modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least sixteen modified nucleotides.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least seventeen modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least eighteen modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least nineteen modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least twenty modified nucleotides.
  • the isolated oligonucleotide comprises more than one modified nucleotide
  • at least a first nucleotide comprises a first modification and at least a second nucleotide comprises a second modification.
  • the first modification and second modification are different.
  • the at least first nucleotide and the at least second nucleotide are located on different strands of the isolated oligonucleotide.
  • the at least first nucleotide and the at least second nucleotide are located on the same strand of the isolated oligonucleotide.
  • the isolated oligonucleotide comprises more than one modified nucleotide, at least a first modified nucleotide comprises a first modification, and at least a second modified nucleotide comprises a second modification, and at least a third nucleotide comprises a third modification.
  • the isolated oligonucleotide comprises a first, a second, a third and a fourth modifications.
  • the isolated oligonucleotide comprises more than four modifications.
  • all modifications are on the sense strand.
  • all modifications are on the antisense strand. Any combination of locations of the modifications between the sense strand and antisense strand is envisaged within the isolated oligonucleotides of the present disclosure.
  • the modified nucleotides are consecutively located on the sense strand or the antisense strand or both. In some embodiments, some but not all of the modified nucleotides are consecutively located on the sense strand or the antisense strand or both. In some embodiments, the modified nucleotides on the sense strand or the antisense strand or both are not consecutively located.
  • Envisaged within the present disclosure is an isolated oligonucleotide, wherein any nucleotide on the sense strand or antisense strand can be modified. In some embodiments, any nucleotide on the antisense strand can be modified. In some embodiments, any nucleotide on the antisense strand can be modified.
  • the sense strand or the antisense strand or both comprise one or more modified nucleotide (s) .
  • the sense strand comprises one modified nucleotide.
  • only the sense strand comprises one or more modified nucleotide (s) .
  • only the antisense strand comprises one modified nucleotide.
  • only the antisense strand comprises one or more modified nucleotide (s) .
  • the isolated oligonucleotides of the present disclosure comprises at least one modified nucleotide (s) .
  • the one or more modified nucleotide (s) increases the stability or potency or both of the isolated oligonucleotide.
  • the one or more modified nucleotide (s) increases the stability of the RNA duplex, and siRNA.
  • RNA stability examples include, but are not limited to, locked nucleic acids.
  • locked nucleic acid or “LNA” includes, but is not limited to, a modified RNA nucleotide in which the ribose moiety comprises a methylene bridge connecting the 2’ oxygen and the 4’ carbon. This methylene bridge locks the ribose in the 3’-endo confirmation, also known as the north confirmation, that is found in A-form RNA duplexes.
  • LNA locked nucleic acid
  • LNAs having a 2′-4′cyclic linkage as described in the International Patent Application WO 99/14226, WO 00/56746, WO 00/56748, and WO 00/66604, the contents of which are incorporated herein by reference.
  • the sense strand or the antisense strand or both comprise at least one nucleotide having a modified phosphate backbone.
  • the sense strand of the isolated oligonucleotide comprises at least one nucleotide having a modified phosphate backbone.
  • the antisense strand of the isolated oligonucleotide comprises at least one nucleotide having a modified phosphate backbone.
  • the modified phosphate backbone comprises a modified phosphodiester bond.
  • the modified phosphodiester bond is modified by replacing one or more oxygen atoms with a moiety, wherein the moiety is bonded to the phosphorus atom in the phosphodiester bond with a carbon, nitrogen, or sulfur atom in the moiety, or by forming a 2’-5’ linkage.
  • the modified phosphodiester bond comprises phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate diester, mesyl phosphoramidate, or phosphonoacetate.
  • the isolated oligonucleotide of the present disclosure comprises one or more non-natural base-containing nucleotide, a locked nucleotide, or an abasic nucleotide.
  • the one or more modified nucleotide comprises a phosphorothioate derivative or an acridinine substituted nucleotide.
  • the isolated oligonucleotides of the present disclosure comprise a phosphate mimic at the 5’-terminus of antisense strand, including but not limited to vinylphosphonate or other phosphate analogues.
  • the 5’-phosphate mimic is ethylphosphonate, vinylphosphonate or an analog thereof.
  • the modified nucleotide comprises 5-fluorouracil , 5-bromouracil , 5-chlorouracil , 5-iodouracil , hypoxanthine , xanthine , 4-acetylcytosine , 5- (carboxyhydroxylmethyl) uracil , 5-carboxymethylaminomethyl-2-thiouridine , 5-carboxymethylaminomet-hyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methyl-aminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-
  • the phosphate mimic is linked to the 5’-terminus of the isolated oligonucleotides (e.g., siRNAs) as shown in the following structures of Table 1: Table 1: Phosphate Mimic Structure
  • the sense strand or the antisense strand or both comprise a terminal or internal nucleotide linked to one or more targeting ligands.
  • the terminal or internal nucleotide is linked to the one or more targeting ligands directly.
  • the terminal or internal nucleotide is linked to the one or more targeting ligands indirectly by a linker.
  • the one or more targeting ligands linked directly or indirectly to the terminal or internal nucleotide can further comprise a PK modulator.
  • the PK modulator is a competitive modulator, a positive allosteric modulator, a negative allosteric modulator or a neutral allosteric modulator.
  • the targeting ligand is selected from one or more of a carbohydrate, a peptide, a lipid, an antibody or a fragment thereof, an aptamer, an albumin, a fibrinogen, and a folate.
  • an isolated oligonucleotide comprising: (a) a sense strand comprising X1 nucleotides, wherein at least one nucleotide is modified with a first modification, each of the remaining nucleotides is independently modified with a second modification, and X1 is an integer selected from 13-36, wherein the first modification and the second modification are different; and (b) an antisense strand comprising X2 nucleotides, wherein at least one nucleotide is modified with a third modification, each of the remaining nucleotides is independently modified with a fourth modification, and X2 is an integer selected from 18-31, wherein the third modification and the fourth modification are different.
  • the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure is 18-21 and the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure is 20-23. In some embodiments, the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure, is 20 or 21 and the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure is 22 or 23.
  • the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure equals the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure plus 2. In some embodiments, the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure is 21 and the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure is 23.
  • the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure is 20 and the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure is 22.
  • the isolated oligonucleotide comprises: (a) a sense strand comprising 20 nucleotides, wherein at least one nucleotide is modified with a first modification, each of the remaining nucleotides is independently modified with a second modification, wherein the first modification and the second modification are the same or different; and (b) an antisense strand comprising 22 nucleotides, wherein at least one nucleotide is modified with a third modification, each of the remaining nucleotides is independently modified with a fourth modification, wherein the third modification and the fourth modification are the same or different.
  • the first modification is modification of the sugar moiety of the at least one nucleotide at the 2’-position selected from 2’-F modification, 2’-CN modification, 2’-N 3 modification, 2’-deoxy modification, and an equivalent thereof, and a combination thereof.
  • the first modification is 2’-F modification, 2’-CN modification, 2’-N 3 modification, or 2’-deoxy modification, or a stereoisomer thereof.
  • the first modification is 2’-F modification, 2’-CN modification, or 2’-N 3 modification, or a stereoisomer thereof.
  • the first modification is 2’-F modification or a stereoisomer thereof.
  • the second modification is modification of the sugar moiety of one or more of the remaining nucleotides at the 2’-position selected from 2’-C 1 -C 6 alkyl, 2’-OR modification wherein R is C 1 -C 6 alkyl optionally substituted with C 1 -C 6 alkoxy, acetamide, phenyl, or heteroaryl comprising a 5-or 6-membered ring and 1 or 2 heteroatoms selected from N, O, and S, 2’-amino, and morpholino replacement, and an equivalent thereof, and a combination thereof.
  • the second modification is 2’-OR modification, or morpholino replacement, or a combination thereof.
  • the second modification is 2’-OR modification.
  • the second modification is 2’-O-methyl modification or 2’-methoxyethoxy modification.
  • the second modification is 2’-O-methyl modification.
  • the second modification is morpholino replacement.
  • the first modification is 2’-F modification or a stereoisomer thereof, and the second modification is 2’-O-methyl modification or 2’-methoxyethoxy modification. In some embodiments, the first modification is 2’-F modification or a stereoisomer thereof, and the second modification is 2’-O-methyl modification.
  • the third modification is modification of the sugar moiety of the at least one nucleotide at the 2’-position selected from 2’-F modification, 2’-CN modification, 2’-N 3 modification, 2’-deoxy modification, and an equivalent thereof, and a combination thereof.
  • the third modification is 2’-F modification, 2’-CN modification, 2’-N 3 modification, or 2'-deoxy modification, or a stereoisomer thereof.
  • the third modification is 2’-F modification, 2’-CN modification, or 2’-N 3 modification, or a stereoisomer thereof.
  • the third modification is 2’-F modification or a stereoisomer thereof.
  • the fourth modification is modification of the sugar moiety of one or more of the remaining nucleotides at the 2’-position selected from 2’-C 1 -C 6 alkyl, 2’-OR modification wherein R is C 1 -C 6 alkyl optionally substituted with C 1 -C 6 alkoxy, acetamide, phenyl, or heteroaryl comprising a 5-or 6-membered ring and 1 or 2 heteroatoms selected from N, O, and S, 2’-amino, and morpholino replacement, and an equivalent thereof, and a combination thereof.
  • the fourth modification is 2’-OR modification, or morpholino replacement, or a combination thereof.
  • the fourth modification is 2’-OR modification.
  • the fourth modification is 2’-O-methyl modification or 2’-methoxyethoxy modification.
  • the fourth modification is 2’-O-methyl modification.
  • the fourth modification is morpholino replacement.
  • the third modification is 2’-F modification or a stereoisomer thereof, and the fourth modification is 2’-O-methyl modification or 2’-methoxyethoxy modification. In some embodiments, the third modification is 2’-F modification or a stereoisomer thereof, and the fourth modification is 2’-O-methyl modification.
  • the isolated oligonucleotide of the present disclosure comprising a sense and an antisense strand
  • at least three nucleotides are modified with the first modification.
  • at least two of the at least three nucleotides modified with the first modification are consecutively located.
  • at least three of the at least three nucleotides modified with the first modification are consecutively located.
  • At least four nucleotides are modified with the first modification. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least three of the at least four nucleotides modified with the first modification are consecutively located. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least four of the at least four nucleotides modified with the first modification are consecutively located.
  • At least five nucleotides are modified with the first modification. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least three of the at least five nucleotides modified with the first modification are consecutively located. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least four of the at least five nucleotides modified with the first modification are consecutively located.
  • the at least three nucleotides, the at least four nucleotides, or the at least five nucleotides modified with the first modification are located from position 10 to position 15 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand.
  • two of the at least three nucleotides modified with the first modification are located at positions selected from position 10, 11, 12, and 13 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand.
  • three of the at least three nucleotides modified with the first modification are located at positions selected from position 10, 11, 12, and 13 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand.
  • one of the at least three nucleotides modified with the first modification is located at position 11 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand.
  • three of the at least three nucleotides modified with the first modification are located at positions 11, 12 and 13 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, three of the at least three nucleotides modified with the first modification are located at positions 12, 13 and 14 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense.
  • three of the at least three nucleotides modified with the first modification are located at positions 10, 11 and 12 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand.
  • one of the at least four nucleotides modified with the first modification is located at position 10 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of the at least four nucleotides modified with the first modification is located at position 11 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand.
  • one of the at least four nucleotides modified with the first modification is located at position 12 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of the at least four nucleotides modified with the first modification is located at position 13 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand.
  • one of the at least four nucleotides modified with the first modification is located at position 14 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of the at least four nucleotides modified with the first modification is located at position 15 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand.
  • the at least four nucleotides modified with the first modification are located at positions 10, 11, 12 and 13 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand.
  • the at least five nucleotides modified with the first modification are located at positions 10, 11, 12, 13 and 15 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand.
  • the sense strand comprises five nucleotides modified with the first modification, wherein the five nucleotides modified with the first modification are located at positions 10, 11, 12, 13 and 15 from the nucleotide complementary to the first nucleotide at the 5’-terminus of the antisense strand.
  • the at least three nucleotides, the at least four nucleotides, or the at least five nucleotides modified with the first modification are consecutively located.
  • the at least three nucleotides, the at least four nucleotides, or the at least five nucleotides are modified with 2’-F modification.
  • the sense strand of the isolated oligonucleotide of the present disclosure comprises nucleotides modified with 2’-F modification ( “F” ) , and nucleotides modified with 2’-O-methyl modification ( “M” ) , according to the formula: 5’ (M) g (F) f (M) e (F) d (M) c (F) b (M) a 3’, wherein M is 2’-O-methyl modified nucleotide, F is 2’-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5’ (M) 0 (F) 0 (M) 5 (F) 1 (M) 1 (F) 4 (M) 9 3’.
  • the sense strand of the isolated oligonucleotide comprises nucleotides modified with 2’-F modification ( “F” ) , and nucleotides modified with 2’-O-methyl modification ( “M” ) , according to the formula: 5’ (M) g (F) f (M) e (F) d (M) c (F) b (M) a 3’, wherein M is 2’-O-methyl modified nucleotide, F is 2’-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5’ (M) 0 (F) 0 (M) 5 (F) 1 (M) 1 (F) 4 (M) 9 3’.
  • the antisense strand of the isolated oligonucleotide of the present disclosure at most seven nucleotides are modified with the third modification.
  • the antisense strand of the isolated oligonucleotide of the present disclosure at most four of the at most seven nucleotides modified with the third modification are located from position 2 to position 8 from the first nucleotide at the 5’-terminus of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at least one of the at most seven nucleotides are modified with the third modification is located at position 2 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the antisense strand of the isolated oligonucleotide of the present disclosure at most two of the at most seven nucleotides modified with the third modification are consecutively located. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, the at most two consecutively located of the at most seven nucleotides modified with the third modification are located at positions 2 and 3 from the first nucleotide at the 5’-terminus of the antisense strand.
  • At least one of the at most seven nucleotides modified with the third modification is located at position 14 from the first nucleotide at the 5’-terminus of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, two or three of the at most seven nucleotides modified with the third modification are located at positions selected from position 2, 3, 5, and 6 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the antisense strand of the isolated oligonucleotide of the present disclosure in some embodiments, three of the at most seven nucleotides modified with the third modification are located at positions selected from position 2, 3, 5, and 6 from the first nucleotide at the 5’-terminus of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, two of the at most seven nucleotides modified with the third modification are located at positions 2 and 5 from the first nucleotide at the 5’-terminus of the antisense strand.
  • two of the at most seven nucleotides modified with the third modification are located at positions 2 and 3 from the first nucleotide at the 5’-terminus of the antisense strand.
  • three of the at most seven nucleotides modified with the third modification are located at positions 2, 3 and 5 from the first nucleotide at the 5’-terminus of the antisense strand.
  • one or two of the at most seven nucleotides modified with the third modification are located at positions selected from position 14 and 16 from the first nucleotide at the 5’-terminus of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, two of the at most seven nucleotides modified with the third modification are located at positions 14 and 16 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the at most seven nucleotides are modified with 2’-F modification.
  • one of the at most seven nucleotides modified with the third modification is located at position 14 from the first nucleotide at the 5’-terminus of the antisense strand.
  • two of the at most seven nucleotides modified with the third modification is located at positions 14 and 16 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the antisense strand comprises at most seven nucleotides modified with the third modification
  • the at most seven nucleotides are modified with 2’-F modification.
  • one of the at most seven nucleotides modified with the third modification is located at position 2 from the first nucleotide at the 5’-terminus of the antisense strand.
  • one of the at most seven nucleotides modified with the third modification is located at position 3 from the first nucleotide at the 5’-terminus of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, one of the at most seven nucleotides modified with the third modification is located at position 5 from the first nucleotide at the 5’-terminus of the antisense strand.
  • one of the at most seven nucleotides modified with the third modification is located at position 7 from the first nucleotide at the 5’-terminus of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, one of the at most seven nucleotides modified with the third modification is located at position 10 from the first nucleotide at the 5’-terminus of the antisense strand.
  • one of the at most seven nucleotides modified with the third modification is located at position 14 from the first nucleotide at the 5’-terminus of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, one of the at most seven nucleotides modified with the third modification is located at position 16 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the at most seven nucleotides modified with the third modification are located at positions 2, 3, 5, 7, 10, 14 and 16 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the antisense strand comprises nucleotides modified with 2’-F modification ( “F” ) , and nucleotides modified with 2’-O-methyl modification ( “M” ) , according to the formula: 3’ (M) a (F) b (M) c (F) d (M) e (F) f (M) g (F) h (M) i (F) j (M) k (F) l (M) m (F) n (M) o 5’, wherein M is 2’-O-methyl modified nucleotide, F is 2’-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is
  • a terminal or internal nucleotide is linked to a targeting ligand.
  • the targeting ligand is attached to one or more nucleotides at the 5’ end of the sense strand of the isolated oligonucleotide of the present disclosure.
  • the targeting ligand is attached to one or more nucleotides at the 3’ end of the sense strand of the isolated oligonucleotide of the present disclosure.
  • the targeting ligand is attached to one or more nucleotides at the 5’ end of the antisense strand of the isolated oligonucleotide of the present disclosure. In some embodiments, the targeting ligand is attached to one or more nucleotides at the 3’ end of the antisense strand of the isolated oligonucleotide of the present disclosure. In some embodiments, the targeting ligand is attached to one or more nucleotides of the at least two single-stranded nucleotides at the 3’-terminus of the antisense strand of the isolated oligonucleotide of the present disclosure.
  • the targeting ligand is selected from one or more of a carbohydrate, a peptide, a lipid, an antibody or a fragment thereof, an aptamer, an albumin, a fibrinogen, and a folate.
  • the targeting ligand binds to a surface protein on a cell expressing a target mRNA of the isolated oligonucleotide of the present disclosure.
  • the targeting ligand mediates entry of the isolated oligonucleotide of the present disclosure, into a cell expressing a target mRNA of the isolated oligonucleotide of the present disclosure.
  • the targeting ligand is a therapeutic ligand. In some embodiments, the targeting ligand is a therapeutic antibody.
  • the targeting ligand is attached to the isolated oligonucleotide of the present disclosure by a linker.
  • the linker is any one or a protein, a DNA, an RNA or a chemical compound.
  • the isolated oligonucleotide, the linker and the targeting ligand, of the present disclosure form a scaffold.
  • the term “scaffold” refers to a compound or complex that comprises a linker of the present disclosure, wherein the linker is covalently attached to either a ligand or an isolated oligonucleotide or both.
  • the isolated oligonucleotide, the linker and the targeting ligand, of the present disclosure form a conjugate.
  • conjugate refers to a compound or complex that comprises an isolated oligonucleotide being covalently attached to a ligand via a linker of the present disclosure.
  • targeting ligand refers to a moiety that, when being covalently attached to GalNAc an oligonucleotide) , is capable of mediating its entry into, or facilitating or allowing its delivery to, a target site (e.g., a target cell or tissue) .
  • the targeting ligand comprises a sugar ligand moiety (e.g., N-acetylgalactosamine (GalNAc) ) which may direct uptake of an oligonucleotide into the liver.
  • GalNAc N-acetylgalactosamine
  • the targeting ligand binds to the asialoglycoprotein receptor (ASGPR) . In some embodiments, the targeting ligand binds to (e.g., through ASGPR) the liver, such as the parenchymal cells of the liver.
  • ASGPR asialoglycoprotein receptor
  • the targeting ligand binds to (e.g., through ASGPR) the liver, such as the parenchymal cells of the liver.
  • Suitable targeting ligands include, but are not limited to, the ligands disclosed in Winkler (Ther. Deliv., 2013, 4 (7) : 791-809) , PCT Patent Appl’n Pub. Nos. WO/2017/100401, WO/2012/089352, and WO/2009/082607, and U.S. Patent Appl’n Pub. Nos. 2009/0239814, 2012/0136042, 2013/0158824, and 2009/0247608, each of which is incorporated by reference.
  • the targeting ligand comprises a carbohydrate moiety.
  • carbohydrate moiety refers to a moiety which comprises one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched or cyclic) , with an oxygen, nitrogen or sulfur atom bonded to each carbon atom.
  • the carbohydrate moiety comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.
  • the carbohydrate moiety comprises an oligosaccharide containing from about 4-9 monosaccharide units.
  • the carbohydrate moiety comprises a polysaccharide (e.g., a starch, a glycogen, a cellulose, or a polysaccharide gum) .
  • the carbohydrate moiety comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide. In some embodiments, the carbohydrate moiety comprises an oligosaccharide (e.g., containing from about four to about nine monosaccharide units) . In some embodiments, the carbohydrate moiety comprises a polysaccharide (e.g., a starch, a glycogen, a cellulose, or a polysaccharide gum) .
  • the ligand is capable of binding to a human asialoglycoprotein receptor (ASGPR) , e.g., human asialoglycoprotein receptor 2 (ASGPR2) .
  • ASGPR human asialoglycoprotein receptor 2
  • the carbohydrate moiety comprises a sugar (e.g., one, two, or three sugar) .
  • the carbohydrate moiety comprises galactose or a derivative thereof (e.g., one, two, or three galactose or the derivative thereof) .
  • the carbohydrate moiety comprises N-acetylgalactosamine or a derivative thereof (e.g., one, two, or three N-acetylgalactosamine or the derivative thereof) .
  • the carbohydrate moiety comprises N-acetyl-D-galactosylamine or a derivative thereof (e.g., one, two, or three N-acetyl-D-galactosylamine or the derivative thereof) .
  • the carbohydrate moiety comprises N-acetylgalactosamine (e.g., one, two, or three N-acetylgalactosamine) . In some embodiments, the carbohydrate moiety comprises N-acetyl-D-galactosylamine (e.g., one, two, or three N-acetyl-D-galactosylamine) .
  • the carbohydrate moiety comprises mannose or a derivative thereof (e.g., mannose-6-phosphate) .
  • the carbohydrate moiety further comprises a linking moiety that connects the one or more sugar (e.g., N-acetyl-D-galactosylamine) with a linker.
  • the linker comprises thioether (e.g., thiosuccinimide, or the hydrolysis analogue thereof) , disulfide, triazole, phosphorothioate, phosphodiester, ester, amide, or any combination thereof.
  • the linker is a triantennary linking moiety.
  • Suitable targeting ligands include, but are not limited to, the ligands disclosed in PCT Appl’n Pub. Nos. WO/2015/006740, WO/2017/100401, WO/2017/214112, WO/2018/039364, and WO/2018/045317, each of which is incorporated herein by reference.
  • the targeting ligand comprises a lipid or a lipid moiety (e.g., one, two, or three lipid moiety) .
  • the lipid moiety comprises (e.g., one, two, of three of) C8-C24 fatty acid, cholesterol, vitamin, sterol, phospholipid, or any combination thereof.
  • the targeting ligand comprises a peptide or a peptide moiety (e.g., one, two, or three peptide moiety) .
  • the peptide moiety comprises (e.g., one, two, or three of) integrin, insulin, glucagon-like peptide, or any combination thereof.
  • the targeting ligand comprises an antibody or an antibody moiety (e.g., transferrin) .
  • the targeting ligand comprises one, two, or three antibody moieties (e.g., transferrin) .
  • the targeting ligand comprises an oligonucleotide (e.g., aptamer or CpG) . In some embodiments, the targeting ligand comprises one, two, or three oligonucleotides (e.g., aptamer or CpG) .
  • the ligand comprises: one, two, or three sugar (e.g., N-acetyl-D-galactosylamine) ; one, two, or three lipid moieties; one, two, or three peptide moieties; one, two, or three antibody moieties; one, two, or three oligonucleotides; or any combination thereof.
  • sugar e.g., N-acetyl-D-galactosylamine
  • the linker is attached to the isolated oligonucleotide of the present disclosure, via a phosphate group, or an analog of a phosphate group, in the isolated oligonucleotide.
  • the ligand comprises a sugar ligand moiety (e.g., N-acetylgalactosamine (GalNAc) ) which may direct uptake of an oligonucleotide into the liver.
  • a sugar ligand moiety e.g., N-acetylgalactosamine (GalNAc)
  • GalNAc N-acetylgalactosamine
  • the ligand comprises GalNAc, or a derivative thereof. In some embodiments, the ligand comprises a GalNAc G1b structure shown below: wherein each independently indicates the point of attachment to the oligonucleotide compound or the other GalNAc G1b moiety, GalNAc G1b
  • the ligand comprises three GalNAc moieties, or three derivatives thereof.
  • the targeting ligand is linked by a G1b linker moiety.
  • the ligand comprises three GalNAc G1b moieties.
  • the GalNAc G1b moieties are consecutively located.
  • the consecutively located GalNAc G1b moieties are located on the 3’ end of the sense strand.
  • the first GalNAc G1b moiety is linked to the second GalNAc G1b moiety and the second GalNAc G1b is linked to the third GalNAc G1b moiety.
  • the first GalNAc G1b moiety is linked to the sense strand of the oligonucleotide compound of the present disclosure.
  • the ligand comprises a [GalNAc G1b] [GalNAc G1b] [GalNAc G1b] moiety, with structure shown below:
  • the ligand comprises three GalNAc G1b moieties, wherein the first GalNAc G1b moiety is linked to the sense strand of the isolated oligonucleotide, the first GalNAc G1b moiety is also linked to the second GalNAc G1b moiety, and the second GalNAc G1b is linked to the third GalNAc G1b moiety.
  • the ligand comprises three GalNAc G1b moieties
  • the three GalNAc G1b moieties are consecutively located on the 3’ end of the sense strand.
  • the isolated oligonucleotide is linked to the ligand (e.g., GalNAc G1b, or three GalNAc G1b moieties) .
  • the isolated oligonucleotide is linked to the ligand via an internal or terminal nucleotide of the isolated oligonucleotide.
  • the isolated oligonucleotide is linked to the ligand via a ligand linker. In some embodiments, the
  • the ligand is linked to a terminal nucleotide on the sense strand of the isolated oligonucleotide.
  • the ligand is linked to a terminal nucleotide on the sense strand via a ligand linker.
  • the ligand linker is a monovalent linker.
  • the ligand linker is a bivalent linker.
  • the ligand linker is a trivalent linker.
  • the linkage at the 3’ end of the isolated oligonucleotide of the present disclosure may be directly via 5’, 3’ or 2’ hydroxyl groups, or indirectly, via a non-nucleotide linker or a nucleoside, utilizing either the 2’ or 3’ hydroxyl positions of the nucleoside.
  • Linkages may also utilize a functionalized sugar or nucleobase of a 3’ terminal nucleotide.
  • the ligand described herein can be attached to the isolated oligonucleotide of the present disclosure with various ligand linkers that can be cleavable or non-cleavable.
  • the present disclosure further provides oligonucleotides and conjugates containing modified phosphate groups (also referred to as phosphate mimics or phosphate derivatives) for nucleic acid delivery.
  • modified phosphate groups also referred to as phosphate mimics or phosphate derivatives
  • the present disclosure also relates to uses of oligonucleotides and conjugates containing modified phosphate groups, e.g., in delivering nucleic acid and/or treating or preventing diseases.
  • the present disclosure provides phosphate mimics of 5’-terminal nucleotides.
  • the phosphate mimics could improve the Ago2 binding/loading and enhance the metabolic stability of the oligonucleotides, thus enhancing the potency and duration of the isolated oligonucleotides (e.g., dsRNA or siRNA) .
  • the oligonucleotides comprise 5’-terminal nucleotide modifications.
  • the 5’-terminal modifications provide the functional effect of a phosphate group, but are more stable in the environmental conditions that the oligonucleotide will be exposed to when administered to a subject.
  • the isolated oligonucleotide comprises phosphate mimics that are more resistant to phosphatases and other enzymes while minimizing negative impact on the oligonucleotide's function (e.g., minimizing any reduction in gene target knockdown when used as an RNAi inhibitor molecule) .
  • the 5’-terminal modification is a chemical modification.
  • the chemical modification enhances stability against nucleases or other enzymes that degrade or interfere with the structure or activity of the isolated oligonucleotide.
  • the sense or antisense strand of the isolated oligonucleotides of the present disclosure comprise a 5’-terminal phosphate group.
  • the 5’-terminal phosphate group comprises a modified phosphate.
  • the modified phosphate is referred to as a “phosphate mimic” .
  • aryl as used herein, includes groups with aromaticity, including “conjugated, ” or multicyclic systems with one or more aromatic rings and do not contain any heteroatom in the ring structure.
  • aryl includes both monovalent species and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl and the like.
  • alkyl or “C 1 -C 6 alkyl” , as used herein, is intended to include C 1 , C 2 , C 3 , C 4 , C 5 or C 6 straight chain (linear) saturated aliphatic hydrocarbon groups and C 3 , C 4 , C 5 or C 6 branched saturated aliphatic hydrocarbon groups.
  • C 1 -C 6 alkyl is intended to include C 1 , C 2 , C 3 , C 4 , C 5 and C 6 alkyl groups.
  • alkyl examples include, moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, or n-hexyl.
  • a straight chain or branched alkyl has six or fewer carbon atoms (e.g., C 1 -C 6 for straight chain, C 3 -C 6 for branched chain) , and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms.
  • the straight chain alkyl has one carbon atom. In some embodiments, the straight chain alkyl has two carbon atoms.
  • the phosphate mimic is linked to the 5’-terminus of the isolated oligonucleotides (e.g., siRNAs) as shown in the following formula: wherein: B is H or a nucleobase moiety; X is O or S; R 1 is H or C 1 -C 6 alkyl; R 2 is H or C 1 -C 6 alkyl; Y 1 is O or S; Y 2 is O or S; Z is H, halogen, or -OR Z ; R Z is H, C 1 -C 6 alkyl, or - (C 1 -C 6 alkyl) - (C 6 -C 10 aryl) , wherein the C 1 -C 6 alkyl or - (C 1 - C 6 alkyl) - (C 6 -C 10 aryl) is optionally substituted with one or more R Za ; each R Za independently is halogen, C 1 -C 6 alkyl, or -O- (C 1 )
  • the phosphate mimic is linked to the 5’-terminus of the isolated oligonucleotides (e.g., siRNAs) as shown in the following formula: wherein: B is H or a nucleobase moiety; X is O or S; R 1 is H or C 1 -C 6 alkyl; R 2 is H or C 1 -C 6 alkyl; Y 1 is O or S; Y 2 is O or S; and indicates an attachment to a nucleotide of the isolated oligonucleotide (e.g., siRNA) .
  • B is H or a nucleobase moiety
  • X is O or S
  • R 1 is H or C 1 -C 6 alkyl
  • R 2 is H or C 1 -C 6 alkyl
  • Y 1 is O or S
  • Y 2 is O or S
  • indicates an attachment to a nucleotide of the isolated oligonucleotide e.g., siRNA
  • the phosphate mimic is linked to the 5’-terminus of the isolated oligonucleotides (e.g., siRNAs) as shown in the following formula: wherein: B is H or a nucleobase moiety; X is O or S; R 1 is H or C 1 -C 6 alkyl; R 2 is H or C 1 -C 6 alkyl; and indicates an attachment to a nucleotide of the isolated oligonucleotide (e.g., siRNA) .
  • B is H or a nucleobase moiety
  • X is O or S
  • R 1 is H or C 1 -C 6 alkyl
  • R 2 is H or C 1 -C 6 alkyl
  • indicates an attachment to a nucleotide of the isolated oligonucleotide e.g., siRNA
  • X is O.
  • X is S.
  • R 1 is H.
  • R 1 is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) .
  • R 1 is methyl
  • R 2 is H.
  • R 2 is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) .
  • R 2 is methyl
  • Y 1 is O.
  • Y 1 is S.
  • Y 2 is O.
  • Y 2 is S.
  • Z is H.
  • Z is not H.
  • Z is halogen (e.g., F, Cl, Br, or I) .
  • Z is F or Cl.
  • Z is F
  • Z is -OR Z .
  • Z is -OH.
  • Z is not -OH.
  • Z is -O- (C 1 -C 6 alkyl) (e.g., wherein the C 1 -C 6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) .
  • Z is -OCH 3 .
  • Z is -O- (C 1 -C 6 alkyl) -O- (C 1 -C 6 alkyl) (e.g., wherein the C 1 -C 6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) .
  • Z is -OCH 2 CH 2 OCH 3 .
  • Z is -O- (C 1 -C 6 alkyl) - (C 6 -C 10 aryl) optionally substituted with one or more R Za .
  • Z is -O- (C 1 -C 6 alkyl) - (C 6 -C 10 aryl) .
  • Z is
  • Z is optionally substituted with one or more R Za .
  • Z is optionally substituted with one or more halogen.
  • Z is optionally substituted with one or more C 1 -C 6 alkyl or -O- (C 1 -C 6 alkyl) , wherein the C 1 -C 6 alkyl or -O- (C 1 -C 6 alkyl) is optionally substituted with one or more halogen.
  • R Z is H.
  • R Z is not H.
  • R Z is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more R Za .
  • R Z is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I) or -O- (C 1 -C 6 alkyl) (e.g., wherein the C 1 -C 6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen.
  • halogen e.g., F, Cl, Br, or I
  • C 1 -C 6 alkyl e.g., where
  • R Z is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) .
  • R Z is methyl, ethyl, or propyl.
  • R Z is methyl
  • R Z is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogen (e.g., F, Cl, Br, or I) .
  • halogen e.g., F, Cl, Br, or I
  • R Z is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more -O- (C 1 -C 6 alkyl) (e.g., wherein the C 1 -C 6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) , wherein the -O- (C 1 -C 6 alkyl) is optionally substituted with one or more halogen.
  • C 1 -C 6 alkyl e.g., methyl, ethyl, n-propyl, i-prop
  • R Z is - (C 1 -C 6 alkyl) - (C 6 -C 10 aryl) optionally substituted with one or more R Za .
  • R Z is - (C 1 -C 6 alkyl) - (C 6 -C 10 aryl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I) , C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) , or -O- (C 1 -C 6 alkyl) (e.g., wherein the C 1 -C 6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) , wherein the C 1 -C 6 alkyl or
  • R Z is - (C 1 -C 6 alkyl) - (C 6 -C 10 aryl) .
  • At least one R Za is halogen (e.g., F, Cl, Br, or I) .
  • At least one R Za is F or Cl.
  • At least one R Za is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I) .
  • halogen e.g., F, Cl, Br, or I
  • At least one R Za is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) .
  • At least one R Za is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogen (e.g., F, Cl, Br, or I) .
  • halogen e.g., F, Cl, Br, or I
  • At least one R Za is -O- (C 1 -C 6 alkyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I) .
  • halogen e.g., F, Cl, Br, or I
  • At least one R Za is -O- (C 1 -C 6 alkyl) .
  • At least one R Za is -O- (C 1 -C 6 alkyl) substituted with one or more halogen (e.g., F, Cl, Br, or I) .
  • halogen e.g., F, Cl, Br, or I
  • B is H.
  • B is a nucleobase moiety.
  • nucleobase moiety refers to a nucleobase that is attached to the rest of the isolated oligonucleotides (e.g., dsRNA or siRNA) of the present disclosure, e.g., via an atom of the nucleobase or a functional group thereof.
  • the nucleobase moiety is adenine (A) , cytosine (C) , guanine (G) , thymine (T) , or uracil (U) .
  • the nucleobase moiety is uracil (U) .
  • the phosphate mimic is linked to the 5’-terminus of the isolated oligonucleotides as shown in the following formula: wherein: B is a nucleobase moiety, wherein the nucleobase moiety is uracil (U) , wherein the uracil is at position 1 from the 5’-terminus of the sense strand or at position 1 from the 5’-terminus of the antisense strand; X is O; R 1 is C 1 alkyl; R 2 is H; and indicates an attachment to a nucleotide of the isolated oligonucleotide (e.g., siRNA) .
  • B is a nucleobase moiety, wherein the nucleobase moiety is uracil (U) , wherein the uracil is at position 1 from the 5’-terminus of the sense strand or at position 1 from the 5’-terminus of the antisense strand
  • X is O
  • R 1 is C 1 alkyl
  • R 2 is
  • the phosphate mimic is attached to the 5’-terminus of the antisense strand of the isolated oligonucleotide.
  • the phosphate mimic is attached to a 5’-terminal uridine of the antisense strand of the isolated oligonucleotide, having the following structure (5’-MeEPmU) .
  • 5’-MeEPmU 5’-terminal uridine of the antisense strand of the isolated oligonucleotide
  • mU is a 2’-O-methyl modified uridine nucleotide
  • MeEP is a mono methyl protected phosphate mimic.
  • the phosphate mimic is attached to a 5’-terminal uridine of the antisense strand of the isolated oligonucleotide, having the following structure (5’-MeEPmUs) .
  • mU is a 2’-O-methyl modified uridine nucleotide
  • MeEP is a mono methyl protected phosphate mimic
  • s is a phosphorothioate internucleotide linkage.
  • the phosphate mimic is attached to a 5’-terminal uridine of the antisense strand of the isolated oligonucleotide, having the following structure (5’-EPmUs) .
  • mU is a 2’-O-methyl modified uridine nucleotide
  • EP is a phosphate mimic
  • s is a phosphorothioate internucleotide linkage.
  • the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 774 to 799; and b) 4602 to 4625, from the 5’ end of an INHBE mRNA sequence according to SEQ ID NO: 1, and the antisense strand is substantially complementary to the sense strand such that the sense strand and the antisense strand together form a double stranded region, the antisense strand comprises a mono methyl protected phosphate mimic (MeEP) .
  • the MeEP is linked to the 5’ end of the antisense strand (5’-MeEP) .
  • the phosphate mimic is attached to a 5’-terminal uridine of the antisense strand.
  • the 5’-terminal uridine is a 2’-O-methyl modified nucleotide.
  • the sense strand or the antisense strand or both comprise at least one nucleotide having a modified phosphate backbone.
  • the sense strand of the isolated oligonucleotide comprises at least one nucleotide having a modified phosphate backbone.
  • the antisense strand of the isolated oligonucleotide comprises at least one nucleotide having a modified phosphate backbone.
  • the modified phosphate backbone comprises a modified phosphodiester bond.
  • a phosphodiester bond comprises a linkage having the formula: wherein denotes attachment to a 3’ carbon of a first nucleotide in the isolated oligonucleotide of the present disclosure; and denotes attachment to a 5’ carbon of a second nucleotide in the isolated oligonucleotide of the present disclosure.
  • the phosphodiester bond is unmodified, wherein Z 1 is O and Z 2 is OH or O – .
  • the phosphodiester bond is modified, wherein Z 1 is O, S, NH, or N (C 1 -C 6 alkyl) and Z 2 is OH, SH, NH 2 , NH (C 1 -C 6 alkyl) , O – , S – , HN – , or (C 1 -C 6 alkyl) N – , and wherein when Z 1 is O, Z 2 is not OH or O – .
  • Z 1 is O.
  • Z 1 is S.
  • Z 1 is NH
  • Z 1 is N (C 1 -C 6 alkyl) .
  • Z 2 is OH
  • Z 2 is SH.
  • Z 2 is NH 2 .
  • Z 2 is NH (C 1 -C 6 alkyl) .
  • Z 2 is SH, NH 2 , or NH (C 1 -C 6 alkyl) .
  • Z 2 is O – .
  • Z 2 is S – .
  • Z 2 is HN – .
  • Z 2 is (C 1 -C 6 alkyl) N – .
  • Z 2 is S – , HN – , or (C 1 -C 6 alkyl) N – .
  • Z 1 is O and Z 2 is SH.
  • Z 1 is O and Z 2 is NH 2 .
  • Z 1 is O and Z 2 is NH (C 1 -C 6 alkyl) .
  • Z 1 is S and Z 2 is OH.
  • Z 1 is S and Z 2 is SH.
  • Z 1 is S and Z 2 is NH 2 .
  • Z 1 is S and Z 2 is NH (C 1 -C 6 alkyl) .
  • Z 1 is NH and Z 2 is OH.
  • Z 1 is NH and Z 2 is SH.
  • Z 1 is NH and Z 2 is NH 2 .
  • Z 1 is NH and Z 2 is NH (C 1 -C 6 alkyl) .
  • Z 1 is N (C 1 -C 6 alkyl) and Z 2 is OH.
  • Z 1 is N (C 1 -C 6 alkyl) and Z 2 is SH.
  • Z 1 is N (C 1 -C 6 alkyl) and Z 2 is NH 2 .
  • Z 1 is N (C 1 -C 6 alkyl) and Z 2 is NH (C 1 -C 6 alkyl) .
  • Z 1 is O and Z 2 is S – .
  • Z 1 is O and Z 2 is HN – .
  • Z 1 is O and Z 2 is (C 1 -C 6 alkyl) N – .
  • Z 1 is S and Z 2 is O – .
  • Z 1 is S and Z 2 is S – .
  • Z 1 is S and Z 2 is HN – .
  • Z 1 is S and Z 2 is (C 1 -C 6 alkyl) N – .
  • Z 1 is NH and Z 2 is O – .
  • Z 1 is NH and Z 2 is S – .
  • Z 1 is NH and Z 2 is HN – .
  • Z 1 is NH and Z 2 is (C 1 -C 6 alkyl) N – .
  • Z 1 is N (C 1 -C 6 alkyl) and Z 2 is O – .
  • Z 1 is N (C 1 -C 6 alkyl) and Z 2 is S – .
  • Z 1 is N (C 1 -C 6 alkyl) and Z 2 is HN – .
  • Z 1 is N (C 1 -C 6 alkyl) and Z 2 is (C 1 -C 6 alkyl) N – .
  • the modified phosphodiester bond comprises a phosphorothioate internucleotide linkage.
  • the modified phosphodiester bond comprises wherein denotes attachment to a 3’ carbon of a first nucleotide in the isolated oligonucleotide of the present disclosure; and denotes attachment to a 5’ carbon of a second nucleotide in the isolated oligonucleotide of the present disclosure.
  • the modified phosphodiester bond comprises wherein denotes attachment to a 3’ carbon of a first nucleotide in the isolated oligonucleotide of the present disclosure; and denotes attachment to a 5’ carbon of a second nucleotide in the isolated oligonucleotide of the present disclosure.
  • the modified phosphodiester bond comprises wherein denotes attachment to a 3’ carbon of a first nucleotide in the isolated oligonucleotide of the present disclosure; and denotes attachment to a 5’ carbon of a second nucleotide in the isolated oligonucleotide of the present disclosure.
  • the isolated oligonucleotide of the present disclosure comprises at least one modified phosphodiester bond (s) .
  • the sense strand or the antisense strand or both comprise one or more modified phosphodiester bonds.
  • only the sense strand comprises one or more modified phosphodiester bonds.
  • only the antisense strand comprises one or more modified phosphodiester bonds.
  • both the sense strand and antisense strand comprise one or more modified phosphodiester bonds.
  • the isolated oligonucleotide comprises at least two modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least three modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least four modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least five modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least six modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least seven modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least eight modified phosphodiester bonds.
  • the isolated oligonucleotide comprises at least nine modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least ten modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least eleven modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least twelve modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least thirteen modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least fourteen modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least fifteen modified phosphodiester bonds.
  • the isolated oligonucleotide comprises at least sixteen modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least seventeen modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least eighteen modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least nineteen modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises at least twenty modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises more than twenty modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises between twenty and thirty modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises between thirty and forty modified phosphodiester bonds. In some embodiments, the isolated oligonucleotide comprises between forty and fifty modified phosphodiester bonds.
  • the isolated oligonucleotide comprises at least two phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least three phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least four phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least five phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least six phosphorothioate internucleotide linkages.
  • the isolated oligonucleotide comprises at least seven phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least eight phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least nine phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least ten phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least eleven phosphorothioate internucleotide linkages.
  • the isolated oligonucleotide comprises at least twelve phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least thirteen phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least fourteen phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least fifteen phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least sixteen phosphorothioate internucleotide linkages.
  • the isolated oligonucleotide comprises at least seventeen phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least eighteen phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises at least nineteen phosphorothioate internucleotide linkages. some embodiments, the isolated oligonucleotide comprises at least twenty phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises more than twenty phosphorothioate internucleotide linkages.
  • the isolated oligonucleotide comprises between twenty and thirty phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises between thirty and forty phosphorothioate internucleotide linkages. In some embodiments, the isolated oligonucleotide comprises between forty and fifty phosphorothioate internucleotide linkages.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least one modified phosphodiester bond (s) . In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least two modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least three modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least four modified phosphodiester bonds.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least five modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least six modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least seven modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least eight modified phosphodiester bonds.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least nine modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least ten modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least eleven modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least twelve modified phosphodiester bonds.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least thirteen modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least fourteen modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least fifteen modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least sixteen modified phosphodiester bonds.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least seventeen modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least eighteen modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least nineteen modified phosphodiester bonds. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least twenty modified phosphodiester bonds.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least one phosphorothioate internucleotide linkage (s) . In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least two phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least three phosphorothioate internucleotide linkages.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least four phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least five phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least six phosphorothioate internucleotide linkages.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least seven phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least eight phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least nine phosphorothioate internucleotide linkages.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least ten phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least eleven phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least twelve phosphorothioate internucleotide linkages.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least thirteen phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least fourteen phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least fifteen phosphorothioate internucleotide linkages.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least sixteen phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least seventeen phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least eighteen phosphorothioate internucleotide linkages.
  • the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least nineteen phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least twenty phosphorothioate internucleotide linkages.
  • the modified phosphodiester bonds are consecutively located on the sense strand or the antisense strand or both. In some embodiments, some but not all of the modified phosphodiester bonds are consecutively located on the sense strand or the antisense strand or both. In some embodiments, the modified phosphodiester bonds on the sense strand or the antisense strand or both are not consecutively located.
  • Envisaged within the present disclosure is an isolated oligonucleotide, wherein any phosphodiester bond on the sense strand or antisense strand can be modified. In some embodiments, any phosphodiester bond on the antisense strand can be modified. In some embodiments, any phosphodiester bond on the antisense strand can be modified.
  • the antisense strand comprises between one and twenty, between one and fifteen, between one and ten, between one and five, or less than five modified phosphodiester bonds. In some embodiments, the between one and twenty, between one and fifteen, between one and ten, between one and five, or less than five modified phosphodiester bonds comprise phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises less than five modified phosphodiester bonds. In some embodiments, the antisense strand comprises one, two, three, or four modified phosphodiester bonds.
  • the antisense strand comprises one, two, three, or four modified phosphodiester bonds
  • the one, two, three, or four modified phosphodiester bonds comprise phosphorothioate internucleotide linkages.
  • the antisense strand comprises four modified phosphodiester bonds.
  • the antisense strand comprises four modified phosphodiester bonds.
  • the modified phosphodiester bonds comprise phosphorothioate.
  • the antisense strand comprises at least one, at least two, at least three, or at least four phosphorothioate internucleotide linkages
  • the phosphorothioate internucleotide linkages connect the nucleotides at position 1 and position 2 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the antisense strand comprises at least one, at least two, at least three, or at least four phosphorothioate internucleotide bonds
  • the phosphorothioate internucleotide linkages connect the nucleotides at position 2 and position 3 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the antisense strand comprises at least one, at least two, at least three, or at least four phosphorothioate internucleotide bonds
  • the phosphorothioate internucleotide linkages connect the nucleotides at position 20 and position 21 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the antisense strand comprises at least one, at least two, at least three, or at least four phosphorothioate internucleotide bonds
  • the phosphorothioate internucleotide linkages connect the nucleotides at position 21 and position 22 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the antisense strand comprises at least one, at least two, at least three, or at least four modified phosphodiester bonds, wherein the modified phosphodiester bonds comprise phosphorothioate internucleotide linkages, the phosphorothioate internucleotide linkages are located between nucleotides at position 1 and 2, position 2 and 3, position 20 and 21, and position 21 and 22 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the antisense strand comprises at least one, at least two, at least three, or at least four phosphorothioate internucleotide linkages
  • the phosphorothioate internucleotide linkages are located between nucleotides at position 1 to 3 and nucleotides at position 20 to 22 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the antisense strand comprises at least four phosphorothioate internucleotide linkages
  • the phosphorothioate internucleotide linkages are located between nucleotides at position 1 to 3 and nucleotides at position 20 to 22 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the antisense strand comprises four phosphorothioate internucleotide linkages. In some embodiments, wherein the antisense strand comprises four phosphorothioate internucleotide linkages, the phosphorothioate internucleotide linkages are located between nucleotides at position 1 to 3 and nucleotides at position 20 to 22 from the first nucleotide at the 5’-terminus of the antisense strand.
  • the sense strand comprises between one and twenty, between one and fifteen, between one and ten, between one and five, or less than five modified phosphodiester bonds. In some embodiments, the between one and twenty, between one and fifteen, between one and ten, between one and five, or less than five modified phosphodiester bonds comprise phosphorothioate internucleotide linkages. In some embodiments, the sense strand comprises less than five modified phosphodiester bonds. In some embodiments, wherein the sense strand comprises less than five modified phosphodiester bonds, the sense strand comprises one, two, three, or four modified phosphodiester bonds.
  • the sense strand comprises one, two, three, or four modified phosphodiester bonds
  • the one, two, three, or four modified phosphodiester bonds comprise phosphorothioate internucleotide linkages.
  • the sense strand comprises four modified phosphodiester bonds.
  • the modified phosphodiester bonds comprise phosphorothioate internucleotide linkages.
  • the sense strand comprises at least one, at least two, at least three, or at least four modified phosphodiester bonds
  • the phosphodiester bonds comprise phosphorothioate internucleotide linkages.
  • the sense strand comprises at least one, at least two, at least three, or at least four phosphorothioate internucleotide linkages
  • the phosphorothioate internucleotide linkages connect the nucleotides at position 1 and position 2 from the first nucleotide at the 5’-terminus of the sense strand.
  • the sense strand comprises at least one, at least two, at least three, or at least four phosphorothioate internucleotide linkages
  • the phosphorothioate internucleotide linkages connect the nucleotides at position 2 and position 3 from the first nucleotide at the 5’-terminus of the sense strand.
  • the sense strand comprises at least one, at least two, at least three, or at least four phosphorothioate internucleotide linkages
  • the phosphorothioate internucleotide linkages connect the nucleotides at position 18 and position 19 from the first nucleotide at the 5’-terminus of the sense strand.
  • the sense strand comprises at least one, at least two, at least three, or at least four phosphorothioate internucleotide linkages
  • the phosphorothioate internucleotide linkages connect the nucleotides at position 19 and position 20 from the first nucleotide at the 5’-terminus of the sense strand.
  • the sense strand comprises at least one, at least two, at least three, or at least four modified phosphodiester bonds, wherein the modified phosphodiester bonds comprise phosphorothioate internucleotide linkages, the phosphorothioate internucleotide linkages are located between nucleotides at position 1 and 2, position 2 and 3, position 18 and 19, and position 19 and 20 from the first nucleotide at the 5’-terminus of the sense strand.
  • the sense strand comprises at least one, at least two, at least three, or at least four phosphorothioate internucleotide linkages
  • the phosphorothioate internucleotide linkages are located between nucleotides at position 1 to 3 and nucleotides at position 18 to 20 from the first nucleotide at the 5’-terminus of the sense strand.
  • the sense strand comprises at least four phosphorothioate internucleotide linkages
  • the at least four phosphorothioate internucleotide linkages are located between nucleotides at position 1 to 3 and nucleotides at position 18 to 20 from the first nucleotide at the 5’-terminus of the sense strand.
  • the sense strand comprises four phosphorothioate internucleotide linkages. In some embodiments, wherein the sense strand comprises four phosphorothioate internucleotide linkages, the phosphorothioate internucleotide linkages are located between nucleotides at position 1 to 3 and nucleotides at position 18 to 20 from the first nucleotide at the 5’-terminus of the sense strand.
  • the antisense strand and the sense strand comprise four phosphorothioate internucleotide linkages
  • the antisense strand comprises phosphorothioate internucleotide linkages located between nucleotides at position 1 to 3 and nucleotides at position 20 to 22 from the first nucleotide at the 5’-terminus of the antisense strand
  • the sense strand comprises phosphorothioate internucleotide linkages located between nucleotides at position 1 to 3 and nucleotides at position 18 to 20 from the first nucleotide at the 5’-terminus of the sense strand.
  • the present disclosure also provides a vector encoding at least one isolated oligonucleotide disclosed herein.
  • the vector is any one of a plasmid, a cosmid or a viral vector.
  • the vector is an adenoviral vector.
  • the vector is a lentiviral vector.
  • the plasmid is an expression plasmid.
  • the vector encodes one isolated oligonucleotide disclosed herein.
  • the vector encodes more than one isolated oligonucleotide disclosed herein.
  • the vector encodes, two, three, four, or five isolated oligonucleotides disclosed herein.
  • the vector encodes more than five isolated oligonucleotides disclosed herein.
  • the disclosure provides nucleic acids comprising the sequences encoding the isolated oligonucleotides (e.g., dsRNAs or siRNAs) targeting INHBE described herein.
  • isolated oligonucleotides e.g., dsRNAs or siRNAs
  • the nucleic acids are ribonucleic acids (RNAs) . In some embodiments, the nucleic acids are deoxyribonucleic acids (DNAs) .
  • the DNAs may be a vector or a plasmid, e.g., an expression vector.
  • a “vector” is any nucleic acid molecule for the cloning of and/or transfer of a nucleic acid into a cell.
  • a vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence.
  • a “replicon” can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo, i.e., capable of replication under its own control.
  • vector includes both viral and nonviral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo.
  • viral and nonviral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo.
  • a large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc.
  • the insertion of the nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini.
  • the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini
  • Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have incorporated the nucleic acid of the vector into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker.
  • a “recombinant” vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences (i.e., transgenes) , e.g., two, three, four, five or more heterologous nucleotide sequences.
  • heterologous nucleotide sequences i.e., transgenes
  • telomere By the term “express” or “expression” of a polynucleotide coding sequence, it is meant that the sequence is transcribed, and optionally, translated. Typically, according to the present disclosure, expression of a coding sequence of the disclosure will result in production of the polypeptide of the disclosure. The entire expressed polypeptide or fragment can also function in intact cells without purification.
  • the vector is an expression vector for manufacturing siRNAs of the disclosure.
  • exemplary expression vectors may comprise a sequence encoding the sense and/or antisense strand of the isolated oligonucleotide of the present disclosure, under the control of a suitable promoter for transcription.
  • Interfering RNAs may be expressed from a variety of eukaryotic promoters known to those of ordinary skill in the art, including pol III promoters, such as the U6 or H1 promoters, or pol II promoters, such as the cytomegalovirus promoter. Those of skill in the art will recognize that these promoters can also be adapted to allow inducible expression of the interfering RNA.
  • the isolated oligonucleotide of the present disclosure can be expressed endogenously from plasmid or viral expression vectors, or from minimal expression cassettes, for example, PCR generated fragments comprising one or more promoters and an appropriate template or templates for transcribing the siRNA.
  • plasmid-based expression vectors for shRNA include members of the pSilencer series (Ambion) and pCpG-siRNA (InvivoGen) .
  • kits for production of PCR-generated shRNA expression cassettes include Silencer Express (Ambion) and siXpress (Mirus)
  • Viral vectors for the in vivo expression of the isolated oligonucleotides are also contemplated as within the scope of the instant disclosure.
  • Viral vectors may be derived from a variety of viruses including adenovirus, adeno-associated virus, lentivirus (e.g., HIV, FIV, and EIAV) , and herpes virus.
  • examples of commercially available viral vectors for shRNA expression include pSilencer adeno (Ambion) and pLenti6/BLOCK-iT TM -DEST (Invitrogen) . Selection of viral vectors, methods for expressing the siRNA from the vector and methods of delivering the viral vector, for example incorporated within a nanoparticle, are within the ordinary skill of one in the art.
  • any suitable vector can be used to deliver the isolated oligonucleotides of the present dislclosre (e.g., dsRNAs or siRNAs) described herein to a cell or subject.
  • the vector can be delivered to cells in vivo. In other embodiments, the vector can be delivered to cells ex vivo, and then cells containing the vector are delivered to the subject.
  • the choice of delivery vector can be made based on a number of factors known in the art, including age and species of the target host, in vitro versus in vivo delivery, level and persistence of expression desired, intended purpose (e.g., for therapy or screening) , the target cell or organ, route of delivery, size of the isolated polynucleotide, safety concerns, and the like.
  • the present disclosure also provides a delivery system comprising at least one isolated oligonucleotide disclosed herein or vector of the present disclosure encoding at least one isolated oligonucleotide disclosed herein.
  • the delivery system is any one of a liposome, a nanoparticle, a polymer based delivery system or a ligand-conjugate delivery system.
  • the ligand-conjugate delivery system comprises one or more of an antibody, a peptide, a sugar moiety or a combination thereof.
  • the polymer-based nanoparticle comprises a multiblock copolymer a diblock copolymer. In some embodiments, the polymer-based nanoparticle is pH responsive. In some embodiments, the polymer-based nanoparticle further comprises a buffering component.
  • the delivery system comprises a liposome.
  • Liposomes are spherical vesicles having at least one lipid bilayer, and in some embodiments, an aqueous core.
  • the lipid bilayer of the liposome may comprise phospholipids.
  • An exemplary but non-limiting example of a phospholipid is phosphatidylcholine, but the lipid bilayer may comprise additional lipids, such as phosphatidylethanolamine.
  • Liposomes may be multilamellar, i.e. consisting of several lamellar phase lipid bilayers, or unilamellar liposomes with a single lipid bilayer.
  • Liposomes can be made in a particular size range that makes them viable targets for phagocytosis. Liposomes can range in size from 20 nm to 100 nm, 100 nm to 400 nm, 1 ⁇ M and larger, or 200 nm to 3 ⁇ M. Examples of lipidoids and lipid-based formulations are provided in U.S. Published Application 20090023673. In other embodiments, the one or more lipids are one or more cationic lipids. One skilled in the art will recognize which liposomes are appropriate for siRNA encapsulation.
  • the liposome or the nanoparticle of the present disclosure comprises a micelle.
  • a micelle is an aggregate of surfactant molecules.
  • An exemplary micelle comprises an aggregate of amphiphilic macromolecules, polymers or copolymers in aqueous solution, wherein the hydrophilic head portions contact the surrounding solvent, while the hydrophobic tail regions are sequestered in the center of the micelle.
  • the nanoparticle comprises a nanocrystal.
  • Exemplary nanocrystals are crystalline particles with at least one dimension of less than 1000 nanometers, preferably of less than 100 nanometers.
  • the nanoparticle comprises a polymer-based nanoparticle.
  • the polymer comprises a multiblock copolymer, a diblock copolymer, a polymeric micelle or a hyperbranched macromolecule.
  • the particle comprises one or more cationic polymers.
  • the cationic polymer is chitosan, protamine, polylysine, polyhistidine, polyarginine or poly (ethylene) imine.
  • the one or more polymers contain the buffering component, degradable component, hydrophilic component, cleavable bond component or some combination thereof.
  • the nanoparticles or some portion thereof are degradable. In other embodiments, the lipids and/or polymers of the nanoparticles are degradable.
  • any of these delivery systems of the present disclosure can comprise a buffering component.
  • any of the of the present disclosure can comprise a buffering component and a degradable component.
  • any of the of the present disclosure can comprise a buffering component and a hydrophilic component.
  • any of the of the present disclosure can comprise a buffering component and a cleavable bond component.
  • any of the of the present disclosure can comprise a buffering component, a degradable component and a hydrophilic component.
  • any of the of the present disclosure can comprise a buffering component, a degradable component and a cleavable bond component.
  • any of the of the present disclosure can comprise a buffering component, a hydrophilic component and a cleavable bond component.
  • any of the of the present disclosure can comprise a buffering component, a degradable component, a hydrophilic component and a cleavable bond component.
  • the particle is composed of one or more polymers that contain any of the aforementioned combinations of components.
  • the delivery system comprises a ligand-conjugate delivery system.
  • the ligand-conjugate delivery system comprises one or more of an antibody, a peptide, a sugar moiety, lipid or a combination thereof
  • the isolated oligonucleotide of the present disclosure targeting an INHBE mRNA is conjugated to, complexed to, or encapsulated by the one or more lipids or polymers of the delivery system.
  • the isolated oligonucleotide of the present disclosure targeting an INHBE mRNA e.g., siRNA or dsRNA
  • the isolated oligonucleotide of the present disclosure targeting an INHBE mRNA can be incorporated into the lipid or polymer-based shell of the delivery system, for example via intercalation.
  • the isolated oligonucleotide of the present disclosure targeting an INHBE mRNA e.g., siRNA or dsRNA
  • the isolated oligonucleotide of the present disclosure targeting an INHBE mRNA e.g., siRNA or dsRNA
  • the ligand conjugate delivery system further comprises a targeting agent.
  • the targeting agent comprises a peptide ligand, a nucleotide ligand, a polysaccharide ligand, a fatty acid ligand, a lipid ligand, a small molecule ligand, an antibody, an antibody fragment, an antibody mimetic or an antibody mimetic fragment.
  • the isolated oligonucleotide disclosed herein may further comprise a ligand that facilitates delivery or uptake of the isolated oligonucleotide to a particular tissue or cell, such as a liver cell.
  • the ligand targets delivery of the RNAi construct to hepatocytes.
  • the ligand may comprise galactose, galactosamine or N-acetyl-galactosamine (GalNAc) .
  • the ligand comprises a multivalent galactose or multivalent GalNAc moiety, such as a trivalent or tetravalent galactose or GalNAc moiety.
  • the ligand can be covalently attached to the 5 'or 3' end of the sense strand of the RNAi construct, optionally via a linker.
  • the targeting agent comprises a binding partner for a cell surface protein that is upregulated or overexpressed or normally expressed in a target cell encoding INHBE mRNA and expressing INHBE protein, e.g., Activin E.
  • the binding partner can be a transmembrane peptidoglycan expressed on the surface of many types of such cells. Targeting of cell surface protein by the delivery system of the present disclosure thus provides superior delivery and specificity of the compositions of the disclosure to target cells.
  • the target cell can be any one of an intestinal cell, an arterial cell, a cell of the cardiovascular system, a hepatocyte, a pancreatic cell or a combination thereof.
  • the delivery system of the present disclosure comprises a polymer based delivery system.
  • polymer based delivery system comprises a blending polymer.
  • the blending polymer is a copolymer comprising a degradable component and hydrophilic component.
  • the degradable component of the blending polymer is a polyester, poly (ortho ester) , poly (ethylene imine) , poly (caprolactone) , polyanhydride, poly (acrylic acid) , polyglycolide or poly (urethane) .
  • the degradable component of the blending polymer is poly (lactic acid) (PLA) or poly (lactic-co-glycolic acid) (PLGA) .
  • the hydrophilic component of the blending polymer is a polyalkylene glycol or a polyalkylene oxide.
  • the polyalkylene glycol is polyethylene glycol (PEG) .
  • the polyalkylene oxide is polyethylene oxide (PEO) .
  • the delivery system of the present disclosure is a polymer-based nanoparticle.
  • Polymer based nanoparticles comprise one or more polymers.
  • the one or more polymers comprise a polyester, poly (ortho ester) , poly (ethylene imine) , poly (caprolactone) , polyanhydride, poly (acrylic acid) , polyglycolide or poly (urethane) .
  • the one or more polymers comprise poly (lactic acid) (PLA) or poly (lactic-co-glycolic acid) (PLGA) .
  • the one or more polymers comprise poly (lactic-co-glycolic acid) (PLGA) .
  • the one or more polymers comprise poly (lactic acid) (PLA) .
  • the one or more polymers comprise polyalkylene glycol or a polyalkylene oxide.
  • the polyalkylene glycol is polyethylene glycol (PEG) or the polyalkylene oxide is polyethylene oxide (PEO) .
  • the polymer-based nanoparticle comprises poly (lactic-co-glycolic acid) PLGA polymers.
  • the PLGA nanoparticle further comprises a targeting agent, as described herein.
  • the delivery system of the present disclosure is a nanoparticle of average characteristic dimension of less than about 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, 180 nm, 150 nm, 120 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm or 20 nm.
  • the nanoparticle has an average characteristic dimension of 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 150 nm, 180 nm, 200 nm, 250 nm or 300 nm.
  • the nanoparticle has an average characteristic dimension of 10-500 nm, 10-400 nm, 10-300 nm, 10-250 nm, 10-200 nm, 10-150 nm, 10-100 nm, 10-75 nm, 10-50 nm, 50-500 nm, 50-400 nm, 50-300 nm, 50-200 nm, 50-150 nm, 50-100 nm, 50-75 nm, 100-500 nm, 100-400 nm, 100-300 nm, 100-250 nm, 100-200 nm, 100-150 nm, 150-500 nm, 150-400 nm, 150-300 nm, 150-250 nm, 150-200 nm, 200-500 nm, 200-400 nm, 200-300 nm, 200-250 nm, 200-500 nm, 200-400 nm, 200-300 nm, 200-250 nm, 200-500 nm, 200
  • the delivery system of the present disclosure is administered with one or more additional therapeutic agents.
  • the additional therapeutic agents can be a steroid, an anti-inflammatory agent, an antibody, a fusion protein, a small molecule, or combination thereof.
  • the additional therapeutic agent is incorporated into a delivery system of the present disclosure comprising at least one isolated oligonucleotide targeting INHBE disclosed herein.
  • the additional therapeutic agent is conjugated to, complexed to, or encapsulated by the one or more lipids or polymers of the delivery system. Additional therapeutic agents can be encapsulated in the hollow core of delivery system.
  • additional therapeutic agents can be incorporated into the lipid or polymer-based shell of the delivery system, for example via intercalation.
  • additional therapeutic agents can be attached to the surface of the delivery system.
  • the additional therapeutic agents are conjugated to one or more lipids or polymers of the delivery system, e.g. via covalent attachment.
  • the additional therapeutic agent and the delivery system comprising at least one isolated oligonucleotide targeting INHBE disclosed herein are formulated in the same composition.
  • the delivery system comprising at least one isolated oligonucleotide of the present disclosure targeting INHBE and the additional therapeutic agent can be formulated in the same pharmaceutical composition.
  • compositions of the disclosure can optionally comprise therapeutic agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.
  • the pharmaceutical composition comprises a therapeutic agent, such as a chemotherapeutic agent.
  • the therapeutic agent is formulated in the delivery system comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE of the present disclosure.
  • an additional therapeutic agent is not formulated in the delivery system comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE of the present disclosure, but both the delivery system and the therapeutic agent are formulated in the same pharmaceutical composition.
  • an additional therapeutic agent is not formulated in the delivery system comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE of the present disclosure, and the delivery system and the therapeutic agent are formulated in separate pharmaceutical compositions.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • a “pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient.
  • a pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation) , transdermal (topical) , intraperitoneal (into the body cavity) and transmucosal administration.
  • compositions containing the nanoparticles described herein may be manufactured in a manner that is generally known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and/or auxiliaries that facilitate processing of the active agents into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required nanoparticle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol and sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Oral compositions generally include an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active age can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the agents in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or agents of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the agents are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • pharmaceutically acceptable salts refer to derivatives of the compounds of the present disclosure wherein the parent compound is modified by making acid or base salts thereof.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • compositions of the disclosure can be found in Remington: the Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, PA (1995) .
  • oligonucleotides of e.g., dsRNAs or siRNAs
  • delivery systems comprising same.
  • the one or more oligonucleotides of (e.g., dsRNAs or siRNAs) targeting INHBE of the present disclosure may be generated exogenously by chemical synthesis, by in vitro transcription, or by cleavage of longer double-stranded RNA with Dicer or another appropriate nuclease with similar activity.
  • Chemically synthesized siRNAs produced from protected ribonucleoside phosphoramidites using a conventional DNA/RNA synthesizer, may be obtained from commercial suppliers.
  • the siRNAs can be purified by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof, for example. Alternatively, siRNAs may be used with little if any purification to avoid losses due to sample processing.
  • the one or more oligonucleotides of (e.g., dsRNAs or siRNAs) targeting INHBE of the present disclosure can be incorporated in a delivery system of the present disclosure (e.g., a nanoparticle) .
  • Sterile injectable solutions comprising a delivery system of the disclosure can be prepared by incorporating the one or more isolated oligonucleotides (e.g. dsRNA and siRNA) targeting INHBE disclosed herein, in the delivery systems (e.g. nanoparticle) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Alternatively, or in addition, sterilization can be achieved through other means such as radiation or gas. Generally, dispersions are prepared by incorporating the delivery particles into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the present disclosure also provides a method of inhibiting or downregulating the expression or level of INHBE in a subject in need thereof, wherein the method comprises administering to the subject an effective amount at least one isolated oligonucleotide disclosed herein, at least one vector disclosed herein, at least one delivery system disclosed herein, or at least one pharmaceutical composition disclosed herein.
  • the present disclosure also provides a method of treating or preventing a disease or disorder associated with aberrant or increased expression or activity of INHBE or a disease or disorder where INHBE plays a role in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of at least one isolated oligonucleotide disclosed herein, at least one vector disclosed herein, at least one delivery system disclosed herein, or at least one pharmaceutical composition disclosed herein.
  • the present disclosure also provides at least one isolated oligonucleotide disclosed herein, a vector of the of the present disclosure encoding at least one isolated oligonucleotide disclosed herein, a delivery system of the present disclosure, or a pharmaceutical composition of the present disclosure, for use in treatment or prevention of a disease or disorder associated with aberrant or increased expression or activity of INHBE or a disease or disorder where INHBE plays a role, in a subject in need thereof.
  • the present disclosure also provides use of at least one isolated oligonucleotide disclosed herein, a vector of the of the present disclosure encoding at least one isolated oligonucleotide disclosed herein, a delivery system of the present disclosure, or a pharmaceutical composition of the present disclosure, in the manufacture of a medicament for treatment or prevention of a disease or disorder associated with aberrant or increased expression or activity of INHBE or a disease or disorder where INHBE plays a role in a subject in need thereof.
  • oligonucleotides e.g., dsRNA or siRNA
  • the one or more oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE as described herein can reduce or inhibit INHBE activity through the RNAi pathway.
  • the cell can be in vitro, in vivo or ex vivo.
  • the cell can be from a cell line, or in vivo in a subject in need thereof.
  • the one or more oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE as described herein are capable of inducing RNAi-mediated degradation of an INHBE mRNA in a cell of a subject.
  • dsRNA or siRNA of the present disclosure or a nucleic acid molecule encoding a dsRNA or siRNA of this disclosure is delivered to a cell in order to inhibit or alter or modify expression of a target gene.
  • the dsRNA may be administered in a number of ways including, but not limited to, direct introduction into a cell (i.e., intracellularly) and/or extracellular introduction into a cavity, interstitial space, or into the circulation of the organism.
  • “Introducing” in the context of a cell or organism means presenting the nucleic acid molecule to the organism and/or cell in such a manner that the nucleic acid molecule gains access to the interior of a cell. Where more than one nucleic acid molecule is to be introduced these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, these polynucleotides can be introduced into cells in a single transformation event or in separate transformation events. Thus, the term “transformation” as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient.
  • inhibitor or “reduce” or grammatical variations thereof, as used herein, refer to a decrease or diminishment in the specified level or activity of at least about 5%, about 10%, about 15%, about 25%, about 35%, about 40%, about 50%, about 60%, about 75%, about 80%, about 90%, about 95%or more. In some embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10%or even 5%) .
  • IC50 refers to the concentration of an agent where cell viability is reduced by half.
  • the IC 50 is thus a measure of the effectiveness of an agent in inhibiting a biological process.
  • cell lines are cultured using standard techniques, treated with any of the one or more oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE as described herein, and the IC 50 value of the oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE is calculated after 24, 48 and/or 72 hours to determine its effectiveness in downregulating or inhibiting the level of INHBE mRNA or protein to 50%, as compared to the level of INHBE mRNA or protein in an untreated cell or in the same cell before initiation of treatment with the isolated oligonucleotide.
  • oligonucleotides e.g., dsRNA or siRNA
  • INHBE mRNA and/or protein expression can be used to characterize gene silencing, and to determine the effectiveness of the compositions described herein.
  • Expression of INHBE may be evaluated by any technique known in the art. Examples thereof include immunoprecipitations methods, utilizing INHBE antibodies in assays such as ELISAs, western blotting, or immunohistochemistry to visualize INHBE protein (e.g., Activin E) expression in cells, or flow cytometry.
  • INHBE protein e.g., Activin E
  • Additional methods include various hybridization methods utilizing a nucleic acid that specifically hybridizes with a nucleic acid encoding INHBE or a unique fragment thereof, or a transcription product (e.g., mRNA) or splicing product of said nucleic acid, northern blotting methods, Southern blotting methods, and various PCR-based methods such as RT-PCR, qPCR or digital droplet PCR.
  • INHBE mRNA expression may additionally be assessed using high throughput sequencing techniques.
  • Methods of assaying the effect of individual isolated oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE include transfecting representative cell lines with isolated oligonucleotides and measuring viability.
  • cells from representative cell lines can be transfected using methods known in the art, such as the RNAiMAX Lipofectamine kit (Invitrogen) and cultured using any suitable technique known in the art.
  • additional therapeutic agents as described herein can be added at variable concentrations to cell culture media following transfection.
  • cell viability can be measured using methods such as Cell Titer Glo 2.0 (Promega) to determine cell viability, and/or INHBE mRNA and protein levels can be assessed using the methods described herein.
  • the method comprises administering the at least one isolated oligonucleotide, the vector, the delivery system, or the pharmaceutical composition, in combination with at least a second therapeutic agent.
  • the second therapeutic agent is an antibody, a small molecule drug, a peptide, a nucleotide molecule, or a combination thereof.
  • the second therapeutic agent is an isolated oligonucleotide of the present disclosure.
  • the present disclosure also provides a method of inhibiting or downregulating the expression or level of INHBE in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a first and at least a second oligonucleotides disclosed herein, wherein the first and at least second oligonucleotides comprise different sequences.
  • the first and at least second oligonucleotides are administered simultaneously.
  • the first and at least second oligonucleotides are administered sequentially.
  • the subject is a human. In some embodiments of the methods of inhibiting or downregulating INHBE expression or activity in a cell of the present disclosure, the subject experiences symptoms of or suffers from an INHBE-associated condition.
  • the subject is a human.
  • the disease or disorder is an INHBE-associated condition.
  • the subject is a human.
  • the disease or disorder is an INHBE-associated condition.
  • the subject is a human. In some embodiments, the subject is not a human. In some embodiments, the disease or disorder is an INHBE-associated condition.
  • INHBE-associated condition is intended to include any condition in which decreasing the expression of INHBE is beneficial. Such a condition may be caused, for example, by excessive production of INHBE, INHBE gene mutations that increase INHBE levels.
  • An INHBE-associated condition includes but is not limited to a metabolic disease or metabolic syndrome.
  • Exemplary metabolic disease include but are not limited to diabetes, type I diabetes, type II diabetes, galactosemia, polycystic ovary syndrome, stroke, muscle wasting or atrophy hereditary fructose intolerance, fructose 1, 6-diphosphatase deficiency, glycogen storage disorders, congenital disorders of glycosylation, insulin resistance, insulin insufficiency, hyperinsulinemia, impaired glucose tolerance (IGT) , abnormal glycogen metabolism; disorders of amino acid metabolism, e.g., maple syrup urine disease (MSUD) , or homocystinuria; disorder of organic acid metabolism, e.g., methylmalonic aciduria, 3-methylglutaconic aciduria -Barth syndrome, glutaric aciduria or 2-hydroxyglutaric aciduria –D and L forms; disorders of fatty acid beta-oxidation, e.g., medium-chain acyl-CoA dehydrogenase deficiency (MCAD) , long-chain 3-hydroxyacyl-
  • metabolic disorders are associated with body fat distribution and include, but are not limited to metabolic syndrome, type 2 diabetes, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C) , apolipoprotein B or other lipid fractions) , obesity (particularly abdominal obesity) , lipodystrophy (such as an inability to deposit fat in adipose depots regionally (partial lipodystrophy) or in the whole body (lipoatrophy) ) , insulin resistance or higher or altered insulin levels at fasting or during a metabolic challenge, liver fat deposition or fatty liver disease and their complications (such as, for example, cirrhosis, fibrosis, or inflammation of the liver) , nonalcoholic steatohepatitis, hepatic steatosis, inflammatory and fibrotic liver diseases, such as nonalcoholic steatohepatitis
  • Exemplary metabolic syndrome includes but is not limited to a clustering of components that reflect overnutrition, sedentary lifestyles, genetic factors, increasing age, and resultant excess adiposity.
  • Metabolic syndrome includes the clustering of abdominal obesity, insulin resistance, dyslipidemia, and elevated blood pressure and is associated with other comorbidities including the prothrombotic state, proinflammatory state, nonalcoholic fatty liver disease, and reproductive disorders.
  • the prevalence of the metabolic syndrome has increased to epidemic proportions not only in the United States and the remainder of the urbanized world but also in developing nations. Metabolic syndrome is associated with an approximate doubling of cardiovascular disease risk and a 5-fold increased risk for incident type 2 diabetes mellitus.
  • Nanoparticles comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE mRNA of the present disclosure can be administered to a subject by many of the well-known methods currently used for therapeutic treatment.
  • a compositions comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE of the present disclosure may be injected directly into cells, injected into the blood stream or body cavities, taken orally or applied through the skin with patches.
  • the dose chosen should be sufficient to constitute effective treatment but not so high as to cause unacceptable side effects.
  • the state of the disease condition and the health of the patient should preferably be closely monitored during and for a reasonable period after treatment.
  • compositions comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE mRNA of the present disclosure can be administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperintoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and parenterally.
  • the parenteral administration comprises intramuscular, intraperitoneal, subcutaneous or intravenous administration.
  • compositions of the disclosure may be administered parenterally.
  • Systemic administration of compositions comprising nanoparticles of the disclosure can also be by intravenous, transmucosal, subcutaneous, intraperitoneal, intramuscular or transdermal means.
  • compositions comprising nanoparticles may be administered by injection or by infusion.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the dosages of the pharmaceutical compositions used in accordance with the disclosure vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage.
  • the dose should be sufficient to result in slowing, and preferably regressing or treatment of the condition or symptom associated with expression or activity of INHBE. Dosages may vary depending on the age and size of the subject and the type and severity of the disease or disorder associated with INHBE expression.
  • an effective amount or “therapeutically effective amount” refers to an amount of a pharmaceutical agent to treat, ameliorate, inhibit, downregulate or control the expression of INHBE or symptoms associated with aberrant or abnormal expression of INHBE in a subject, or to exhibit a detectable therapeutic or inhibitory effect in a subject.
  • the effect can be detected by any assay method known in the art.
  • the precise effective amount for a subject will depend upon the subject’s body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration.
  • Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
  • the therapeutically effective amount can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration.
  • a standard xenograft or patient derived xenograft mouse model can be used to determine the effectiveness of the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE mRNA of the present disclosure.
  • Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., the maximum tolerated dose and no observable adverse effect dose. Pharmaceutical compositions that exhibit large therapeutic windows are preferred. The dosage may vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active agent (s) or to maintain the desired effect.
  • Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination (s) , reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • the dosage of nanoparticles comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE mRNA of the present disclosure depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Wide variations in the needed dosage are to be expected in view of the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection (e.g., 2-, 3-, 4-, 6-, 8-, 10-; 20-, 50-, 100-, 150-, or more fold) .
  • intravenous injection e.g., 2-, 3-, 4-, 6-, 8-, 10-; 20-, 50-, 100-, 150-, or more fold
  • Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art.
  • Administrations can be single or multiple.
  • Encapsulation of the inhibitor in a suitable delivery vehicle e.g., capsules or implantable devices
  • a therapeutically effective dose of the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE mRNA of the present disclosure can optionally be combined with approved amounts of therapeutic agents, and described herein.
  • kits comprising at least one isolated oligonucleotide disclosed herein, a vector of the present disclosure encoding an isolated oligonucleotide disclosed herein, a delivery system of the present disclosure, or a pharmaceutical composition of the present disclosure.
  • the vector encodes one isolated oligonucleotide disclosed herein. In some embodiments, the vector encodes more than one isolated oligonucleotide disclosed herein.
  • kits are for use in the treatment of diseases related to abnormal or aberrant expression of INHBE.
  • the kits are for use in downregulating or inhibiting expression of INHBE partially or completely.
  • the kit is for use in the treatment of disease or downregulating or inhibiting expression of INHBE in a mammal.
  • the mammal is a human, a mouse, a rat, a rabbit, a pig, a bovine, a canine, a feline, an ungulate, an ape, a monkey or an equine species.
  • the mammal is a human
  • the kit comprises nanoparticles.
  • Nanoparticles comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting INHBE mRNA of the present disclosure, can be lyophilized before being packaged in the kit, or can be provided in solution with a pharmaceutically acceptable carrier, diluent of excipient.
  • the kit comprises a therapeutically effective amount of a composition comprising the delivery system of the present disclosure comprising one or more of the isolated oligonucleotides of the present disclosure targeting INHBE (dsRNA or siRNA) , and instructions for use of the same.
  • the kit further comprises at least one additional therapeutic agents, as described herein.
  • Articles of manufacture of the present disclosure include, but are not limited to, instructions for use of the kit in treating diseases related to abnormal or aberrant expression of INHBE.
  • kits further comprise instructions for administering the isolated oligonucleotides, the vector, the delivery systems and the pharmaceutical compositions of the disclosure.
  • EXAMPLE 1 Design and testing of siRNA compounds against human INHBE mRNA.
  • siRNAs compounds against human INHBE transcript were designed (see Table 2) .
  • Oligonucleotides were prepared by solid-phase synthesis according to standard protocols. Briefly, oligonucleotide synthesis was conducted on a solid support to incorporate each nucleoside phosphoramidites from 3’-end to 5’-end to prepare oligo single strands. ETT or BTT was used as an activator for the coupling reaction. Iodine in water/pyridine/THF was used to oxidize phosphite-triester (P (III) ) to afford phosphate backbones and DDTT was used for the preparation of phosphorothioate linkages. Aqueous ammonium was used to cleave oligos from solid support and to remove protecting groups globally.
  • the oligonucleotide crude was then concentrated by Genevac and purified by AEX-HPLC.
  • the pure fractions were combined and concentrated, and their purity was analyzed by LC-MS.
  • the oligonucleotides were then dialyzed against water using MidiTrap G-25 column, concentrated, and their OD amounts were measured.
  • siRNA duplexes To prepare siRNA duplexes, the sense and antisense strands were annealed at 95 °C for 10 min, based on equal molar amounts, and cooled down to room temperature. The duplex purity was determined by AEX-HPLC, and the solutions were lyophilized to afford the desired siRNA duplex powder.
  • 6-to 8-week-old female BALB/c mice were administered subcutaneously with a single dose of 0.5 mg/kg of selected siRNA compounds for one dose screening (Table 7) , or with a single dose of 0.5, 1, or 2 mg/kg of selected siRNA compounds for dose-response study (Table 8) .
  • animals were transiently transfected in vivo with INHBE-expressing plasmid by hydrodynamic tail vein injection (HDI) .
  • HDI hydrodynamic tail vein injection
  • animals were sacrificed and liver tissues were isolated and stored in RNA later at 4°C. RNA in the liver were extracted using MNTR/FX96 (LR) kit (GeneOn BioTech) .
  • cDNA was generated from the isolated RNA using High-Capacity cDNA Reverse Transcription Kits with RNase Inhibitor and a TaqMan RT-qPCR gene expression assay was conducted to analyze the compound potency in silencing INHBE mRNA. Data is presented as %of INHBE mRNA remaining relative to mock transfection when normalized to NeoR mRNA levels (Mean, +/-SD) . The results of the experiments are shown in Tables 7 and 8. The tested compounds were able to reduce the level of human INHBE mRNA.
  • EXAMPLE 2 Evaluating efficacy of siRNA compounds in primary human and non-human primate hepatocytes
  • siRNA compounds from Table 9 were further evaluated for their efficacy in primary human hepatocytes (PHHs) and primary cynomolgus hepatocytes (PCHs) .
  • siRNA compound duplexes 254 (FIG. 1A) , 279 (FIG. 1B) , 286 (FIG. 1C) , 296 (FIG. 1D) , 297 (FIG. 1E) , and 298 (FIG. 1F) were evaluated for their efficacy in PHHs.
  • PHHs were seeded into 96-well plates at the density of 54,000 cells per well.
  • the siRNA compounds were diluted to the desired concentration (100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM, 0.0001 nM and 0.00001 nM) with PBS.
  • siRNA compounds were added at the same time of seeding cells by free uptake.
  • RNA isolation After 48 hours incubation, the culture medium was removed, and the cells were harvested for RNA isolation. cDNA was generated from the isolated RNA and TaqMan RT-qPCR gene expression assay was conducted to analyze the compound potency in silencing human INHBE mRNA. Data are presented as %of INHBE mRNA remaining relative to mock control when normalized to GAPDH mRNA levels (Mean, +/-SD) . Half maximal inhibitory concentration (IC50) was determined based on the curves generated.
  • siRNA compound duplexes 254 (FIG. 2A) , 296 (FIG. 2B) , and 297 (FIG. 2C) were evaluated for their efficacy in PCHs.
  • PCHs were seeded into 96-well plates at the density of 54,000 cells per well.
  • the siRNA compounds were diluted to the desired concentrations (100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM, 0.0001 nM and 0.00001 nM) with PBS.
  • the siRNA compounds were added at the same time of seeding cells by free uptake. After 48 hours incubation, the culture medium was removed, and the cells were harvested for RNA isolation.
  • cDNA was generated from the isolated RNA and TaqMan RT-qPCR gene expression assay was conducted to analyze the compound potency in silencing Macaca fascicularis INHBE mRNA. Data are presented as %of Macaca fascicularis INHBE mRNA remaining relative to mock control when normalized to Macaca fascicularis GAPDH mRNA levels (Mean, +/-SD) . Half maximal inhibitory concentration (IC50) was determined based on the curves generated.
  • siRNA compound duplex 297 from Table 9 was evaluated for its potency in mice.
  • mice Transgenic 8-9 weeks old male mice expressing human INHBE but not mouse INHBE were used for this study. Animals were randomly divided into four groups according to their body weight levels. siRNA compound duplex 297 was administered at 0.1 mg/kg, 1.0 mg/kg, or 10 mg/kg by single subcutaneous injection. After administration, liver samples were collected from 3 animals in each group at day 6, day 20, and day 34 post dose. The collected liver tissues were first snap-frozen in liquid nitrogen and then transferred to -80°C for storage. RNA in the liver was extracted using MNTR/FX96 (LR) kit. cDNA was generated from the isolated RNA and TaqMan RT-qPCR gene expression assay was conducted to analyze human INHBE mRNA level. Data are presented as %of human INHBE mRNA remaining relative to baseline when normalized to Gapdh mRNA levels (Mean ⁇ SD) .
  • FIG. 3 is a graph showing the in vivo efficacy of siRNA compound duplex 297 in silencing INHBE mRNA of human INHBE knock-in mice.
  • these results indicate that the siRNA compound efficacy is dose-dependent, with higher doses showing more long-term effects.
  • these results suggest that low doses of siRNA duplex, such as 0.1 mg/kg and 1.0 mg/kg in mice, can be administered in multiple doses.
  • these results suggest that high doses of siRNA duplex, such as 10 mg/kg in mice, can be administered as a single dose.
  • siRNA compound duplexes 247, 252, 254, 294, and 295 from Table 9 were evaluated for their potency in Macaca fascicularis.
  • INHBE knockdown was evaluated.
  • Nonterminal studies were implemented using male obesity cynomolgus monkeys, which have been on high fat/sugar diet over 12 months by the time of the studies. Animals were divided into study groups randomly based on body weight, with 3-4 monkeys per group.
  • a liver biopsy was performed 8 or 7 days prior to dosing (Day -8 or Day -7) to establish baseline INHBE mRNA expression levels. Blood samples were collected at the same pre-dose timepoints (Day -8/-7 and Day 0) to determine baseline plasma Activin E protein levels. Animals received a single 3 mg/kg subcutaneous dose on Day 0, 7–8 days after the initial liver biopsy.
  • Follow-up liver biopsies were conducted on Days 14, 28, 42, 56, 70, 84, and 112 post-dose. Liver tissues were analyzed via RT-qPCR to assess residual INHBE mRNA (FIG. 4) .
  • siRNA compound duplexes 247, 252, 254, 294, and 295 showed reduced levels of INHBE mRNA at Day 14, and gradual increases by Day 112 (FIG. 4) .
  • Liver samples from monkeys administered siRNA compound duplex 254 had the lowest level of mRNA by Day 112 (FIG. 4) .
  • siRNA compound duplexes 296, 297, and 298 from Table 9 were evaluated for their potency in Macaca fascicularis.
  • INHBE knockdown and Activin E protein (encoded by the gene INHBE) suppression were evaluated.
  • Nonterminal studies were implemented using male obesity cynomolgus monkeys, which have been on high fat/sugar diet over 12 months by the time of the studies. Animals were divided into study groups randomly based on body weight, with 3-4 monkeys per group. A liver biopsy was performed 8 or 7 days prior to dosing (Day -8 or Day -7) to establish baseline INHBE mRNA expression levels. Blood samples were collected at the same pre-dose timepoints (Day -8/-7) and Day 0 (predose) to determine baseline plasma Activin E protein levels. Animals received a single 3 mg/kg subcutaneous dose on Day 0, 7–8 days after the initial liver biopsy. Following-up liver biopsies were conducted on Days 14, 28, 56, and 84 post-dose, while plasma samples were collected on Days 7, 14, 28, 56, and 84 post-dose.
  • siRNA compound duplexes 296, 297, and 298 also showed reduction of INHBE mRNA at Day 14 (FIG. 5) . These three duplexes further decreased INHBE mRNA levels by Day 28 (FIG. 5) . INHBE mRNA levels remained constant from Day 28 until Day 84 in liver samples from monkeys administered with siRNA compound duplexes 297 and 298. Activin E protein levels in blood serum samples from monkeys administered with siRNA compound duplex 297 showed sustained decrease throughout the study (FIG. 6) , consistent with the reduced mRNA levels observed (FIG. 5) .
  • siRNA compound duplexes can be reduced in vivo, as demonstrated in non-human primates with a dose of 3 mg/kg.
  • the reduced mRNA levels translate to a reduction in protein expression.
  • these data demonstrate the potential for siRNA compound duplexes disclosed herein for reducing INHBE expression for preventing and/or treating conditions or diseases associated with INHBE.

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Abstract

Cette divulgation concerne des oligonucléotides isolés comprenant des régions duplex ciblant l'ARNm d'INHBE humain, ainsi que des systèmes d'apport, des kits et des compositions les comprenant, et des méthodes d'utilisation de ceux-ci pour inhiber ou réguler à la baisse l'expression génique d'INHBE.
PCT/CN2025/092633 2024-04-30 2025-04-30 Petit arn interférant ciblant inhbe et utilisations associées Pending WO2025228441A2 (fr)

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Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
WO1989002439A1 (fr) 1987-09-21 1989-03-23 Ml Technology Ventures, L.P. Reactifs non nucleotides de liaison pour sondes de nucleotide
WO1995006731A2 (fr) 1993-09-02 1995-03-09 Ribozyme Pharmaceuticals, Inc. Acide nucleique enzymatique contenant un non-nucleotide
WO1995011910A1 (fr) 1993-10-27 1995-05-04 Ribozyme Pharmaceuticals, Inc. 2'-amido et 2'-peptido oligonucleotides modifies
WO1999014226A2 (fr) 1997-09-12 1999-03-25 Exiqon A/S Analogues d'oligonucleotides
WO2000056746A2 (fr) 1999-03-24 2000-09-28 Exiqon A/S Synthese perfectionnee de [2.2.1]bicyclo-nucleosides
WO2000056748A1 (fr) 1999-03-18 2000-09-28 Exiqon A/S Analogues de xylo-lna
WO2000066604A2 (fr) 1999-05-04 2000-11-09 Exiqon A/S Analogues de l-ribo-lna
US20050244858A1 (en) 2004-03-15 2005-11-03 City Of Hope Methods and compositions for the specific inhibition of gene expression by double-stranded RNA
US20090023673A1 (en) 2006-10-03 2009-01-22 Muthiah Manoharan Lipid containing formulations
WO2009082607A2 (fr) 2007-12-04 2009-07-02 Alnylam Pharmaceuticals, Inc. Lipides de ciblage
WO2012089352A1 (fr) 2010-12-29 2012-07-05 F. Hoffmann-La Roche Ag Conjugués de petites molécules pour l'administration intracellulaire d'acides nucléiques
US20130158824A1 (en) 2011-12-16 2013-06-20 GM Global Technology Operations LLC Limiting branch pressure to a solenoid valve in a fluid circuit
WO2015006740A2 (fr) 2013-07-11 2015-01-15 Alnylam Pharmaceuticals, Inc. Conjugués ligands d'oligonucléotides et procédé pour leur préparation
WO2016100401A1 (fr) 2014-12-15 2016-06-23 Dicerna Pharmaceuticals, Inc. Acides nucléiques double brin modifiés par un ligand
WO2017214112A1 (fr) 2016-06-06 2017-12-14 Arrowhead Pharmaceuticals, Inc. Nucléotides modifiés 5 '-cyclo-phosphonate
WO2018039364A1 (fr) 2016-08-23 2018-03-01 Dicerna Pharmaceuticals, Inc. Compositions comprenant des oligonucléotides modifiés de manière réversible et leurs utilisations
WO2018045317A1 (fr) 2016-09-02 2018-03-08 Dicerna Pharmaceuticals, Inc. Analogues de 4'-phosphate et oligonucléotides comprenant ceux-ci

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20230119130A (ko) * 2020-12-14 2023-08-16 리제너론 파마슈티칼스 인코포레이티드 인히빈 서브유닛 베타 e(inhbe) 억제제로 대사 장애 및 심혈관 질환을 치료하는 방법
KR20240036041A (ko) * 2021-07-21 2024-03-19 알닐람 파마슈티칼스 인코포레이티드 대사 장애-연관 표적 유전자 iRNA 조성물 및 이의 사용 방법
CN118159654A (zh) * 2021-09-20 2024-06-07 阿尔尼拉姆医药品有限公司 抑制素亚基βE（INHBE）调节剂组合物及其使用方法

Patent Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
WO1989002439A1 (fr) 1987-09-21 1989-03-23 Ml Technology Ventures, L.P. Reactifs non nucleotides de liaison pour sondes de nucleotide
WO1995006731A2 (fr) 1993-09-02 1995-03-09 Ribozyme Pharmaceuticals, Inc. Acide nucleique enzymatique contenant un non-nucleotide
WO1995011910A1 (fr) 1993-10-27 1995-05-04 Ribozyme Pharmaceuticals, Inc. 2'-amido et 2'-peptido oligonucleotides modifies
WO1999014226A2 (fr) 1997-09-12 1999-03-25 Exiqon A/S Analogues d'oligonucleotides
WO2000056748A1 (fr) 1999-03-18 2000-09-28 Exiqon A/S Analogues de xylo-lna
WO2000056746A2 (fr) 1999-03-24 2000-09-28 Exiqon A/S Synthese perfectionnee de [2.2.1]bicyclo-nucleosides
WO2000066604A2 (fr) 1999-05-04 2000-11-09 Exiqon A/S Analogues de l-ribo-lna
US20050244858A1 (en) 2004-03-15 2005-11-03 City Of Hope Methods and compositions for the specific inhibition of gene expression by double-stranded RNA
US20090023673A1 (en) 2006-10-03 2009-01-22 Muthiah Manoharan Lipid containing formulations
WO2009082607A2 (fr) 2007-12-04 2009-07-02 Alnylam Pharmaceuticals, Inc. Lipides de ciblage
US20090239814A1 (en) 2007-12-04 2009-09-24 Alnylam Pharmaceuticals, Inc. Carbohydrate Conjugates as Delivery Agents for Oligonucleotides
US20090247608A1 (en) 2007-12-04 2009-10-01 Alnylam Pharmaceuticals, Inc. Targeting Lipids
US20120136042A1 (en) 2007-12-04 2012-05-31 Alnylam Pharmaceuticals, Inc Carbohydrate conjugates as delivery agents for oligonucleotides
WO2012089352A1 (fr) 2010-12-29 2012-07-05 F. Hoffmann-La Roche Ag Conjugués de petites molécules pour l'administration intracellulaire d'acides nucléiques
US20130158824A1 (en) 2011-12-16 2013-06-20 GM Global Technology Operations LLC Limiting branch pressure to a solenoid valve in a fluid circuit
WO2015006740A2 (fr) 2013-07-11 2015-01-15 Alnylam Pharmaceuticals, Inc. Conjugués ligands d'oligonucléotides et procédé pour leur préparation
WO2016100401A1 (fr) 2014-12-15 2016-06-23 Dicerna Pharmaceuticals, Inc. Acides nucléiques double brin modifiés par un ligand
WO2017214112A1 (fr) 2016-06-06 2017-12-14 Arrowhead Pharmaceuticals, Inc. Nucléotides modifiés 5 '-cyclo-phosphonate
WO2018039364A1 (fr) 2016-08-23 2018-03-01 Dicerna Pharmaceuticals, Inc. Compositions comprenant des oligonucléotides modifiés de manière réversible et leurs utilisations
WO2018045317A1 (fr) 2016-09-02 2018-03-08 Dicerna Pharmaceuticals, Inc. Analogues de 4'-phosphate et oligonucléotides comprenant ceux-ci

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"Guide to Huge Computers", 1994, ACADEMIC PRESS
"Remington: the Science and Practice of Pharmacy", 1995, MACK PUBLISHING CO.
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RES, vol. 25, 1997, pages 3389 - 3402
CARILLO, H.LIPTON, D., APPLIED MATH, vol. 48, 1988, pages 1073
FERENTZ ET AL., J. AM. CHEM. SOC, vol. 113, 1991, pages 4000 - 4002
JAESCHKE ET AL., TETRAHEDRON LETT., vol. 34, 1993, pages 301
KARLIN ET AL., PNAS, vol. 90, 1993, pages 5873 - 5877
SEELA ET AL., NUCLEIC ACIDS RESEARCH, vol. 15, 1987, pages 3113 - 3129
WINKLER, THER. DELIV., vol. 4, no. 7, 2013, pages 791 - 809

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