DOUBLE STRANDED RNA TARGETING 17-BETA HYDROXYSTEROIDDEHYDROGENASE 13 (HSD17B13) AND METHODS OF USE THEREOF

BACKGROUND

17-beta hydroxysteroid dehydrogenase 13 (HSD17B13) is a member of the 17 β-hydroxysteroid dehydrogenases (HSD17Bs) class of enzymes that catalyze the conversion between 17-keto- and 17-hydroxysteroids. The human HSD17B13 gene is located on chromosome 4 (4q22.1), and its expression is highly restricted to liver, particularly hepatocytes, but not to other cell types of liver. The human HSD17B13 gene encodes a 300 amino acid protein, which is localized on lipid droplets and is a novel liver-specific Lipid Droplet (LD)-related protein.

HSD17B13 is a hepatic retinol dehydrogenase associated with histological features of non-alcoholic fatty liver disease. HSD17B13 expression has been shown to be significantly upregulated in non-alcoholic fatty liver patients and promoting lipid accumulation in the liver. HSD17B13 plays an important role as a liver-specific LD protein in regulating liver lipid homeostasis and lipid metabolism and may be a novel target for the treatment of nonalcoholic fatty liver disease (NAFLD) and related liver diseases. NAFLD and related liver diseases like non-alcoholic steatohepatitis (NASH) are common causes of chronic liver disease. Accordingly, there is a need in the art for alternative therapies and combination therapies for subjects having a HSD17B13-associated disease.

SUMMARY

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) 229 to 249; b) 344 to 364; c) 474 to 496; d) 669 to 741; e) 882 to 947; f) 999 to 1032; g) 1101 to 1216; h) 1297 to 1326; and i) 1421 to 1507, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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) 229 to 249; b) 344 to 364; c) 474 to 496; d) 669 to 741; e) 882 to 947; f) 999 to 1032; g) 1101 to 1216; h) 1297 to 1326; and i) 1421 to 1507, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) mRNA sequence according to SEQ ID NO: 1, and the anti-sense strand is substantially complementary to the sense strand such that the sense strand and the anti-sense strand together form a double stranded region.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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) 229 to 249; b) 344 to 364; c) 474 to 496; d) 669 to 741; e) 882 to 947; f) 999 to 1032; g) 1101 to 1216; h) 1297 to 1326; and i) 1421 to 1507, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 927 to 947; b) 1007 to 1032; c) 1194 to 1216; and d) 1421 to 1445, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, from the 5′ end of an HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 927 to 947; b) 1007 to 1032; c) 1194 to 1216; and d) 1421 to 1445, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 344 to 364; b) 476 to 496; c) 669 to 741; d) 882 to 915; e) 999 to 1030; f) 1101 to 1121; g) 1297 to 1326; and h) 1487 to 1507, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 344 to 364; b) 476 to 496; c) 669 to 741; d) 882 to 915; e) 999 to 1030; f) 1101 to 1121; g) 1297 to 1326; and h) 1487 to 1507, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a sequence that substantially identical to a region comprising the sequence between any one of the nucleotide positions selected from: a) 229 to 249; and b) 474 to 494, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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) 229 to 249; and b) 474 to 494, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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) 229 to 249; and b) 474 to 494, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the single stranded RNA molecule of the antisense strand comprises a 3′ overhang. In some embodiments, in the single stranded RNA molecule of the sense strand, the 3′ overhang comprise at least one nucleotide. In some embodiments, in the single stranded RNA molecule of the sense strand, the 3′ overhang comprise two nucleotides.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises an RNA sequence of at least 20 nucleotides in length. In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises an RNA sequence of 20 nucleotides in length.

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises an RNA sequence of at least 22 nucleotides in length. In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises an RNA sequence of 22 nucleotides in length.

In some embodiments of the isolated oligonucleotide of the present disclosure, the double stranded region is between 19 and 21 nucleotides in length. In some embodiments of the isolated oligonucleotide of the present disclosure, the double stranded region is 20 nucleotides in length.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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 Tables 1-4, as described in the detailed description.

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 2-30.

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: 31-60.

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 2-30; and the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 31-60, wherein the anti sense strand and the sense strand sequences have sufficient complementarity to allow formation of a double stranded region between the antisense and the sense strand.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 927 to 947; b) 1007 to 1032; c) 1194 to 1216; and d) 1421 to 1445, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, from the 5′ end of an HSD17B13 mRNA sequence according to SEQ ID NO: 1, the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UUAUUCAUUUCAUUUUGAUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ AAUCAAAAUGAAAUGAAUAA 3′); or ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UAAUGUGAAAUAAAGCUUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CAAAGCUUUAUUUCACAUUA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UGAAAAAAUGUGAAAUAAAGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUUUAUUUCACAUUUUUUCA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UAUCUUAAAGAAAACCUUUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ AAAAGGUUUUCUUUAAGAUA 3′); v) an antisense strand of nucleic acid sequence according to SEQ CD NO: 6 (5′ UAUAUCUUAAAGAAAACCUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ AAGGUUUUCUUUAAGAUAUA 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UUUUCAAAUGCUGAAUCUUAAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ UAAGAUUCAGCAUUUGAAAA 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUCUUUCAAAUGCUGAAUCUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GAUUCAGCAUUUGAAAGAUA 3′); or viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAAUCUUUCAAAUGCUGAAUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ AUUCAGCAUUUGAAAGAUUA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein 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) 229 to 249; and b) 474 to 494, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ UUAUUAAUAUCCCACAGAACCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ GUUCUGUGGGAUAUUAAUAA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ UGAUCCAAAAAUGUCCUAGGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 59 (5′ CCUAGGACAUUUUUGGAUCA 3′); or iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ UGAUCCAAAAAUGUCCUAGGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO:60 (5′ CCUAGGACAUUUUUGIAUCA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand 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) 229 to 249; b) 669 to 689; and c) 1007 to 1027, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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) 229 to 249; b) 669 to 689; and c) 1007 to 1027, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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 anti sense strand together form a double stranded region.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 229 to 249; b) 669 to 689; and c) 1007 to 1027, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is substantially identical to a region between the nucleotide positions 1007 to 1027, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, from the 5′ end of an HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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 the nucleotide positions 1007 to 1027, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between the nucleotide positions a) 1007 to 1027, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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 the nucleotide positions 669 to 689, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between the nucleotide positions 669 to 689, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a sequence that substantially identical to a region between the nucleotide positions 229 to 249, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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 between the nucleotide positions 229 to 249, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a sequence that is identical to a region between the nucleotide positions 229 to 249, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the isolated oligonucleotide is capable of inducing degradation of the HSD17B13 mRNA.

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 3, 12 or 29.

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: 32, 41 or 58.

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 3, 12 or 29; and the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 32, 41 or 58, wherein the antisense strand and the sense strand sequences have sufficient complementarity to allow formation of a double stranded region between the anti sense and the sense strand.

In some embodiments of the isolated oligonucleotide of the present disclosure, the double stranded region comprises an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UAAUGUGAAAUAAAGCUUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CAAAGCUUUAUUUCACAUUA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure, the double stranded region comprises an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UGAGACAUGAGGUUUUGAUACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ UAUCAAAACCUCAUGUCUCA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure, the double stranded region comprises an antisense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ UUAUUAAUAUCCCACAGAACCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ GUUCUGUGGGAUAUUAAUAA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand or the antisense strand or both comprise one or more modified nucleotide(s).

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises a mono methyl protected phosphate mimic (5′-McEP).

In some embodiments of the isolated oligonucleotide of the present disclosure, in the sense strand or the antisense strand or both, a terminal or internal nucleotide is linked to a targeting ligand.

In some embodiments of the isolated oligonucleotide of the present disclosure, the targeting ligand comprises at least one GalNAc Glb moiety.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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)₀(F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁(M)₁(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁5′.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises nucleotides modified with 2′-F modification (“F”), and nucleotides modified with 2′-O-methyl modification (“M”), according to the formula:

5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉3′.

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises any one of: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 440 (5′ [MeEPmUs][fAs][fA][mU][fG][mU][ftr][mA][mA][fA][mU][mA][mA][fA][mG][ft][mU][mU][mU][mGs][mCs][mA] 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 442 (5′ [McEPmUs][fUs][fA][mG][fA][mC][fA][mU][mG][fA][mG][mG][mU][fU][mU][fU][mG][mA][mU][mAs][mCs][mC] 3′); or iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 444 (5′ [McEPmUs][fUs][fA][mU][fU][mA][fA][mU][mA][fU][mC][mC][mC][fA][mC][fA][mG][mA][mA][mCs][mCs][mA] 3′), wherein “m” is a 2′-O-methyl modified nucleotide, “f” is a 2′-F modified nucleotide, “s” is a phosphorothioate internucleotide linkage, “MeEP” is a mono methyl protected phosphate mimic.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises any one of: i) a sense strand of nucleic acid sequence according to SEQ ID NO: 441 (5′ [mCs][mAs][mA][mA][mG][fC][mU][fU][fU][fA][fU][mU][mU][mC][mA][mC][mA][mUs][m Us][mA][Glb][Glb][Glb] 3′); ii) a sense strand of nucleic acid sequence according to SEQ ID NO: 443 (5′ [mUs][mAs][mU][mC][mA][fA][mA][fA][ft][fC][fU][mC][mA][mU][mG][mU][mC][mUs][m Cs][mA][Glb][Glb][Glb] 3′); or iii) a sense strand of nucleic acid sequence according to SEQ ID NO: 445 (5′ [mGs][mUs][mU][mC][mU][fG][mU][fG][fn][M][fA][mU][mA][mU][mU][mA][mA][mUs][m As][mA][Glb][Glb][Glb] 3′), wherein “m” is a 2′-O-methyl modified nucleotide, “f” is a 2′-F modified nucleotide, “s” is a phosphorothioate internucleotide linkage, and “Glb” is a GalNac Glb moiety.

The present disclosure also provides a vector encoding an isolated oligonucleotide disclosed herein.

The present disclosure also provides a delivery system comprising an isolated oligonucleotide or vector disclosed herein.

The present disclosure also provides a pharmaceutical composition comprising an isolated oligonucleotide, vector or delivery system disclosed herein, and a pharmaceutically acceptable carrier, diluent or excipient.

The present disclosure also provides a kit comprising an isolated oligonucleotide, vector, delivery system or a pharmaceutical composition disclosed herein.

The present disclosure also provides a method of inhibiting or downregulating the expression or level of HSD17B13 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 present disclosure also provides a method of treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 or a disease or disorder where HSD17B13 plays a role in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of an isolated oligonucleotide, vector, delivery system or a pharmaceutical composition disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are a western blot and line graph depicting in vivo potency of siRNA compounds listed in Table 4 in reducing cynomolgus HSD17B13 protein in Macaca fascicularis after a 3 mg/kg single subcutaneous dosing. FIG. 1A is a western blot showing HSD17B13 protein levels remaining in cynomolgus livers at two timepoints (D-7, Day −7, 7 days before dosing; D85, Day 85, 85 days after dosing). FIG. 1B is a graph depicting relative quantification of HSD17B13 protein levels remaining in cynomolgus livers over time. Data is presented as HSD17B13 remaining relative to baseline protein level one week before dosing (Day −7) (Mean, +/−SD).

FIG. 2 is a graph depicting the ex vivo potency of siRNA compounds listed in Table 4 in silencing human HSD17B13 mRNA in primary human hepatocytes (PHH) compared to PBS control. Compounds were directly added to the cultured PHH at 2 doses (10 nM and 100 nM). 48 hrs later, the cells were harvested for mRNA analysis through RT-qPCR. Data is presented as % HSD17B13 mRNA remaining when normalized to PBS control (Mean, +/−SD).

FIGS. 3A-3B are a series of graphs depicting in vim potency of si RNA compounds listed in Table 2 with GalNac conjugations in reducing human HSD17B13 mRNA in HDI mouse liver after subcutaneous dosing of siRNA compound at multiple doses, as indicated. FIG. 3A is a graph showing human HSD17B13 mRNA remaining in HDI mouse liver using a single dose of 1 mg/kg. FIG. 3B is a graph showing human HSD17B13 mRNA remaining in HDI mouse liver using a dose of 0.25 mg/kg (squares), 0.5 mg/kg (circles) and 1 mg/kg (triangles). Data is represented as % of human HSD17B13 mRNA remaining relative to PBS groups when normalized to Neomycin-resistant (NeoR) gene mRNA levels (Mean, +/−SD).

DETAILED DESCRIPTION

The present disclosure provides isolated oligonucleotides (oligonucleotide(s)) that form a double stranded region, preferably small interfering RNAs (siRNAs), that can decrease HSD17B13 mRNA expression, in turn leading to a decrease in the degree of HSD17B13 protein expression in target cells. The oligonucleotides disclosed herein can have therapeutic application in regulating the expression of HSD17B13, for treatment of diseases, including but not limited to nonalcoholic fatty liver disease (NAFLD) and related liver diseases. NAFLD and related liver diseases like non-alcoholic steatohepatitis (NASH).

The present disclosure has identified specific regions within the HSD17B13 mRNA, that provide targets for binding double stranded oligonucleotides, e.g., siRNA, leading to reduction in level of expression of the HSD17B13 mRNA.

The HSD17B13 mRNA sequence described herein, is an mRNA sequence of HSD17B13 according to accession no. NM_178135.5:

- 1 acacaaggac tgaaccagaa ggaagaggac agagcaaagc catgaacatc atcctagaaa
- 61 tccttctgct tctgatcacc atcatctact cctacttgga gtcgttggtg aagtttttca
- 121 ttcctcagag gagaaaatct gtggctgggg agattgttct cattactgga gctgggcatg
- 181 gaataggcag gcagactact tatgaatttg caaaacgaca gagcatattg gttctgtggg
- 241 atattaataa gcgcggtgtg gaggaaactg cagctgagtg ccgaaaacta ggcgtcactg
- 301 cgcatgcgta tgtggtagac tgcagcaaca gagaagagat ctatcgctct ctaaatcagg
- 361 tgaagaaaga agtgggtgat gtaacaatcg tggtgaataa tgctgggaca gtatatccag
- 421 ccgatcttct cagcaccaag gatgaagaga ttaccaagac atttgaggtc aacatcctag
- 481 gacatttttg gatcacaaaa gcacttcttc catcgatgat ggagagaaat catggccaca
- 541 tcgtcacagt ggcttcagtg tgcggccacg aagggattcc ttacctcatc ccatattgtt
- 601 ccagcaaatt tgccgctgtt ggctttcaca gaggtctgac atcagaactt caggccttgg
- 661 gaaaaactgg tatcaaaacc tcatgtctct gcccagtttt tgtgaatact gggttcacca
- 721 aaaatccaag cacaagatta tggcctgtat tggagacaga tgaagtcgta agaagtctga
- 781 tagatggaat acttaccaat aagaaaatga tttttgttcc atcgtatatc aatatctttc
- 841 tgagactaca gaagtttctt cctgaacgcg cctcagcgat tttaaatcgt atgcagaata
- 901 ttcaatttga agcagtggtt ggccacaaaa tcaaaatgaa atgaataaat aagctccagc
- 961 cagagatgta tgcatgataa tgatatgaat agtttcgaat caatgctgca aagctttatt
- 1021 tcacattttt tcagtcctga taatattaaa aacattggtt tggcactagc agcagtcaaa
- 1081 cgaacaagat taattacctg tcttcctgtt tctcaagaat atttacgtag tttttcatag
- 1141 gtctgttttt cctttcatgc ctcttaaaaa cttctgtgct tacataaaca tacttaaaag
- 1201 gttttcttta agatatttta tttttccatt taaaggtgga caaaagctac ctccctaaaa
- 1261 gtaaatacaa agagaactta tttacacagg gaaggtttaa gactgttcaa gtagcattcc
- 1321 aatctgtagc catgccacag aatatcaaca agaacacaga atgagtgcac agctaagaga
- 1381 tcaagtttca gcaggcagct ttatctcaac ctggacatat tttaagattc agcatttgaa
- 1441 agatttccct agcctcttcc tttttcatta gcccaaaacg gtgcaactct attctggact
- 1501 ttattacttg attctgtctt ctgtataact ctgaagtcca ccaaaagtgg accctctata
- 1561 tttcctccct ttttatagtc ttataagata cattatgaaa ggtgaccgac tctattttaa
- 1621 atctcagaat tttaagttct agccccatga taaccttttt ctttgtaatt tatgctttca
- 1681 tatatccttg gtcccagaga tgtttagaca attttaggct caaaaattaa agctaacaca
- 1741 ggaaaaggaa ctgtactggc tattacataa gaaacaatgg acccaagaga agaaaaggaa
- 1801 gaaagaaagg ttttttggtt tttgttttgt tttgttttgt tttttgtttt tttgagatgg
- 1861 agtctcactc tttcgcccag gctggagtgc agtggtatga tctcagctca ctgcaagctc
- 1921 cacctcccgg gttcacgcca ttctcctgcc tcagcctcct gagtagctgg gactacaggc
- 1981 gcccgccacc acacccggct aattttttgt attttttgta gagacggggt ttcaccatgt
- 2041 tagccaagat ggtctcgatc tcctgacctc gtgatccacc tgcctcggcc tcccaaagtg
- 2101 ctgggattac gggtgtgagc caccgtgccc agcctttttt tttttaatag aaaaaataat
- 2161 ccgactccca ctacatcaag actaatcttg ttttgtgtgt ttttcacatg tattatagaa
- 2221 tgcttttgca tggactatcc tcttgttttt attaaaaaca aatgattttt ttaaaagtca
- 2281 caaaaacaat tcactaaaaa taaatatgtc attgtgcttt aaaaaaataa cctcttgtag
- 2341 ttataaaata aaacgtttga cttctaaa (SEQ ID NO: 1).

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) 229 to 249; b) 344 to 364; c) 474 to 496; d) 669 to 741; e) 882 to 947; f) 999 to 1032; g) 1101 to 1216; h) 1297 to 1326; and i) 1421 to 1507, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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) 229 to 249; b) 344 to 364; c) 474 to 496; d) 669 to 741; e) 882 to 947; f) 999 to 1032; g) 1101 to 1216; h) 1297 to 1326; and i) 1421 to 1507, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 927 to 947; b) 1007 to 1032; c) 1194 to 1216; and d) 1421 to 1445, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 927 to 947; b) 1007 to 1027; c) 1012 to 1032; c) 1194 to 1214; d) 1196 to 1216; e) 1421 to 1441; f) 1424 to 1444 or g) 1425 to 1445, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, from the 5′ end of an HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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) 927 to 947; b) 1007 to 1027; c) 1012 to 1032; c) 1194 to 1214; d) 1196 to 1216; e) 1421 to 1441; f) 1424 to 1444 or g) 1425 to 1445, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, from the 5′ end of an HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 927 to 947; b) 1007 to 1027; c) 1012 to 1032; c) 1194 to 1214; d) 1196 to 1216; e) 1421 to 1441; f) 1424 to 1444 or g) 1425 to 1445, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, from the 5′ end of an HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 344 to 364; b) 476 to 496; c) 669 to 741; d) 882 to 915; e) 999 to 1030; f) 1101 to 1121; g) 1297 to 1326; and h) 1487 to 1507, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 344 to 364; b) 476 to 496; c) 669 to 741; d) 882 to 915; e) 999 to 1030; f) 1101 to 1121; g) 1297 to 1326; and h) 1487 to 1507, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 344 to 364; b) 476 to 496; c) 669 to 689; d) 721 to 741; e) 882 to 902; f) 888 to 908; g) 891 to 911; h) 895 to 915; i) 999 to 1019; f) 1000 to 1120; g) 1004 to 1024; h) 1008 to 1028; i) 1009 to 1029; j) 1010 to 1030; k) 1101 to 1121; l) 1297 to 1317; m) 1302 to 1322; n) 1306 to 1326; and o) 1487 to 1507, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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) 344 to 364; b) 476 to 496; c) 669 to 689; d) 721 to 741; e) 882 to 902; f) 888 to 908; g) 891 to 911; h) 895 to 915; i) 999 to 1019; f) 1000 to 1120; g) 1004 to 1024; h) 1008 to 1028; i) 1009 to 1029; j) 1010 to 1030; k) 1101 to 1121; l) 1297 to 1317; m) 1302 to 1322; n) 1306 to 1326; and o) 1487 to 1507, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ED NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 344 to 364; b) 476 to 496; c) 669 to 689; d) 721 to 741; e) 882 to 902; f) 888 to 908; g) 891 to 911; h) 895 to 915; i) 999 to 1019; f) 1000 to 1121; g) 1004 to 1024; h) 1008 to 1028; i) 1009 to 1029; j) 1010 to 1030; k) 1101 to 1121; l) 1297 to 1317; m) 1302 to 1322; n) 1306 to 1326; and o) 1487 to 1507, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 229 to 249; b) 669 to 689; and c) 1007 to 1027, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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) 229 to 249; b) 669 to 689; and c) 1007 to 1027, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 229 to 249; b) 669 to 689; and c) 1007 to 1027, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is substantially identical to a region between the nucleotide positions 1007 to 1027, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, from the 5′ end of an HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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 the nucleotide positions 1007 to 1027, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between the nucleotide positions 1007 to 1027, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is substantially identical to a region between the nucleotide positions 669 and 689, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, from the 5′ end of an HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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 the nucleotide positions 669 and 689, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, from the 5′ end of an HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between the nucleotide positions 669 and 689, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ CD NO: 1, from the 5′ end of an HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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 the nucleotide positions 229 to 249, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between the nucleotide positions 229 to 249, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

The HSD17B13 mRNA sequence according to SEQ ID NO: 1, as described herein, is any heterologous mRNA sequence with sufficient identity to an HSD17B13 according to accession no. NM_178135.5, as described herein, that allows binding to the sense strand of the oligonucleotides of the present disclosure.

In some embodiments of the isolated oligonucleotide of the present disclosure, the isolated oligonucleotide is capable of inducing degradation of the HSD17B13 mRNA.

In some embodiments, the isolated oligonucleotide of the present disclosure is a small interfering RNA (siRNA). Accordingly, 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 a HSD17B13mRNA sequence.

Definitions

“RNAi” or “RNA interference” refers to the process of sequence-specific post-transcriptional gene silencing, mediated by double-stranded RNA (dsRNA). Duplex RNA siRNA (small interfering RNA), miRNA (micro RNA), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA), pi RNA (Piwi-interacting RNA), or rasiRNA (repeat associated siRNA) and modified forms thereof are all capable of mediating RNA interference. These 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, PNA, or a combination thereof. These 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. These dsRNA molecules can also include one or more modified nucleotides, as described herein, which can be incorporated on either strand.

In the RNAi gene silencing or knockdown process, 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. After introduction into the 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.

Certain dsRNAs in cells can undergo the action of Dicer enzyme, a ribonuclease III enzyme. Dicer can process the dsRNA into shorter pieces of dsRNA, i.e. siRNAs. RNAi also involves an endonuclease complex known as the RNA induced silencing complex (RISC). Following cleavage by Dicer, 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.

As used herein, “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. To target a gene, for example using an siRNA, 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. Once introduced into a cell, the siRNA directs the RISC complex to cleave an RNA comprising a target sequence, thereby degrading the RNA.

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. 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.

The term “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). Moreover, 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.

The term “region” or “fragment” is used interchangeably and as applied to an oligonucleotide.

The HSD17B13 mRNA sequence, as described herein, will be understood to mean a full length HSD17B13 mRNA nucleotide sequence, unless indicated otherwise. In some embodiments, the HSD17B13 mRNA sequence can be a nucleotide sequence of reduced length relative to a reference nucleic acid or a nucleotide sequence of the HSD17B13 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. Such a nucleic acid fragment according to the disclosure may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, 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.

As used herein, “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.

As used herein, 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. As used herein, the term “substantially complementary” means that two nucleic acid sequences are complementary at least at about 90%, 95% or 99% of their nucleotides.

In some embodiments, 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.

The term “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. The term “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.

As used herein, 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.

As used herein, 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. In order to obtain a gapped alignment, as described here, 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) can be used. 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). After the alignment, 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.

Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo, H., and Lipton, D., (Applied Math48:1073(1988)). More particularly, preferred computer programs for determining sequence identity include but are not limited to the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; (Altschul et al., J. Mol. Biol. 215:403-410 (1990)); version 2.0 or higher of BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and, for polynucleotide sequence BLASTN can be used to determine sequence identity. 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.

As used herein, “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. Thus, 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. In addition, 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 HSD17B13

The disclosure provides isolated oligonucleotides comprising a double stranded RNAs (dsRNAs) duplex region which target a HSD17B13 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. In some embodiments, the double stranded RNA molecule is a small interfering RNA (siRNA). In other embodiments, the double stranded RNA molecule is a short hairpin RNA (shRNA) molecule. In other embodiments, the double stranded RNA molecule is a Dicer substrate that is processed in a cell to produce an siRNA. In other embodiments the double stranded RNA molecule is part of a microRNA precursor molecule.

In some embodiments, the dsRNA is a small interfering RNA (siRNA) which targets a HSD17B13 mRNA sequence for degradation. In some embodiments, the siRNA targeting HSD17B13 is packaged in a delivery system described herein (e.g., nanoparticle).

The isolated oligonucleotides of the present disclosure targeting HSD17B13 for degradation can comprise a sense strand at least 70% identical to any fragment of a HSD17B13 mRNA, for example the HSD17B13 mRNA of SEQ ID NO: 1. In some embodiments, 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 HSD17B13 for degradation can comprise an antisense strand at least 70% identical to a sequence complementary to any fragment of a HSD17B13 mRNA, for example the HSD17B13 mRNA of SEQ ID NO: 1. In some embodiments, 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. In some embodiments, the sense region and antisense regions are complementary, and base pair to form an RNA duplex structure. The fragment of the HSD17B13 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.

In some embodiments, 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 a HSD17B13 mRNA sequence. In some embodiments, the sense region is identical to a HSD17B13 mRNA sequence.

As used herein, 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 a HSD17B13 mRNA sequence. In some cases, the sense region may have 100% identity, i.e. complete identity or homology, to the target nucleic acid sequence. In other cases, there may be one or more mismatches between the sense region and the target nucleic acid sequence. For example, there may be 1, 2, 3, 4, 5, 6, or 7 mismatches between the sense region and the target nucleic acid sequence.

As used herein, the term “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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, the sense region comprises a sequence that is identical to a sequence of SEQ ID NO: 1 or a region of SEQ CD NO: 1, as disclosed herein. In some embodiments, 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.

In some embodiments, the sense region of the isolated oligonucleotides of the present disclosure targeting HSD17B13 has one or more mismatches between the sequence of the isolated oligonucleotides and the HSD17B13 sequence. For example, 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 HSD17B13 sequence. In some embodiments, the HSD17B13 sequence is an HSD17B133′ 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. Further, the isolated oligonucleotides RNAs may be tolerant of mismatches outside the seed region. As used herein, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 HSD17B13 targeting isolated oligonucleotide of the present disclosure is complementary to the sense region. In some embodiments, the sense region and the antisense region are fully complementary (no mismatches). In some embodiments 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.

In general, 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand is a single stranded RNA molecule. In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand is a single stranded RNA molecule. In some embodiments of the isolated oligonucleotide of the present disclosure, both the sense strand and the antisense strand are single stranded RNA molecules. In some embodiments of the isolated oligonucleotide of the present disclosure is an siRNA targeting HSD17B13, 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.

In some embodiments, 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. An overhang can be a 3′ overhang, wherein the 3′-end of a strand has a single strand region of from one to eight nucleotides. An 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 of the present disclosure can be the same length, or can be different lengths.

In some embodiments of the isolated oligonucleotide of the present disclosure, the single stranded RNA molecule of the sense strand comprises a 3′ overhang. In some embodiments, in the single stranded RNA molecule of the sense strand, the 3′ overhang comprise at least one nucleotide. In some embodiments, in the single stranded RNA molecule of the sense strand, the 3′ overhang comprise two nucleotides.

In some embodiments of the isolated oligonucleotide of the present disclosure, the single stranded RNA molecule of the antisense strand comprises a 3′ overhang. In some embodiments, in the single stranded RNA molecule of the antisense strand, the 3′ overhang comprise at least one nucleotide. In some embodiments, in the single stranded RNA molecule of the antisense strand, the 3′ overhang comprise two nucleotides.

In additional embodiments, both ends of isolated oligonucleotide of the present disclosure have an overhang, for example, a 3′ dinucleotide overhang on each end. The overhangs at the 5′- and 3′-ends may be of different lengths, or be the same length.

An overhang of an isolated oligonucleotide of the present disclosure can contain one or more deoxyribonucleotides, one or more ribonucleotides, or a combination of deoxyribonucleotides and ribonucleotides. In some embodiments, one, or both, of the overhang nucleotides of an siRNA may be 2′-deoxyribonucleotides.

In some embodiments, the first single stranded RNA molecule comprises a first 3′ overhang. In some embodiments, the second single stranded RNA molecule comprises a second 3′ overhang. In some embodiments, the first and second 3′ overhangs comprise a dinucleotide.

In some embodiments of the isolated oligonucleotide of the present disclosure, the 3′ overhang comprises any one of thymidine-thymidine (dTdT), Adenine-Adenine (AA), Cysteine-Cysteine (CC), Guanine-Guanine (GG) or Uracil-Uracil (UU). In some embodiments, the isolated oligonucleotide of the present disclosure, the 3′ overhang comprises a thymidine-thymidine (dTdT) or a Uracil-Uracil (UU) overhang. In some embodiment, the 3′ overhang comprises a Uracil-Uracil (UU) overhang. Without wishing to be bound by theory, it is thought that 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.

In some embodiments, 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. In some embodiments, 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. For example, the 5′ end of the siRNA can be blunt and the 3′ end of the same isolated oligonucleotide comprise an overhang, or vice versa.

In some embodiments, both ends of the isolated oligonucleotide of the present disclosure are blunt ends.

In some embodiments of the isolated oligonucleotide of the present disclosure, the double stranded region comprises an antisense strand and a sense strand, according to any one of the pairs of anti sense strand and sense strand sequences in Table 1, as described below.

TABLE 1

Exemplary Sequences of the Present Application

Guide strand

Guide
Guide
Passenger

Passenger
Passenger

sequence
SEQ ID
start
end
strand
SEQ ID
start
end

(5′-3′)
NO
position
position
sequence (5′-3′)
NO
position
position

UUAUUCAUUUC
2
927
947
AAUCAAAAUGA
31
929
947

AUUUUGAUUUU

AAUGAAUAA

UAAUGUGAAAU
3
1007
1027
CAAAGCUUUAU
32
1009
1027

AAAGCUUUGCA

UUCACAUUA

UGAAAAAAUGU
4
1012
1032
CUUUAUUUCAC
33
1014
1032

GAAAUAAAGCU

AUUUUUUCA

UAUCUUAAAGA
5
1194
1214
AAAAGGUUUUC
34
1196
1214

AAACCUUUUAA

UUUAAGAUA

UAUAUCUUAAA
6
1196
1216
AAGGUUUUCU
35
1198
1216

GAAAACCUUUU

UUAAGAUAUA

UUUUCAAAUGC
7
1421
1441
UAAGAUUCAGC
36
1423
1441

UGAAUCUUAAA

AUUUGAAAA

UAUCUUUCAAA
8
1424
1444
GAUUCAGCAUU
37
1426
1444

UGCUGAAUCUU

UGAAAGAUA

UAAUCUUUCAA
9
1425
1445
AUUCAGCAUUU
38
1427
1445

AUGCUGAAUCU

GAAAGAUUA

UUUCACCUGAU
10
344
364
GCUCUCUAAAU
39
346
364

UUAGAGAGCGA

CAGGUGAAA

UGUGAUCCAAA
11
476
496
UAGGACAUUU
40
478
496

AAUGUCCUAGG

UUGGAUCACA

UGAGACAUGAG
12
669
689
UAUCAAAACCU
41
671
689

GUUUUGAUACC

CAUGUCUCA

UAUAAUCUUGU
13
721
741
AAUCCAAGCAC
42
723
741

GCUUGGAUUUU

AAGAUUAUA

UAAUAUUCUGC
14
882
902
AAAUCGUAUGC
43
884
902

AUACGAUUUAA

AGAAUAUUA

UAAAUUGAAUA
15
888
908
UAUGCAGAAUA
44
890
908

UUCUGCAUACG

UUCAAUUUA

UUUCAAAUUGA
16
891
911
GCAGAAUAUUC
45
893
911

AUAUUCUGCAU

AAUUUGAAA

UCUGCUUCAAA
17
895
915
AAUAUUCAAUU
46
897
915

UUGAAUAUUCU

UGAAGCAGA

UAUAAAGCUUU
18
999
1019
CAAUGCUGCAA
47
1001
1019

GCAGCAUUGAU

AGCUUUAUA

UAAUAAAGCUU
19
1000
1020
AAUGCUGCAAA
48
1002
1020

UGCAGCAUUGA

GCUUUAUUA

UGUGAAAUAAA
20
1004
1024
CUGCAAAGCUU
49
1006
1024

GCUUUGCAGCA

UAUUUCACA

UAAAUGUGAAA
21
1008
1028
AAAGCUUUAUU
50
1010
1028

UAAAGCUUUGC

UCACAUUUA

UAAAAUGUGAA
22
1009
1029
AAGCUUUAUU
51
1011
1029

AUAAAGCUUUG

UCACAUUUUA

UAAAAAUGUGA
23
1010
1030
AGCUUUAUUU
52
1012
1030

AAUAAAGCUUU

CACAUUUUUA

UUAUUCUUGAG
24
1101
1121
UUCCUGUUUC
53
1103
1121

AAACAGGAAGA

UCAAGAAUAA

UAUGCUACUUG
25
1297
1317
AAGACUGUUCA
54
1299
1317

AACAGUCUUAA

AGUAGCAUA

UUUGGÅAUGCU
26
1302
1322
UGUUCAAGUA
55
1304
1322

ACUUGAACAGU

GCAUUCCAAA

UCAGAUUGGAA
27
1306
1326
CAAGUAGCAUU
56
1308
1326

UGCUACUUGAA

CCAAUCUGA

UGUAAUAAAGU
28
1487
1507
CUAUUCUGGAC
57
1489
1507

CCAGAAUAGAG

UUUAUUACA

UUAUUAAUAUC
29
229
249
GUUCUGUGGG
58
231
249

CCACAGAACCA

AUAUUAAUAA

UGAUCCAAAAA
30
474
494
CCUAGGACAUU
59
476
494

UGUCCUAGGAU

UUUGGAUCA

UGAUCCAAAAA
30
474
494
CCUAGGACAUU
60
476
494

UGUCCUAGGAU

UUUGIAUCA

In some embodiments, the sense region comprises a sequence selected from any one of the group of sense strand/passenger strand sequences listed in Tables 1-5. In some embodiments, the antisense region comprises a sequence selected from any one of the group of antisense strand/guide strand sequences listed in Tables 1-5. In some embodiments, the sense and antisense regions comprise complementary sequences selected from the group listed in Tables 1-5.

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 2-30.

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: 31-60.

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 2-30; and the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 31-60, 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 927 to 947; b) 1007 to 1032; c) 1194 to 1216; and d) 1421 to 1445, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ CD NO: 1, from the 5′ end of an HSD17B13 mRNA sequence according to SEQ ID NO: 1, the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UUAUUCAUUUCAUUUUGAUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ AAUCAAAAUGAAAUGAAUAA 3′); or ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UAAUGUGAAAUAAAGCUUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CAAAGCUUUAUUUCACAUUA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UGAAAAAAUGUGAAAUAAAGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUUUAUUUCACAUUUUUUCA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UAUCUUAAAGAAAACCUUUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ AAAAGGUUUUCUUUAAGAUA 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UAUAUCUUAAAGAAAACCUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ AAGGUUUUCUUUAAGAUAUA 3′); vi) an anti sense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UUUUCAAAUGCUGAAUCUUAAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ UAAGAUUCAGCAUUUGAAAA 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUCUUUCAAAUGCUGAAUCUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GAUUCAGCAUUUGAAAGAUA 3′); or viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAAUCUUUCAAAUGCUGAAUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ AUUCAGCAUUUGAAAGAUUA 3′).

20-50% knockdown of HSD17B13 at 0.05 nM

At Least 50% Knockdown of HSD17B13 at 0.05 nM

At Least 50% Knockdown of HSD17B13 at 0.5 nM

In some embodiments of the isolated oligonucleotide of the present disclosure 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) 229 to 249; b) 344 to 364; c) 474 to 496; d) 669 to 741; e) 882 to 947; f) 999 to 1032; g) 1101 to 1216; h) 1297 to 1326; and i) 1421 to 1507, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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, that attenuate expression of the HSD17B13 mRNA by at least 50%, at a dose of 0.5 nM, the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UUAUUCAUUUCAUUUUGAUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ AAUCAAAAUGAAAUGAAUAA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UAAUGUGAAAUAAAGCUUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CAAAGCUUUAUUUCACAUUA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UGAAAAAAUGUGAAAUAAAGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUUUAUUUCACAUUUUUUCA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UAUCUUAAAGAAAACCUUUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ AAAAGGUUUUCUUUAAGAUA 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UAUAUCUUAAAGAAAACCUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ AAGGUUUUCUUUAAGAUAUA 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UUUUCAAAUGCUGAAUCUUAAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ UAAGAUUCAGCAUUUGAAAA 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUCUUUCAAAUGCUGAAUCUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GAUUCAGCAUUUGAAAGAUA 3′); viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAAUCUUUCAAAUGCUGAAUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ AUUCAGCAUUUGAAAGAUUA 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UUUCACCUGAUUUAGAGAGCGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ GCUCUCUAAAUCAGGUGAAA 3′); x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UGUGAUCCAAAAAUGUCCUAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ UAGGACAUUUUUGGAUCACA 3′); xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UGAGACAUGAGGUUUUGAUACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ UAUCAAAACCUCAUGUCUCA 3′); xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UAUAAUCUUGUGCUUGGAUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ AAUCCAAGCACAAGAUUAUA 3′); xiii) an anti sense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UAAUAUUCUGCAUACGAUUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ AAAUCGUAUGCAGAAUAUUA 3′); xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAAAUUGAAUAUUCUGCAUACG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ UAUGCAGAAUAUUCAAUUUA 3′); xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UUUCAAAUUGAAUAUUCUGCAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ GCAGAAUAUUCAAUUUGAAA 3′); xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UCUGCUUCAAAUUGAAUAUUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ AAUAUUCAAUUUGAAGCAGA 3′); xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAUAAAGCUUUGCAGCAUUGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CAAUGCUGCAAAGCUUUAUA 3′); xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UAAUAAAGCUUUGCAGCAUUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ AAUGCUGCAAAGCUUUAUUA 3′); xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UGUGAAAUAAAGCUUUGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ CUGCAAAGCUUUAUUUCACA 3′); xx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UAAAUGUGAAAUAAAGCUUUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ AAAGCUUUAUUUCACAUUUA 3′); xxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UAAAAUGUGAAAUAAAGCUUUG3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ AAGCUUUAUUUCACAUUUUA 3′); xxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAAAAAUGUGAAAUAAAGCUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 52 (5′ AGCUUUAUUUCACAUUUUUA 3′); xxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UUAUUCUUGAGAAACAGGAAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUCCUGUUUCUCAAGAAUAA 3′); xxiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAUGCUACUUGAACAGUCUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ AAGACUGUUCAAGUAGCAUA 3′); xxv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUUGGAAUGCUACUUGAACAGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 55 (5′ UGUUCAAGUAGCAUUCCAAA3′); xxvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ UCAGAUUGGAAUGCUACUUGAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ CAAGUAGCAUUCCAAUCUGA 3′) (79.9); xxvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ UGUAAUAAAGUCCAGAAUAGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ CUAUUCUGGACUUUAUUACA 3′); xxviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ UUAUUAAUAUCCCACAGAACCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ GUUCUGUGGGAUAUUAAUAA 3′); xxix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ UGAUCCAAAAAUGUCCUAGGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 59 (5′ CCUAGGACAUUUUUGGAUCA 3′); or xxx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ UGAUCCAAAAAUGUCCUAGGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO:60 (5′ CCUAGGACAUUUUUGIAUCA 3′).

The present disclosure also provides an isolated oligonucleotide comprising a sense strand 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) 57 to 91; b) 230 to 368; c) 394 to 428; d) 432 to 520; e) 523 to 765; f) 766 to 811; g) 881 to 912; h) 1094 to 1123; i) 1138 to 1171; j) 1198 to 1245; k) 1304 to 1324; j) 1345 to 1377; l) 1422 to 1442; m) 1479 to 1506; and n) 1538 to 1577, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 57 to 88; b) 230 to 250; c) 267 to 290; d) 339 to 368; e) 396 to 428; f) 432 to 454; g) 459 to 517; h) 630 to 679; i) 701 to 765; j) 766-912; k) 1094 to 1123; l) 1138 to 1171; m) 1198 to 1218; n) 1225-1245; o) 1304 to 1324; p) 1345 to 1377; q) 1422 to 1442; r) 1479 to 1506; and s) 1538 to 1577, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 57 to 88; b) 230 to 250; c) 267 to 290; d) 339 to 368; e) 396 to 428; f) 432 to 454; g) 459 to 517; h) 630 to 679; i) 701 to 765; j) 766-912; k) 1094 to 1123; l) 1138 to 1171; m) 1198 to 1218; n) 1225-1245; o) 1304 to 1324; p) 1345 to 1377; q) 1422 to 1442; r) 1479 to 1506; and s) 1538 to 1577, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) mRNA sequence according to SEQ ID NO: 1, and the anti-sense strand is substantially complementary to the sense strand such that the sense strand and the anti-sense strand together form a double stranded region.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 57 to 88; b) 230 to 250; c) 267 to 290; d) 339 to 368; e) 396 to 428; f) 432 to 454; g) 459 to 517; h) 630 to 679; i) 701 to 765; j) 766-912; k) 1094 to 1123; l) 1138 to 1171; m) 1198 to 1218; n) 1225-1245; o) 1304 to 1324; p) 1345 to 1377; q) 1422 to 1442; r) 1479 to 1506; and s) 1538 to 1577, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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, and that attenuates expression of the HSD17B13 mRNA by 20% to 50% at a dose of 0.05 nM, and by at least 50% at a dose of 0.5 nM, the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 64 (5′ UUAGAUGAUGGUGAUCAGAAGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 252 (5′ UUCUGAUCACCAUCAUCUAA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUUAUUAAUAUCCCACAGAACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 258 (5′ UUCUGUGGGAUAUUAAUAAA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 75 (5′ UUAGUUUUCGGCACUCAGCUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 263 (5′ AGCUGAGUGCCGAAAACUAA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 80 (5′ UCUGAUUUAGAGAGCGAUAGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 268 (5′ CUAUCGCUCUCUAAAUCAGA 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 81 (5′ UCUUCACCUGAUUUAGAGAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 269 (5′ CUCUCUAAAUCAGGUGAAGA 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 82 (5′ UUUUCUUCACCUGAUUUAGAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 270 (5′ UCUAAAUCAGGUGAAGAAAA 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 89 (5′ UUAAUCUCUUCAUCCUUGGUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 277 (5′ ACCAAGGAUGAAGAGAUUAA 3′); viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 90 (5′ UGUAAUCUCUUCAUCCUUGGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 278 (5′ CCAAGGAUGAAGAGAUUACA 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 98 (5′ UUUGUGAUCCAAAAAUGUCCUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 286 (5′ GGACAUUUUUGGAUCACAAA 3′); x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 102 (5′ UGAAGAAGUGCUUUUGUGAUCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 290 (5′ AUCACAAAAGCACUUCUUCA 3′); xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 111 (5′ UAAGUUCUGAUGUCAGACCUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 299 (5′ AGGUCUGACAUCAGAACUUA 3′); xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 112 (5′ UUUUUGAUACCAGUUUUUCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 300 (5′ GGAAAAACUGGUAUCAAAAA 3′); xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 113 (5′ UGUUUUGAUACCAGUUUUUCCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 301 (5′ GAAAAACUGGUAUCAAAACA 3′); xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 132 (5′ UUUGUGCUUGGAUUUUUGGUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 320 (5′ ACCAAAAAUCCAAGCACAAA 3′); xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 135 (5′ UUACAGGCCAUAAUCUUGUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 323 (5′ CACAAGAUUAUGGCCUGUAA 3′); xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 142 (5′ UUUCAUCUGUCUCCAAUACAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 330 (5′ UGUAUUGGAGACAGAUGAAA 3′); xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 143 (5′ UCUUCAUCUGUCUCCAAUACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 331 (5′ GUAUUGGAGACAGAUGAAGA 3′) (27.5/70.7); xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 144 (5′ UCAUCUAUCAGACUUCUUACGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 332 (5′ GUAAGAAGUCUGAUAGAUGA 3′); xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 146 (5′ UAAGUAUUCCAUCUAUCAGACU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 334 (5′ UCUGAUAGAUGGAAUACUUA 3′); xx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 147 (5′ UUUAUUGGUAAGUAUUCCAUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 335 (5′ AUGGAAUACUUACCAAUAAA 3′); xxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 149 (5′ UUUCUUAUUGGUAAGUAUUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 337 (5′ GAAUACUUACCAAUAAGAAA 3′); xxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 152 (5′ UAUAUUCUGCAUACGAUUUAAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 340 (5′ UAAAUCGUAUGCAGAAUAUA 3′); xxiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 153 (5′ UCUUCAAAUUGAAUAUUCUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 341 (5′ CAGAAUAUUCAAUUUGAAGA 3′); xxv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 154 (5′ UGAGAAACAGGAAGACAGGUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 342 (5′ ACCUGUCUUCCUGUUUCUCA 3′); xxvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 156 (5′ UCUUGAGAAACAGGAAGACAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 344 (5′ UGUCUUCCUGUUUCUCAAGA 3′); xxvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 159 (5′ UAAUAUUCUUGAGAAACAGGAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 347 (5′ CCUGUUUCUCAAGAAUAUUA 3′); xxviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 160 (5′ UAUGAAAGGAAAAACAGACCUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 348 (5′ GGUCUGUUUUUCCUUUCAUA 3′); xxix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 162 (5′ UUUUUUAAGAGGCAUGAAAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 350 (5′ CUUUCAUGCCUCUUAAAAAA 3′); xxx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 164 (5′ UAAAUAUCUUAAAGAAAACCUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 352 (5′ GGUUUUCUUUAAGAUAUUUA 3′); xxxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 166 (5′ UUUUUGUCCACCUUUAAAUGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 354 (5′ CAUUUAAAGGUGGACAAAAA 3′); xxxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 167 (5′ UGAUUGGAAUGCUACUUGAACA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 355 (5′ UUCAAGUAGCAUUCCAAUCA 3′); xxxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 168 (5′ UCUCAUUCUGUGUUCUUGUUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 356 (5′ AACAAGAACACAGAAUGAGA 3′); xxxiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 169 (5′ UUUAGCUGUGCACUCAUUCUGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 357 (5′ AGAAUGAGUGCACAGCUAAA 3′); xxxv) an anti sense strand of nucleic acid sequence according to SEQ ID NO: 171 (5′ UCUUUCAAAUGCUGAAUCUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 359 (5′ AAGAUUCAGCAUUUGAAAGA 3′); xxxvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 174 (5′ UUAAAGUCCAGAAUAGAGUUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 362 (5′ AACUCUAUUCUGGACUUUAA 3′); xxxvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 176 (5′ UUAAUAAAGUCCAGAAUAGAGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 364 (5′ UCUAUUCUGGACUUUAUUAA 3′); xxxviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 184 (5′ UAAGGGAGGAAAUAUAGAGGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 372 (5′ CCUCUAUAUUUCCUCCCUUA 3′); xxxix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 185 (5′ UAAAGGGAGGAAAUAUAGAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 373 (5′ CUCUAUAUUUCCUCCCUUUA 3′); XL) an antisense strand of nucleic acid sequence according to SEQ ID NO: 186 (5′ UAAAAGGGAGGAAAUAUAGAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 374 (5′ UCUAUAUUUCCUCCCUUUUA 3′); XLi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 187 (5′ UUAAAAAGGGAGGAAAUAUAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 375 (5′ UAUAUUUCCUCCCUUUUUAA 3′); or xLii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 189 (5′ UUAUAAAAAGGGAGGAAAUAUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 377 (5′ UAUUUCCUCCCUUUUUAUAA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 57 to 88; b) 230 to 250; c) 267 to 290; d) 339 to 368; e) 396 to 428; f) 432 to 454; g) 459 to 517; h) 630 to 679; i) 701 to 765; j) 766-912; k) 1094 to 1123; l) 1138 to 1171; m) 1198 to 1218; n) 1225-1245; o) 1304 to 1324; p) 1345 to 1377; q) 1422 to 1442; r) 1479 to 1506; and s) 1538 to 1577, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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, and that attenuates expression of the HSD17B13 mRNA by at least 50% at a dose of 0.5 nM, the double stranded region comprises: (i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 62 (5′ UGAUCAGAAGCAGAAGGAUUUC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 250 (5′ AAUCCUUCUGCUUCUGAUCA 3′); (ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 63 (5′ UAUGGUGAUCAGAAGCAGAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 251 (5′ UUCUGCUUCUGAUCACCAUA 3′); (iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 73 (5′ UUUUUCGGCACUCAGCUGCAGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 261 (5′ UGCAGCUGAGUGCCGAAAAA 3′); (iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 84 (5′ UAUAUACUGUCCCAGCAUUAUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 272 (5′ UAAUGCUGGGACAGUAUAUA 3′); (v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 87 (5′ UAAGAUCGGCUGGAUAUACUGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 275 (5′ AGUAUAUCCAGCCGAUCUUA 3′); (vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 88 (5′ UAAUCUCUUCAUCCUUGGUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 276 (5′ CACCAAGGAUGAAGAGAUUA 3′); (vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 91 (5′ UUAGGAUGUUGACCUCAAAUGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 279 (5′ AUUUGAGGUCAACAUCCUAA 3′); (viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 94 (5′ UAAUGUCCUAGGAUGUUGACCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 282 (5′ GUCAACAUCCUAGGACAUUA 3′); (ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 99 (5′ UUUUGUGAUCCAAAAAUGUCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 287 (5′ GACAUUUUUGGAUCACAAAA 3′); (x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 104 (5′ UAUCGAUGGAAGAAGUGCUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 292 (5′ AAGCACUUCUUCCAUCGAUA 3′); (xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 126 (5′ UUUGGUGAACCCAGUAUUCACA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 314 (5′ UGAAUACUGGGUUCACCAAA 3′); (xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 127 (5′ UUUUGGUGAACCCAGUAUUCAC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 315 (5′ GAAUACUGGGUUCACCAAAA 3′); (xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 128 (5′ UUUUUGGUGAACCCAGUAUUCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 316 (5′ AAUACUGGGUUCACCAAAAA 3); (xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 130 (5′ UGAUUUUUGGUGAACCCAGUAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 318 (5′ ACUGGGUUCACCAAAAAUCA 3′); (xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 131 (5′ UCUUGGAUUUUUGGUGAACCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 319 (5′ GGUUCACCAAAAAUCCAAGA 3′); (xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 133 (5′ UCUUGUGCUUGGAUUUUUGGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 321 (5′ CCAAAAAUCCAAGCACAAGA 3′); (xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 134 (5′ UAUCUUGUGCUUGGAUUUUUGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 322 (5′ AAAAAUCCAAGCACAAGAUA 3′); (xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 136 (5′ UAUACAGGCCAUAAUCUUGUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 324 (5′ ACAAGAUUAUGGCCUGUAUA 3′); (xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 138 (5′ UCAAUACAGGCCAUAAUCUUGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 326 (5′ AAGAUUAUGGCCUGUAUUGA 3′); (xx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 145 (5′ UUAUUCCAUCUAUCAGACUUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 333 (5′ AAGUCUGAUAGAUGGAAUAA 3′); (xxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 148 (5′ UCUUAUUGGUAAGUAUUCCAUC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 336 (5′ UGGAAUACUUACCAAUAAGA 3′); (xxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 150 (5′ UUUUCUUAUUGGUAAGUAUUCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 338 (5′ AAUACUUACCAAUAAGAAAA 3′); (xxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 151 (5′ UAUCAUUUUCUUAUUGGUAAGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 339 (5′ UUACCAAUAAGAAAAUGAUA 3′); (xxiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 157 (5′ UUUCUUGAGAAACAGGAAGACA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 345 (5′ UCUUCCUGUUUCUCAAGAAA 3′); (xxv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 158 (5′ UAUUCUUGAGAAACAGGAAGAC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 346 (5′ CUUCCUGUUUCUCAAGAAUA 3′); (xxvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 161 (5′ UCAUGAAAGGAAAAACAGACCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 349 (5′ GUCUGUUUUUCCUUUCAUGA 3′); (xxvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 163 (5′ UGUUUUUAAGAGGCAUGAAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 351 (5′ UUUCAUGCCUCUUAAAAACA 3′); (xxviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 170 (5′ UCUUAGCUGUGCACUCAUUCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 358 (5′ GAAUGAGUGCACAGCUAAGA 3); (xxix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 173 (5′ UAAAGUCCAGAAUAGAGUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 361 (5′ CAACUCUAUUCUGGACUUUA 3′); (xxx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 175 (5′ UAAUAAAGUCCAGAAUAGAGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 363 (5′ CUCUAUUCUGGACUUUAUUA 3′); (xxxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 178 (5′ UAUAGAGGGUCCACUUUUGGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 366 (5′ CCAAAAGUGGACCCUCUAUA 3′); (xxxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 179 (5′ UUAUAGAGGGUCCACUUUUGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 367 (5′ CAAAAGUGGACCCUCUAUAA 3′); (xxxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 181 (5′ UAAUAUAGAGGGUCCACUUUUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 369 (5′ AAAGUGGACCCUCUAUAUUA 3′); (xxxiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 182 (5′ UAAAUAUAGAGGGUCCACUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 370 (5′ AAGUGGACCCUCUAUAUUUA 3′); (xxxv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 183 (5′ UGAAAUAUAGAGGGUCCACUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 371 (5′ AGUGGACCCUCUAUAUUUCA 3′); (xxxvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 183 (5′ UGAAAUAUAGAGGGUCCACUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 371 (5′ AGUGGACCCUCUAUAUUUCA 3′); or (xxxvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 188 (5′ UAUAAAAAGGGAGGAAAUAUAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 376 (5′ AUAUUUCCUCCCUUUUUAUA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 69 to 91; b) 231 to 310; c) 394 to 426; d) 463 to 648; e) 682 to 724; f) 731 to 762; g) 1096 to 1116; h) 1204 to 1224; i) 1479 to 1499; and j) 1538 to 1561, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 69 to 91; b) 231 to 310; c) 394 to 426; d) 463 to 648; e) 682 to 724; f) 731 to 762; g) 1096 to 1116; h) 1204 to 1224; i) 1479 to 1499; and j) 1538 to 1561, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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, and that attenuates expression of the HSD17B13 mRNA by 20% to 50% at a dose of 0.5 nM, the double stranded region comprises: (i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 65 (5′ UGUAGAUGAUGGUGAUCAGAAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 253 (5′ UCUGAUCACCAUCAUCUACA 3′); (ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 66 (5′ UGAGUAGAUGAUGGUGAUCAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 254 (5′ UGAUCACCAUCAUCUACUCA 3′); (iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 71 (5′ UCUUAUUAAUAUCCCACAGAAC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 259 (5′ UCUGUGGGAUAUUAAUAAGA 3′); (iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 72 (5′ UCACUCAGCUGCAGUUUCCUCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 260 (5′ AGGAAACUGCAGCUGAGUGA 3′); (v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 74 (5′ UGUUUUCGGCACUCAGCUGCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 262 (5′ GCAGCUGAGUGCCGAAAACA 3′); (vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 76 (5′ UCUAGUUUUCGGCACUCAGCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 264 (5′ GCUGAGUGCCGAAAACUAGA 3′); (vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 78 (5′ UCAGUGACGCCUAGUUUUCGGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 266 (5′ CGAAAACUAGGCGUCACUGA 3′); (viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 79 (5′ UUACGCAUGCGCAGUGACGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 267 (5′ GCGUCACUGCGCAUGCGUAA 3′); (ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 83 (5′ UAUACUGUCCCAGCAUUAUUCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 271 (5′ AAUAAUGCUGGGACAGUAUA 3′); (x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 85 (5′ UGAUAUACUGUCCCAGCAUUAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 273 (5′ AAUGCUGGGACAGUAUAUCA 3′); (xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 86 (5′ UGAUCGGCUGGAUAUACUGUCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 274 (5′ ACAGUAUAUCCAGCCGAUCA 3′); (xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 92 (5′ UGUCCUAGGAUGUUGACCUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 280 (5′ GAGGUCAACAUCCUAGGACA 3′); (xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 93 (5′ UAUGUCCUAGGAUGUUGACCUC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 281 (5′ GGUCAACAUCCUAGGACAUA 3′); (xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 95 (5′ UAAAUGUCCUAGGAUGUUGACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 283 (5′ UCAACAUCCUAGGACAUUUA 3′); (xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 96 (5′ UAAAAUGUCCUAGGAUGUUGAC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 284 (5′ CAACAUCCUAGGACAUUUUA 3′); (xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 97 (5′ UAAAAAUGUCCUAGGAUGUUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 285 (5′ AACAUCCUAGGACAUUUUUA 3′); (xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 100 (5′ UUUUUGUGAUCCAAAAAUGUCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 288 (5′ ACAUUUUUGGAUCACAAAAA 3′); (xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 103 (5′ UAUGGAAGAAGUGCUUUUGUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 291 (5′ ACAAAAGCACUUCUUCCAUA 3′); (xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 105 (5′ UAUCAUCGAUGGAAGAAGUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 293 (5′ CACUUCUUCCAUCGAUGAUA 3′); (xx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 106 (5′ UCGAUGUGGCCAUGAUUUCUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 294 (5′ AGAAAUCAUGGCCACAUCGA 3′); (xxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 107 (5′ UGACGAUGUGGCCAUGAUUUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 295 (5′ AAAUCAUGGCCACAUCGUCA 3′); (xxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 108 (5′ UAUGUCAGACCUCUGUGAAAGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 296 (5′ UUUCACAGAGGUCUGACAUA 3′); (xxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 109 (5′ UCUGAUGUCAGACCUCUGUGAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 297 (5′ CACAGAGGUCUGACAUCAGA 3′); (xxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 110 (5′ UGUUCUGAUGUCAGACCUCUGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 298 (5′ AGAGGUCUGACAUCAGAACA 3′); (xxiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 118 (5′ UCAAAAACUGGGCAGAGACAUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 306 (5′ UGUCUCUGCCCAGUUUUUGA 3′); (xxv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 120 (5′ UUUCACAAAAACUGGGCAGAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 308 (5′ UCUGCCCAGUUUUUGUGAAA 3′); (xxvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 121 (5′ UAUUCACAAAAACUGGGCAGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 309 (5′ CUGCCCAGUUUUUGUGAAUA 3′); (xxvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 122 (5′ UUAUUCACAAAAACUGGGCAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 310 (5′ UGCCCAGUUUUUGUGAAUAA 3′); (xxviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 123 (5′ UGUAUUCACAAAAACUGGGCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 311 (5′ GCCCAGUUUUUGUGAAUACA 3′); (xxix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 129 (5′ UUUUUUGGUGAACCCAGUAUUC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 317 (5′ AUACUGGGUUCACCAAAAAA 3′); (xxx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 137 (5′ UAAUACAGGCCAUAAUCUUGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 325 (5′ CAAGAUUAUGGCCUGUAUUA 3′); (xxx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 139 (5′ UCUCCAAUACAGGCCAUAAUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 327 (5′ AUUAUGGCCUGUAUUGGAGA 3′); (xxxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 140 (5′ UAUCUGUCUCCAAUACAGGCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 328 (5′ GCCUGUAUUGGAGACAGAUA 3′); (xxxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 141 (5′ UCAUCUGUCUCCAAUACAGGCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 329 (5′ CCUGUAUUGGAGACAGAUGA 3′); (xxxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 155 (5′ UUUGAGAAACAGGAAGACAGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 343 (5′ CUGUCUUCCUGUUUCUCAAA 3′); (xxxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 165 (5′ UAAAAUAAAAUAUCUUAAAGAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 353 (5′ CUUUAAGAUAUUUUAUUUUA 3′); (xxxiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 172 (5′ UGUCCAGAAUAGAGUUGCACCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 360 (5′ GUGCAACUCUAUUCUGGACA 3′); (xxxv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 177 (5′ UUAGAGGGUCCACUUUUGGUGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 365 (5′ ACCAAAAGUGGACCCUCUAA 3′); (xxxvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 178 (5′ UAUAGAGGGUCCACUUUUGGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 366 (5′ CCAAAAGUGGACCCUCUAUA 3′); or (xxxvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 180 (5′ UAUAUAGAGGGUCCACUUUUGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 368 (5′ AAAAGUGGACCCUCUAUAUA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 3, 12 or 29.

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: 32, 41 or 58.

In some embodiments of the isolated oligonucleotide of the present disclosure, the isolated oligonucleotide comprises: (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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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.

In some embodiments, the sense strand of the isolated oligonucleotide of the present disclosure comprises at least one nucleotide having a modified phosphate backbone. In some embodiments, the antisense strand of the isolated oligonucleotide of the present disclosure comprises at least one nucleotide having a modified phosphate backbone. In some embodiments, in the sense strand or the antisense strand or both sense and antisense strands of the isolated oligonucleotide of the present disclosure, the modified phosphate backbone comprises a modified phosphodiester bond. In some embodiments, 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′-S′ linkage. In some embodiments, the modified phosphodiester bond comprises phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate diester, mesyl phosphoramidate, or phosphonoacetate.

In some embodiments, the isolated oligonucleotide of the present disclosure comprises one or more non-natural base-containing nucleotide, a locked nucleotide, or an abasic nucleotide. In some embodiments, the isolated oligonucleotide of the present disclosure, the terminal nucleotide at the 5′ end comprises a phosphate mimic. In some embodiments, the 5′-phosphate mimic is ethylphosphonate, vinylphosphonate or an analog thereof.

In some embodiments, the antisense strand of the isolated oligonucleotide of the present disclosure comprises at least two single-stranded nucleotides at the 3′-terminus. In some embodiments, the antisense strand of the isolated oligonucleotide of the present disclosure comprises two single-stranded nucleotides at the 3′-terminus.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the sense strand comprises a nucleotide sequence that is identical to a region between the nucleotide positions 1007 to 1027, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, the double stranded region comprises an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UAAUGUGAAAUAAAGCUUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CAAAGCUUUAUUUCACAUUA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the sense strand comprises a sequence that is identical to a region between the nucleotide positions 669 to 689, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, the double stranded region comprises an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UGAGACAUGAGGUUUUGAUACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ UAUCAAAACCUCAUGUCUCA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the sense strand comprises a sequence that is identical to a region between the nucleotide positions 229 to 249, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, the double stranded region comprises an antisense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ UUAUUAAUAUCCCACAGAACCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ GUUCUGUGGGAUAUUAAUAA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 229 to 249; b) 669 to 689; and c) 1007 to 1027, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) mRNA sequence according to SEQ ID NO: 1, and the anti sense strand is substantially complementary to the sense strand such that the sense strand and the antisense strand together form a double stranded region and that attenuates expression of the HSD17B13 mRNA by 20% to 50%, at a dose of 0.05 nM, the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UGAGACAUGAGGUUUUGAUACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ UAUCAAAACCUCAUGUCUCA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ UUAUUAAUAUCCCACAGAACCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ GUUCUGUGGGAUAUUAAUAA 3′).

In some embodiments of the isolated oligonucleotide of the present disclosure wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 229 to 249; b) 669 to 689; and c) 1007 to 1027, from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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, that attenuate expression of the HSD17B13 mRNA by at least 50%, at a dose of 0.5 nM, the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UAAUGUGAAAUAAAGCUUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CAAAGCUUUAUUUCACAUUA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UGAGACAUGAGGUUUUGAUACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ UAUCAAAACCUCAUGUCUCA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ CD NO: 29 (5′ UUAUUAAUAUCCCACAGAACCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ GUUCUGUGGGAUAUUAAUAA 3′).

In some embodiments, the isolated oligonucleotide of the present disclosure can comprises a linker, sometimes referred to as a loop. siRNAs comprising a linker or loop are sometimes referred to as short hairpin RN As (shRNAs). In some embodiments, both the sense and the antisense regions of the siRNA are encoded by one single-stranded RNA. In these embodiments, and 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.

Any suitable nucleotide linker sequence is envisaged as within the scope of the disclosure. 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.

Examples of a non-nucleotide linker 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. Some examples are described in Seela et al., Nucleic Acids Research, 1987, Vol. 15, pp. 3113-3129; Cload et al., J. Am. Chem. Soc, 1991, Vol. 113, pp. 6324-6326; Jaeschke et al., Tetrahedron Lett., 1993, Vol. 34, pp. 301; Arnold et al., WO 1989/002439; Usman et al., WO 1995/006731; Dudycz et al., WO 1995/011910, and Ferentz et al., J. Am. Chem. Soc, 1991, Vol. 113, pp. 4000-4002.

Examples of nucleotide linker sequences include, but are not limited to, AUG, CCC, UUCG, CCACC, AAGCAA, CCACACC and UUCAAGAGA.

In some embodiments, 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 (dsRNA) 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.

In some embodiments, the isolated oligonucleotide of the present disclosure comprises at least one modified nucleotide. In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand or the antisense strand or both comprise one or more modified nucleotide(s). In some embodiments, only the sense strand comprises one or more modified nucleotide(s). In some embodiments, only the antisense strand comprises one or more modified nucleotide(s). In some embodiments, both the sense strand and antisense strand comprise one or more modified nucleotide(s). In some embodiments, the isolated oligonucleotide is partially chemically modified. In some embodiments, the isolated oligonucleotide is fully chemically modified.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, the sense strand and/or the anti sense 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, wherein 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. In some embodiments, the first modification and second modification are different. In some embodiments, the at least first nucleotide and the at least second nucleotide are located on different strands of the isolated oligonucleotide. In some embodiments, the at least first nucleotide and the at least second nucleotide are located on the same strand of the isolated oligonucleotide.

In some embodiments of the isolated oligonucleotide, wherein 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. In some embodiments, the isolated oligonucleotide comprises a first, a second, a third and a fourth modifications. In some embodiments, the isolated oligonucleotide comprises more than four modifications. In some embodiments, all modifications are on the sense strand. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the isolated oligonucleotide of the present disclosure comprises at least one modified nucleotide(s). In some embodiments, the one or more modified nucleotide(s) increases the stability or potency or both of the isolated oligonucleotide. In some embodiments, the one or more modified nucleotide(s) increases the stability of the RNA duplex, and siRNA.

Modifications that increase RNA stability include, but are not limited to locked nucleic acids. As used herein, the term “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. The term inaccessible RNA can be used interchangeably with LNA. 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand or the antisense strand or both comprise at least one nucleotide having a modified phosphate backbone. In some embodiments, the sense strand of the isolated oligonucleotide comprises at least one nucleotide having a modified phosphate backbone. In some embodiments, the antisense strand of the isolated oligonucleotide comprises at least one nucleotide having a modified phosphate backbone. In some embodiments, wherein the isolated oligonucleotide of the present disclosure comprises a modified phosphate backbone, the modified phosphate backbone comprises a modified phosphodiester bond. In some embodiments, 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. In some embodiments, the modified phosphodiester bond comprises phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate diester, mesyl phosphoramidate, or phosphonoacetate.

In some embodiments, the isolated oligonucleotide of the present disclosure comprises one or more non-natural base-containing nucleotide, a locked nucleotide, or an abasic nucleotide. In some embodiments, the one or more modified nucleotide comprises a phosphorothioate derivative or an acridinine substituted nucleotide. In some embodiments, 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. In some embodiments, the 5′-phosphate mimic is ethylphosphonate, vinylphosphonate or an analog thereof.

In some embodiments, 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′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-isopenten-yladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, or 2, 6-diaminopurine.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand or the antisense strand or both comprise a terminal or internal nucleotide linked to one or more targeting ligands. In some embodiments, the terminal or internal nucleotide is linked to the one or more targeting ligands directly. In some embodiments, the terminal or internal nucleotide is linked to the one or more targeting ligands indirectly by a linker. In some embodiments, the one or more targeting ligands linked directly or indirectly to the terminal or internal nucleotide can further comprise a PK modulator. In some embodiments, the PK modulator is a competitive modulator, a positive allosteric modulator, a negative allosteric modulator or a neutral allosteric modulator. In some embodiments, 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.

Modification of the Nucleotides

Provided herein is 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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′-N3 modification, 2′-deoxy modification, and an equivalent thereof, and a combination thereof. In some embodiments, the first modification is 2′-F modification, 2′-CN modification, 2′-N3 modification, or 2′-deoxy modification, or a stereoisomer thereof. In some embodiments, the first modification is 2′-F modification, 2′-CN modification, or 2′-N3 modification, or a stereoisomer thereof. In some embodiments, the first modification is 2′-F modification or a stereoisomer thereof.

In some embodiments, the second modification is modification of the sugar moiety of one or more of the remaining nucleotides at the 2′-position selected from 2′-C₁-C₆alkyl, 2′-OR modification wherein R is C1-C6 alkyl optionally substituted with C1-C6 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. In some embodiments, the second modification is 2′-OR modification, or morpholino replacement, or a combination thereof. In some embodiments, the second modification is 2′-OR modification. In some embodiments, the second modification is 2′-O-methyl modification or 2′-methoxyethoxy modification. In some embodiments, the second modification is 2′-O-methyl modification. In some embodiments, the second modification is morpholino replacement.

In some embodiments, 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.

In some embodiments, 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′-N3 modification, 2′-deoxy modification, and an equivalent thereof, and a combination thereof. In some embodiments, the third modification is 2′-F modification, 2′-CN modification, 2′-N3 modification, or 2′-deoxy modification, or a stereoisomer thereof. In some embodiments, the third modification is 2′-F modification, 2′-CN modification, or 2′-N3 modification, or a stereoisomer thereof. In some embodiments, the third modification is 2′-F modification or a stereoisomer thereof.

In some embodiments, the fourth modification is modification of the sugar moiety of one or more of the remaining nucleotides at the 2′-position selected from 2′-C₁-C₆alkyl, 2′-OR modification wherein R is C₁-C₆alkyl optionally substituted with C₁-C₆alkoxy, 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. In some embodiments, the fourth modification is 2′-OR modification, or morpholino replacement, or a combination thereof. In some embodiments, the fourth modification is 2′-OR modification. In some embodiments, the fourth modification is 2′-O-methyl modification or 2′-methoxyethoxy modification. In some embodiments, the fourth modification is 2′-O-methyl modification. In some embodiments, the fourth modification is morpholino replacement.

In some embodiments, 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.

Sense Strand

In some embodiments of the isolated oligonucleotide of the present disclosure comprising a sense and an antisense strand, in the sense strand of the isolated oligonucleotide of the present disclosure, at least three nucleotides are modified with the first modification. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least two of the at least three 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 three of the at least three nucleotides modified with the first modification are consecutively located.

In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, in the sense strand 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.

In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, in the sense strand 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.

In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, 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.

In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, 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. 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 selected from position 10, 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, 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.

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 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. 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 10, 11 and 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 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. 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 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. 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 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.

In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, 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.

In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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.

In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, not all of the at least three nucleotides, the at least four nucleotides, or 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, the at least three nucleotides, the at least four nucleotides, or the at least five nucleotides are modified with 2′-F modification.

In some embodiments, 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)_a3′, 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)₉3′.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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)_a3′, 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)₉3′.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein 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)_a3′, 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)₉3′, the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 32, 41 or 58.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein 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)_a3′, 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)₉3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 32, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 3.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein 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)_e(F)_b(M)_a3′, 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)₉3′, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 41.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein 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)_a3′, 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)₉3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 41, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 12.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein 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)_a3′, 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)₉3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 58, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 29.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the sense strand comprises a sequence that is identical to a region between the nucleotide positions 229 to 249, from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1, the double stranded region comprises an antisense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ UUAUUAAUAUCCCACAGAACCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO. 58 (5′ GUUCUGUGGGAUAUUAAUAA 3′).

Antisense Strand

In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most seven nucleotides are modified with the third modification.

In some embodiments, in 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.

In some embodiments, in 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.

In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 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. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 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. 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 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, 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.

In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 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. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, the at most seven nucleotides are modified with 2′-F modification. 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 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 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.

In some embodiments of the isolated oligonucleotides of the present disclosure, wherein the antisense strand comprises at most seven nucleotides modified with the third modification, the at most seven nucleotides are modified with 2′-F modification. 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 2 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 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. 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 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. 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 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. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 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.

In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 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)_c(F)_f(M)_g(F)_h(M)_i(F)_j(M)_k(F)_l(M)_m(F)_n(M)_o5′, 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 any one of: 3′(M)₀(F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁5′.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the antisense 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: 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)_o5′, 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, 1, m, n and o is any one of 0-16, wherein the antisense strand is any one of: 3′(M)₀(F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)_t(M)₁(F) (M)₁5′, the anti sense strand comprises a nucleotide sequence according to any one of SEQ ID NOs: 3, 12 or 29.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the antisense 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: 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)_o5′, 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, 1, m, n and o is any one of 0-16, wherein the antisense strand is any one of: 3′(M)₀(F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁5′, the anti sense strand comprises a nucleotide sequence according to SEQ ID NO: 3.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the antisense 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: 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)_o5′, 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 anti sense strand is any one of: 3′(M)₀(F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)_t(M)₁(F)₁(M)₁(F)₂(M)₁5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 3, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 32.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the antisense 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: 3′ (M)_a(F)_b(M)_a(F)_d(M)_e(F)_f(M)_g(F)_h(M)_i(F)_j(M)_k(F)_l(M)_m(F)_n(M)_o5′, 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 anti sense strand is any one of: 3′(M)₀(F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁5′, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 12.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the antisense 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: 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)_o5′, 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, 1, m, n and o is any one of 0-16, wherein the antisense strand is any one of: 3′(M)₀(F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 12, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 41.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the antisense 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: 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)_o5′, 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 any one of: 3′(M)₀(F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁5′, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 29.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the antisense 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: 3′ (M)_a(F)_b(M)_c(F)_d(M)_c(F)_f(M)_g(F)_h(M)_i(F)_j(M)_k(F)_j(M)_l(F)_n(M)_o5′, 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, 1, m, n and o is any one of 0-16, wherein the antisense strand is any one of: 3′(M)₀(F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 29, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 58.

Targeting Ligand

In some embodiments, in the sense strand or the antisense strand or both of the isolated oligonucleotide of the present disclosure, a terminal or internal nucleotide is linked to a targeting ligand. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, the targeting ligand binds to a surface protein on a cell expressing a target mRNA of the isolated oligonucleotide of the present disclosure. In some embodiments, 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.

In some embodiments, the targeting ligand is a therapeutic ligand. In some embodiments, the targeting ligand is a therapeutic antibody.

In some embodiments, the targeting ligand is attached to the isolated oligonucleotide of the present disclosure by a linker. In some embodiments, the linker is any one or a protein, a DNA, an RNA or a chemical compound. In some embodiments, the isolated oligonucleotide, the linker and the targeting ligand, of the present disclosure form a scaffold. As used herein, 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.

In some embodiments, the isolated oligonucleotide, the linker and the targeting ligand, of the present disclosure form a conjugate. As used herein, the term “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.

As used herein, the term “targeting ligand” or “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). In some embodiments, the targeting ligand comprises a sugar ligand moiety (e.g., N-acetylgalactosamine (GalNAc)) which may direct uptake of an oligonucleotide into the liver.

In some embodiments, 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.

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/2016/100401, 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.

In some embodiments, the targeting ligand comprises a carbohydrate moiety.

As used herein, “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. In some embodiments, the carbohydrate moiety comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide. In some embodiments, the carbohydrate moiety comprises an oligosaccharide containing from about 4-9 monosaccharide units. In some embodiments, the carbohydrate moiety comprises a polysaccharide (e.g., a starch, a glycogen, a cellulose, or a polysaccharide gum).

In some embodiments, 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).

In some embodiments, the ligand is capable of binding to a human asialoglycoprotein receptor (ASGPR), e.g., human asialoglycoprotein receptor 2 (ASGPR2).

In some embodiments, the carbohydrate moiety comprises a sugar (e.g., one, two, or three sugar). In some embodiments, the carbohydrate moiety comprises galactose or a derivative thereof (e.g., one, two, or three galactose or the derivative thereof). In some embodiments, the carbohydrate moiety comprises N-acetylgalactosamine or a derivative thereof (e.g., one, two, or three N-acetylgalactosamine or the derivative thereof). In some embodiments, 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).

In some embodiments, 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).

In some embodiments, the carbohydrate moiety comprises mannose or a derivative thereof (e.g., mannose-6-phosphate). In some embodiments, the carbohydrate moiety further comprises a linking moiety that connects the one or more sugar (e.g., N-acetyl-D-galactosylamine) with a linker.

In some embodiments the linker comprises thioether (e.g., thiosuccinimide, or the hydrolysis analogue thereof), disulfide, triazole, phosphorothioate, phosphodiester, ester, amide, or any combination thereof. In some embodiments, 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/2016/100401, WO/2017/214112, WO/2018/039364, and WO/2018/045317, each of which is incorporated herein by reference.

In some embodiments, the targeting ligand comprises a lipid or a lipid moiety (e.g., one, two, or three lipid moiety). In some embodiments the lipid moiety comprises (e.g., one, two, of three of) C8-C24 fatty acid, cholesterol, vitamin, sterol, phospholipid, or any combination thereof.

In some embodiments, the targeting ligand comprises a peptide or a peptide moiety (e.g., one, two, or three peptide moiety). In some embodiments, the peptide moiety comprises (e.g., one, two, or three of) integrin, insulin, glucagon-like peptide, or any combination thereof. In some embodiments, the targeting ligand comprises an antibody or an antibody moiety (e.g., transferrin). In some embodiments, the targeting ligand comprises one, two, or three antibody moieties (e.g., transferrin).

In some embodiments, 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).

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the ligand comprises a sugar ligand moiety (e.g, N-acetylgalactosamine (GalNAc)) which may direct uptake of an oligonucleotide into the liver

In some embodiments, the ligand comprises GalNAc, or a derivative thereof. In some embodiments, the ligand comprises a GalNAc G1b structure shown below.

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In some embodiments, the ligand comprises three GalNAc moieties, or three derivatives thereof. In some embodiments, the ligand comprises three GalNAc Glb moieties. In some embodiments, wherein the ligand comprises three GalNAc G1b moieties, the GalNAc G1b moieties are consecutively located. In some embodiments, the consecutively located GalNAc G1b moieties are located on the 3′ end of the sense strand. In some embodiments, wherein the ligand comprises three GalNAc G1b (“G1b”) moieties that are consecutively located, the first G1 b moiety is linked to the second G1b moiety and the second G1b is linked to the third (i b moiety. In some embodiments, the first GalNAc G1b moiety is linked to the sense strand of the isolated oligonucleotide of the present disclosure.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the ligand comprises three GalNAc G1b (“G1b”) moieties, wherein the first GalNAc G1 b 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 G1b is linked to the third G1b moiety. In some embodiments, wherein the ligand comprises three GalNAc G1b moieties, the three GalNAc G1b moieties are consecutively located on the 3′ end of the sense strand.

In some embodiments of the isolated oligonucleotide of the present disclosure, the isolated oligonucleotide is linked to the ligand (e.g., GalNAc Glb, or three GalNAc Glb moieties). In some embodiments, the isolated oligonucleotide is linked to the ligand via an internal or terminal nucleotide of the isolated oligonucleotide. In some embodiments, the isolated oligonucleotide is linked to the ligand via a ligand linker. In some embodiments, the

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the isolated oligonucleotide comprises a sense and an antisense strand, and wherein the ligand comprises three GalNAc G1b moieties, and the three GalNAc G1b moieties are consecutively located on the 3′ end of the sense strand, the ligand is linked to a terminal nucleotide on the sense strand of the isolated oligonucleotide. In some embodiments, the ligand is linked to a terminal nucleotide on the sense strand via a ligand linker. In some embodiments, the ligand linker is a monovalent linker. In some embodiments, the ligand linker is a bivalent linker. In some embodiments, 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. In some embodiments, 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.

Modification of the Phosphate Groups

Modified Terminal Phosphate Groups

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. 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.

In some embodiments, the present disclosure provides phosphate mimics of 5′-terminal nucleotides. Without wishing to be bound by theory, it is understood that, when being incorporated into oligonucleotides (e.g., at the 5′-terminus of the antisense strand), 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).

In some embodiments of the isolated oligonucleotides of the present disclosure, the oligonucleotides comprise 5′-terminal nucleotide modifications. In some embodiments, 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. In some embodiments, 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).

In some embodiments, the 5′-terminal modification is a chemical modification. In some embodiments, the chemical modification enhances stability against nucleases or other enzymes that degrade or interfere with the structure or activity of the isolated oligonucleotide.

In some embodiments, the sense or antisense strand of the isolated oligonucleotides of the present disclosure comprise a 5′-terminal phosphate group. In some embodiments, the 5′-terminal phosphate group comprises an unmodified phosphate having the formula: —O—P(O)(OH)OH. In some embodiments, the 5′-terminal phosphate group comprises a modified phosphate. In some embodiments, the 5′-terminal phosphate group comprises a modified phosphate having the formula —CH₂—P(═X)(OR¹)OR², wherein X is O or S, R¹is H or C₁-C₆alkyl, and R²is H or C₁-C₆alkyl. In some embodiments, the modified phosphate is referred to as a “phosphate mimic”.

The term, “halo” or “halogen”, as used herein, refers to fluoro, chloro, bromo and iodo.

The term, “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. The term aryl includes both monovalent species and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl and the like. Conveniently, an aryl is phenyl.

The term, “alkyl” or “C₁-C₆alkyl”, as used herein, is intended to include C₁, C₂, C₃, C₄, C₅or C₆straight chain (linear) saturated aliphatic hydrocarbon groups and C₃, C₄, C₅or C₆branched saturated aliphatic hydrocarbon groups. For example, C₁-C₆alkyl is intended to include C₁, C₂, C₃, C₄, C₅and C₆alkyl groups. Examples of alkyl 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. In some embodiments, a straight chain or branched alkyl has six or fewer carbon atoms (e.g., C₁-C₆for straight chain, C₃-C₆for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms. In some embodiments, the straight chain alkyl has one carbon atom. In some embodiments, the straight chain alkyl has two carbon atoms.

In some embodiments, the phosphate mimic is linked to the 5′-terminus of the isolated oligonucleotides (e.g., siRNAs) as shown in the following formula:

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- wherein:
  - B is H or a nucleobase moiety;
  - X is O or S;
  - R¹is H or C₁-C₆alkyl;
  - R²is H or C₁-C₆alkyl;
  - Y¹is O or S;
  - Y²is O or S;
  - Z is H, halogen, or —OR^Z;
  - R^Zis H, C₁-C₆alkyl, or —(C₁-C₆alkyl)-(C₆-C₁₀aryl), wherein the C₁-C₆alkyl or —(C₁-C₆alkyl)-(C₆-C₁₀aryl) is optionally substituted with one or more R^Za;
  - each R^Zaindependently is halogen, C₁-C₆alkyl, or —O—(C₁-C₆alkyl), wherein the C₁-C₆alkyl or —O—(C₁-C₆alkyl) is optionally substituted with one or more halogen; and

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indicates an attachment to a nucleotide of the isolated oligonucleotide (e.g., siRNA).

In some embodiments, the phosphate mimic is linked to the 5′-terminus of the isolated oligonucleotides (e.g., siRNAs) as shown in the following formula:

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- wherein:
  - B is H or a nucleobase moiety;
  - X is O or S;
  - R¹is H or C₁-C₆alkyl;
  - R²is H or C₁-C₆alkyl;
  - Y¹is O or S;
  - Y²is O or S; and

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- - indicates an attachment to a nucleotide of the isolated oligonucleotide (e.g., siRNA).

In some embodiments, the phosphate mimic is linked to the 5′-terminus of the isolated oligonucleotides (e.g., siRNAs) as shown in the following formula:

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- wherein:
  - B is H or a nucleobase moiety;
  - X is O or S;
- R¹is H or C₁-C₆alkyl;
- R²is H or C₁-C₆alkyl; and

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indicates an attachment to a nucleotide of the isolated oligonucleotide (e.g., siRNA).

In some embodiments, X is O.

In some embodiments, X is S.

In some embodiments, R¹is H.

In some embodiments, R¹is C₁-C₆alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

In some embodiments, R¹is methyl.

In some embodiments, R²is H.

In some embodiments, R²is C₁-C₆alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

In some embodiments, R²is methyl.

In some embodiments, Y¹is O.

In some embodiments, Y¹is S.

In some embodiments, Y²is O.

In some embodiments, Y²is S.

In some embodiments, Z is H.

In some embodiments, Z is not H.

In some embodiments, Z is halogen (e.g., F, Cl, Br, or I).

In some embodiments, Z is F or Cl.

In some embodiments, Z is F

In some embodiments, Z is —OR^Z.

In some embodiments, Z is —OH.

In some embodiments, Z is not —OH.

In some embodiments, Z is —O—(C₁-C₆alkyl) (e.g., wherein the C₁-C₆alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

In some embodiments, Z is —OCH₃.

In some embodiments, Z is —O—(C₁-C₆alkyl)-O—(C₁-C₆alkyl) (e.g., wherein the C₁-C₆alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

In some embodiments, Z is —OCH₂CH₂OCH₃.

In some embodiments, Z is —O—(C₁-C₆alkyl)-(C₆-C₁₀aryl) optionally substituted with one or more R^Za.

In some embodiments, Z is —O—(C₁-C₆alkyl)-(C₆-C₁₀aryl).

In some embodiments, Z is

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In some embodiments, Z is

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optionally substituted with one or more R^Za.

In some embodiments, Z is

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optionally substituted with one or more halogen.

In some embodiments, Z is

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optionally substituted with one or more C₁-C₆alkyl or —O—(C₁-C₆alkyl), wherein the C₁-C₆alkyl or —O—C₁-C₆alkyl) is optionally substituted with one or more halogen.

In some embodiments, R^Zis H.

In some embodiments, R^Zis not H.

In some embodiments, R^Zis C₁-C₆alkyl (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.

In some embodiments, R^Zis C₁-C₆alkyl (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—(C₁-C₆alkyl) (e.g., wherein the C₁-C₆alkyl 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.

In some embodiments, R^Zis C₁-C₆alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

In some embodiments, R^Zis methyl, ethyl, or propyl.

In some embodiments, R^Zis methyl.

In some embodiments, R^Zis C₁-C₆alkyl (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).

In some embodiments, R^Zis C₁-C₆alkyl (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—(C₁-C₆alkyl) (e.g., wherein the C₁-C₆alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl), wherein the —O—(C₁-C₆alkyl) is optionally substituted with one or more halogen.

In some embodiments, IV is —(C₁-C₆alkyl)-(C₆-C₁₀aryl) optionally substituted with one or more R^Za.

In some embodiments, R^Zis —(C₁-C₆alkyl)-(C₆-C₁₀aryl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I), C₁-C₆alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl), or —O—(C₁-C₆alkyl) (e.g., wherein the C₁-C₆alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl), wherein the C₁-C₆alkyl or —O—(C₁-C₆alkyl) is optionally substituted with one or more halogen.

In some embodiments, R^Zis —(C₁-C₆alkyl)-(C₆-C₁₀aryl).

In some embodiments, at least one R¹is halogen (e.g., F, Cl, Br, or I).

In some embodiments, at least one R^Zais F or Cl.

In some embodiments, at least one R^Zais C₁-C₆alkyl (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).

In some embodiments, at least one R^Zais C₁-C₆alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

In some embodiments, at least one R^Zais C₁-C₆alkyl (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).

In some embodiments, at least one R^Zais —O—(C₁-C₆alkyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I).

In some embodiments, at least one R^Zais —O—(C₁-C₆alkyl).

In some embodiments, at least one R^Zais —O—(C₁-C₆alkyl) substituted with one or more halogen (e.g., F, Cl, Br, or I).

In some embodiments, B is H.

In some embodiments, B is a nucleobase moiety.

The term “nucleobase moiety”, as used herein, 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.

In some embodiments, the nucleobase moiety is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

In some embodiments, the nucleobase moiety is uracil (U).

In some embodiments, the phosphate mimic is linked to the 5′-terminus of the isolated oligonucleotides as shown in the following formula:

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- 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;
  - R¹is C₁alkyl;
  - R²is H; and

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indicates an attachment to a nucleotide of the isolated oligonucleotide (e.g., siRNA).

In some embodiments of the isolated oligonucleotides of the present disclosure, the phosphate mimic is attached to the 5′-terminus of the antisense strand of the isolated oligonucleotide.

In some embodiments, the phosphate mimic is attached to a 5′-terminal uridine of the anti sense strand of the isolated oligonucleotide, having the following structure (5′-MeEPmU).

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- wherein “mU” is a 2′-O-methyl modified uridine nucleotide and “MeEP” is a mono methyl protected phosphate mimic.

In some embodiments, the phosphate mimic is attached to a 5′-terminal uridine of the anti sense strand of the isolated oligonucleotide, having the following structure (5′-MeEPmUs).

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- wherein “mU” is a 2′-O-methyl modified uridine nucleotide, “MeEP” is a mono methyl protected phosphate mimic, and “s” is a phosphorothioate internucleotide linkage.

The terms “5′-MeEP”, “5′-MeEP”, and “5′ MeEP” are used interchangeably herein.

In some embodiments, wherein the MeEP is linked to the 5′ end of the antisense strand, the phosphate mimic is attached to a 5′-terminal uridine of the antisense strand.

In some embodiments, the 5′-terminal uridine is a 2′-O-methyl modified nucleotide.

Modified Backbone Phosphate/Phosphodiester Bond

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wherein

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denotes attachment to a 3′ carbon of a first nucleotide in the isolated oligonucleotide of the present disclosure; and

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denotes attachment to a 5′ carbon of a second nucleotide in the isolated oligonucleotide of the present disclosure. In some embodiments, the phosphodiester bond is unmodified, wherein Z¹is O and Z²is OH or O⁻. In some embodiments, the phosphodiester bond is modified, wherein Z¹is O, S, NH, or N(C₁-C₆alkyl) and Z²is OH, SH, NH₂, NH(C₁-C₆alkyl), O⁻, S⁻, HN⁻, or (C₁-C₆alkyl)N⁻, and wherein when Z¹is O, Z²is not OH or O⁻.

In some embodiments, Z₁is O.

In some embodiments, Z₁is S.

In some embodiments, Z₁is NH.

In some embodiments, Z₁is N(C₁-C₆alkyl).

In some embodiments, Z₂is OH.

In some embodiments, Z₂is SH.

In some embodiments, Z₂is NH₂.

In some embodiments, Z₂is NH(C₁-C₆alkyl).

In some embodiments, Z₂is SH, NH₂, or NH(C₁-C₆alkyl).

In some embodiments, Z₂is O⁻.

In some embodiments, Z₂is S.

In some embodiments, Z₂is HN⁻.

In some embodiments, Z₂is (C₁-C₆alkyl)N⁻.

In some embodiments, Z₂is S⁻, HN⁻, or (C₁-C₆alkyl)N⁻.

In some embodiments, Z₁is O and Z₂is SH.

In some embodiments, Z₁is O and Z₂is NH₂.

In some embodiments, Z₁is O and Z₂is NH(C₁-C₆alkyl).

In some embodiments, Z₁is S and Z₂is OH.

In some embodiments, Z₁is S and Z₂is SH.

In some embodiments, Z₁is S and Z₂is NH₂.

In some embodiments, Z₁is S and Z₂is NH(C₁-C₆alkyl).

In some embodiments, Z₁is NH and Z₂is OH.

In some embodiments, Z₁is NH and Z₂is SH.

In some embodiments, Z₁is NH and Z₂is NH₂.

In some embodiments, Z₁is NH and Z₂is NH(C₁-C₆alkyl).

In some embodiments, Z₁is N(C₁-C₆alkyl) and Z₂is OH.

In some embodiments, Z₁is N(C₁-C₆alkyl) and Z₂is SH.

In some embodiments, Z₁is N(C₁-C₆alkyl) and Z₂is NH₂.

In some embodiments, Z₁is N(C₁-C₆alkyl) and Z₂is NH(C₁-C₆alkyl).

In some embodiments, Z₁is O and Z₂is S.

In some embodiments, Z₁is O and Z₂is HN⁻.

In some embodiments, Z₁is O and Z₂is (C₁-C₆alkyl)N⁻.

In some embodiments, Z₁is S and Z₂is O⁻.

In some embodiments, Z₁is S and Z₂is S⁻.

In some embodiments, Z₁is S and Z₂is HN⁻.

In some embodiments, Z₁is S and Z₂is (C₁-C₆alkyl)N⁻.

In some embodiments, Z₁is NH and Z₂is O⁻.

In some embodiments, Z₁is NH and Z₂is S⁻.

In some embodiments, Z₁is NH and Z₂is HN⁻.

In some embodiments, Z₁is NH and Z₂is (C₁-C₆alkyl)N⁻.

In some embodiments, Z₁is N(C₁-C₆alkyl) and Z₂is O⁻.

In some embodiments, Z₁is N(C₁-C₆alkyl) and Z₂is S⁻.

In some embodiments, Z₁is N(C₁-C₆alkyl) and Z₂is HN⁻.

In some embodiments, Z₁is N(C₁-C₆alkyl) and Z₂is (C₁-C₆alkyl)N⁻.

In some embodiments, the modified phosphodiester bond comprises a phosphorothioate internucleotide linkage.

In some embodiments, the modified phosphodiester bond comprises

embedded image

wherein

embedded image

denotes attachment to a 3′ carbon of a first nucleotide in the isolated oligonucleotide of the present disclosure; and

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denotes attachment to a 5′ carbon of a second nucleotide in the isolated oligonucleotide of the present disclosure.

In some embodiments, the modified phosphodiester bond comprises

embedded image

wherein

embedded image

denotes attachment to a 3′ carbon of a first nucleotide in the isolated oligonucleotide of the present disclosure; and

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denotes attachment to a 5′ carbon of a second nucleotide in the isolated oligonucleotide of the present disclosure.

In some embodiments, the modified phosphodiester bond comprises

embedded image

wherein

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denotes attachment to a 3′ carbon of a first nucleotide in the denotes attachment to a 5′ carbon of a isolated oligonucleotide of the present disclosure; and

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second nucleotide in the isolated oligonucleotide of the present disclosure.

In some embodiments, the isolated oligonucleotide of the present disclosure comprises at least one modified phosphodiester bond(s). In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand or the antisense strand or both comprise one or more modified phosphodiester bonds. In some embodiments, only the sense strand comprises one or more modified phosphodiester bonds. In some embodiments, only the anti sense strand comprises one or more modified phosphodiester bonds. In some embodiments, both the sense strand and antisense strand comprise one or more modified phosphodiester bonds.

In some embodiments, 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. In some embodiments, 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. some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. some embodiments, 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. In some embodiments, 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 anti sense 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. In some embodiments, 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. In some embodiments, the sense strand and/or the anti sense 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. In some embodiments, 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.

In some embodiments, 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 anti sense 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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. In some embodiments, wherein 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. In some embodiments, the antisense strand comprises four modified phosphodiester bonds. In some embodiments, wherein the antisense strand comprises four modified phosphodiester bonds. the modified phosphodiester bonds comprise phosphorothioate.

In some embodiments, wherein the anti 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 antisense strand. In some embodiments, wherein 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 anti sense strand. In some embodiments, wherein the anti sense 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. In some embodiments, wherein 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. In some embodiments, wherein 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises four phosphorothioate internucleotide linkages. In some embodiments, wherein the anti sense 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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. In some embodiments, wherein 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. In some embodiments, the sense strand comprises four modified phosphodiester bonds. In some embodiments, wherein the sense strand comprises four modified phosphodiester bonds, the modified phosphodiester bonds comprise phosphorothioate internucleotide linkages.

In some embodiments, wherein 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. In some embodiments, wherein 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. In some embodiments, wherein 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. In some embodiments, wherein 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. In some embodiments, wherein 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. In some embodiments, wherein 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein 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, and 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.

In some embodiments of the isolated oligonucleotide of the present disclosure, the antisense strand comprises any one of: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 440 (5′ [McEPmUs][fAs][fA][mU][fG][mU][fG][mA][mA][fA][mU][mA][mA][fA][mG][fC][mU][mU][mU][mGs][mCs][mA] 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 442 (5′ [MeEPmUs][fGs][fA][mG][fA][mC][fA][mU][mG][fA][mG][mG][mU][fU][mU][fU][mG][m A][mU][mAs][mCs][mC] 3′); or iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 444 (5′ [McEPmUs][fUs][fA][mU][fU][mA][fA][mU][mA][fU][mC][mC][mC][fA][mC][fA][mG][mA][mA][mCs][mCs][mA] 3′), wherein “m” is a 2′-O-methyl modified nucleotide, “f” is a 2′-F modified nucleotide, “s” is a phosphorothioate internucleotide linkage, “MeEP” is a mono methyl protected phosphate mimic.

In some embodiments of the isolated oligonucleotide of the present disclosure, wherein the sense strand comprises any one of: i) a sense strand of nucleic acid sequence according to SEQ ID NO: 441 (5′ [mCs][mAs][mA][mA][mG][ft][mU][fU][fU][fA][fU][mU][mU][mC][mA][mC][mA][mUs][m Us][mA][G1b][G1b][G1b] 3′); ii) a sense strand of nucleic acid sequence according to SEQ ID NO: 443 (5′ [mUs][mAs][mU][mC][mA][fA][mA][fA][fC][ft][fU][mC][mA][mU][mG][mU][mC][mUs][m Cs][mA][G1b][G1b][G1b] 3′); or iii) a sense strand of nucleic acid sequence according to SEQ ID NO: 445 (5′ [mGs][mUs][mU][mC][mU][fG][mU][M][fG][fG][fA][mU][mA][mU][mU][mA][mA][mUs][m As][mA][G1b][G1b][G1b] 3′), wherein “m” is a 2′-O-methyl modified nucleotide, “f” is a 2′-F modified nucleotide, “s” is a phosphorothioate internucleotide linkage, and “G1b” is a GalNac G1b moiety.

In some embodiments of the isolated oligonucleotide of the present disclosure is selected from: i) an antisense strand of SEQ ID NO: 440 (5′ [McEPmUs][fAs][fA][mU][fG][mU][fG][mA][mA][fA][mU][mA][mA][fA][mG][fC][mU][mU][mU][mGs][mCs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 441 (5′ [mCs][mAs][mA][mA][mG][fC][mU][fU][fU][fA][fU][mU][mU][mC][mA][mC][mA][mUs][m Us][mA][G1b][G1b][G1b] 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 442 (5′ [McEPmUs][fGs][fA][mG][fA][mC][fA][mU][mG][fA][mG][mG][mU][fU][mU][fU][mG][mA][mU][mAs][mCs][mC] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 443 (5′ [mUs][mAs][mU][mC][mA][fA][mA][fA][fC][ft][fU][mC][mA][mU][mG][mU][mC][mUs][m Cs][mA][G1b][G1b][G1b] 3′); and iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 444 (5′ [MeEPmUs][fUs][fA][mU][fU][mA][fA][mU][mA][fU][mC][mC][mC][fA][mC][fA][mG][mA][mA][mCs][mCs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 445 (5′ [mGs][m Us][mU][mC][mU][fG][mU][fG][fG][fG][fA][mU][mA][mU][mU][mA][mA][mUs][m As][mA][G1b][G1b][G1b] 3′), wherein “m” is a 2′-O-methyl modified nucleotide, “f” is a 2′-F modified nucleotide, “s” is a phosphorothioate internucleotide linkage, “MeEP” is a mono methyl protected phosphate mimic, and “G1b” is a GalNAc G1b moiety.

Nucleic Acids and Vectors

The present disclosure also provides a vector encoding an isolated oligonucleotide disclosed herein. In some embodiments, the vector is any one of a plasmid, a cosmid or a viral vector. In some embodiments, the vector is an adenoviral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the plasmid is an expression plasmid.

The disclosure provides nucleic acids comprising the sequences encoding the isolated oligonucleotides of the present disclosure (e.g., dsRNAs or siRNAs) targeting HSD17B13 described herein.

In some embodiments, 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. The term “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. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. For example, 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. Alternatively, 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.

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.

In some embodiments, 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 (e.g., dsRNAs and siRNAs) 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. Examples of commercially available plasmid-based expression vectors for shRNA include members of the pSilencer series (Ambion, Austin. Tex.) and pCpG-siRNA (InvivoGen. San Diego, Calif.). Examples of kits for production of PCR-generated shRNA expression cassettes include Silencer Express (Ambion, Austin, Tex.) and siXpress (Minis, Madison. Wis.).

Viral vectors for the in vivo expression of the isolated oligonucleotides (e.g., siRNAs and dsRNAs) in eukaryotic cells 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 vino. Examples of commercially available viral vectors for shRNA expression include pSilencer adeno (Ambion, Austin, Tex.) and pLenti6BLOCK-iT™-DEST (Invitrogen, Carlsbad, Calif.). 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.

It will be apparent to those skilled in the art that any suitable vector, optionally incorporated into a nanoparticle, can be used to deliver the isolated oligonucleotides of the present disclosure (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.

Delivery Systems

The present disclosure also provides a delivery system comprising the isolated oligonucleotide disclosed herein or vector of the present disclosure encoding an isolated oligonucleotide disclosed herein. In some embodiments, the delivery system is any one of a liposome, a nanoparticle, a polymer based delivery system or a ligand-conjugate delivery system. In some embodiments, the ligand-conjugate delivery system comprises one or more of an antibody, a peptide, a sugar moiety or a combination thereof.

In some embodiments, the delivery system of the present disclosure comprise nanoparticles comprising the isolated oligonucleotides of the present disclosure (e.g., siRNA or dsRNAs) targeting a HSD17B13 mRNA for degradation.

In some embodiments, the nanoparticle comprises a polymer-based nanoparticle, a lipid-polymer based nanoparticle, a metal based nanoparticle, a carbon nanotube based nanoparticle, a nanocrystal or a polymeric micelle. In some embodiments, the polymer-based nanoparticle comprises a multiblock copolymer, a deblock copolymer, a polymeric micelle or a hyperbranched macromolecule. In some embodiments, 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.

In some embodiments, the delivery system comprises a liposome. Liposomes are spherical vesicles having at least one lipid bilayer, and in some embodiments, an aqueous core. In some embodiments, 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 μM and larger, or 200 nm to 3 μM. 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the nanoparticle comprises a polymer based nanoparticle. In some embodiments, the polymer comprises a multiblock copolymer, a diblock copolymer, a polymeric micelle or a hyperbranched macromolecule. In some embodiments, the particle comprises one or more cationic polymers. In some embodiments, the cationic polymer is chitosan, protamine, polylysine, polyhistidine, polyarginine or poly(ethylene)imine. In other embodiments, the one or more polymers contain the buffering component, degradable component, hydrophilic component, cleavable bond component or some combination thereof.

In some embodiments, the nanoparticles or some portion thereof are degradable. In other embodiments, the lipids and/or polymers of the nanoparticles are degradable.

In some embodiments, any of these delivery systems of the present disclosure can comprise a buffering component. In other embodiments, any of the of the present disclosure can comprise a buffering component and a degradable component. In still other embodiments, any of the of the present disclosure can comprise a buffering component and a hydrophilic component. In yet other embodiments, any of the of the present disclosure can comprise a buffering component and a cleavable bond component. In yet other embodiments, any of the of the present disclosure can comprise a buffering component, a degradable component and a hydrophilic component. In still other embodiments, any of the of the present disclosure can comprise a buffering component, a degradable component and a cleavable bond component. In further embodiments, any of the of the present disclosure can comprise a buffering component, a hydrophilic component and a cleavable bond component. In yet another embodiment, any of the of the present disclosure can comprise a buffering component, a degradable component, a hydrophilic component and a cleavable bond component. In some embodiments, the particle is composed of one or more polymers that contain any of the aforementioned combinations of components.

In some embodiments of the isolated oligonucleotides of the present disclosure, the delivery system comprises a ligand-conjugate delivery system. In some embodiments, the ligand-conjugate delivery system comprises one or more of an antibody, a peptide, a sugar moiety, lipid or a combination thereof

In further embodiments, the isolated oligonucleotide of the present disclosure targeting a HSD17B13 mRNA (e.g., siRNA or dsRNA) is conjugated to, complexed to, or encapsulated by the one or more lipids or polymers of the delivery system. In further embodiments, the isolated oligonucleotide of the present disclosure targeting a HSD17B13 mRNA (e.g., siRNA or dsRNA) can be encapsulated in the hollow core of a nanoparticle. Alternatively, or in addition, the isolated oligonucleotide of the present disclosure targeting a HSD17B13 mRNA (e.g., siRNA or dsRNA) can be incorporated into the lipid or polymer based shell of the delivery system, for example via intercalation. Alternatively, or in addition, the isolated oligonucleotide of the present disclosure targeting a HSD17B13 mRNA (e.g., siRNA or dsRNA) can be attached to the surface of the delivery system. In some embodiments, the isolated oligonucleotide of the present disclosure targeting a HSD17B13 mRNA (e.g., siRNA or dsRNA) is conjugated to one or more lipids or polymers of the delivery system, e.g., via covalent attachment.

In some embodiments, the ligand conjugate delivery system further comprises a targeting agent. In some embodiments, 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.

In some embodiments, 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. In certain embodiments, the ligand targets delivery of the RNAi construct to hepatocytes. In these and other embodiments, the ligand may comprise galactose, galactosamine or N-acetyl-galactosamine (GaLNAc). In certain embodiments, 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.

In some embodiments, 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 HSD17B13 mRNA and expressing HSD17B13 protein. In some embodiments, 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. In some embodiments, 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.

In some embodiments, the delivery system of the present disclosure comprises a polymer based delivery system. In some embodiments, polymer based delivery system comprises a blending polymer. In some embodiments, the blending polymer is a copolymer comprising a degradable component and hydrophilic component. In some embodiments, 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). In some embodiments, the degradable component of the blending polymer is poly(lactic acid) (PLA) or poly(lactic-co-glycolic acid) (PLGA). In some embodiments, the hydrophilic component of the blending polymer is a polyalkylene glycol or a polyalkylene oxide. In some embodiments, the polyalkylene glycol is polyethylene glycol (PEG). In other embodiments, the polyalkylene oxide is polyethylene oxide (PEO).

In some embodiments, the delivery system of the present disclosure is a polymer based nanoparticle. Polymer based nanoparticles comprise one or more polymers. In some embodiments, the one or more polymers comprise a polyester, poly(ortho ester), poly(ethylene imine), poly(caprolactone), polyanhydride, poly(acrylic acid), polyglycolide or poly(urethane). In still other embodiments, the one or more polymers comprise poly(lactic acid) (PLA) or poly(lactic-co-glycolic acid) (PLGA). In some embodiments, the one or more polymers comprise poly(lactic-co-glycolic acid) (PLGA). In some embodiments, the one or more polymers comprise poly(lactic acid) (PLA). In some embodiments, the one or more polymers comprise polyalkylene glycol or a polyalkylene oxide. In some embodiments, the polyalkylene glycol is polyethylene glycol (PEG) or the polyalkylene oxide is polyethylene oxide (PEO).

In some embodiments, the polymer-based nanoparticle comprises poly(lactic-co-glycolic acid) PLGA polymers. In some embodiments, the PLGA nanoparticle further comprises a targeting agent, as described herein.

In some embodiments, 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. In other embodiments, 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. In further embodiments, 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 or 200-300 nm.

Therapeutic Agents

In some embodiments, the delivery system of the present disclosure are administered with one or more additional therapeutic agents. In some embodiments, the additional therapeutic agents can be a steroid, an anti-inflammatory agent, an antibody, a fusion protein, a small molecule or combination thereof.

In some embodiments, the additional therapeutic agent is incorporated into a delivery system of the present disclosure comprising at least one isolated oligonucleotide targeting HSD17B13, disclosed herein. In some embodiments, 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. Alternatively, or in addition, Additional therapeutic agents can be incorporated into the lipid or polymer based shell of the delivery system, for example via intercalation. Alternatively, or in addition, additional therapeutic agents can be attached to the surface of the delivery system. In some embodiments, the additional therapeutic agents are conjugated to one or more lipids or polymers of the delivery system, e.g. via covalent attachment.

In some embodiments, the additional therapeutic agent and the delivery system at least one isolated oligonucleotide targeting HSD17B13, disclosed herein, are formulated in the same composition. For example, the delivery system comprising isolated oligonucleotide of the present disclosure targeting HSD17B13 and the additional therapeutic agent can be formulated in the same pharmaceutical composition.

In some embodiments, the additional therapeutic agent and the delivery system comprises at least one isolated oligonucleotide targeting HSD17B13, disclosed herein are formulated as separate compositions, e.g., for separate administration to a subject.

Pharmaceutical Compositions

The present disclosure also provides a pharmaceutical composition comprising: an isolated oligonucleotide disclosed herein, a vector of the present disclosure encoding an isolated oligonucleotide disclosed herein, or a delivery system of the present disclosure, and a pharmaceutically acceptable carrier, diluent, or excipient.

The pharmaceutical compositions of the disclosure can optionally comprise therapeutic agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.

In some embodiments, the pharmaceutical composition comprises a therapeutic agent, such as a chemotherapeutic agent. In some embodiments, the therapeutic agent is formulated in the delivery system comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 of the present disclosure.

In some embodiments, an additional therapeutic agent is not formulated in the delivery system comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 of the present disclosure, but both the delivery system and the therapeutic agent are formulated in the same pharmaceutical composition. In some embodiments, an additional therapeutic agent is not formulated in the delivery system comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 of the present disclosure, and the delivery system and the therapeutic agent are formulated in separate pharmaceutical compositions.

Pharmaceutical compositions can contain any of the reagents discussed above, and one or more of a pharmaceutically acceptable carrier, a diluent or an excipient.

A pharmaceutical composition is in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler or a vial. The quantity of active ingredient (e.g., a formulation of the disclosed agent) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In one embodiment, the active agent is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, anions, cations, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

“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. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), intraperitoneal (into the body cavity) and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, intraperitoneal or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. These preparations can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The formulations can be presented in unit/dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.

The pharmaceutical 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.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required 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. In many cases, it will be preferable to include 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.

For administration by inhalation, 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.

The pharmaceutical compositions of the present disclosure can be prepared with pharmaceutically acceptable carriers that will protect the one or more isolated oligonucleotides (e.g., dsRNAs or siRNAs) targeting HSD17B13 mRNA of the present disclosure against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art, and the materials can be obtained commercially. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active agent and the particular therapeutic effect to be achieved.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

As used herein, “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. Examples of 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.

Techniques for formulation and administration of the disclosed compositions of the disclosure can be found in Remington: the Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, PA (1995).

All percentages and ratios used herein, unless otherwise indicated, are by weight. Other features and advantages of the present disclosure are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present disclosure. The examples do not limit the claimed disclosure. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present disclosure.

Methods of Making Isolated Oligonucleotides

Provided herein are methods of making the one or more oligonucleotides of (e.g., dsRNAs or siRNAs) targeting HSD17B13 of the present disclosure, and delivery systems comprising same.

The one or more oligonucleotides of (e.g., dsRNAs or siRNAs) targeting HSD17B13 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.

In some embodiments, the one or more oligonucleotides of (e.g., dsRNAs or siRNAs) targeting HSD17B13 of the present disclosure can be produced using an expression vector into which a nucleic acid encoding the double stranded RNA has been cloned, for example under control of a suitable promoter.

In some embodiments, the one or more oligonucleotides of (e.g., dsRNAs or siRNAs) targeting HSD17B13 of the present disclosure can be incorporated in a delivery system of the present disclosure (e.g., a nanoparticle).

Delivery systems comprising dsRNAs or siRNAs of the disclosure can be prepared by any suitable means known in the art. For example, polymeric nanoparticles can be prepared using various methods including, but not limited to, solvent evaporation, spontaneous emulsification, solvent diffusion, desolation, dialysis, ionic gelation, nanoprecipitation, salting out, spray drying and supercritical fluid methods. The dispersion of preformed polymers and the polymerization of monomers are two additional strategies for preparation of polymeric nanoparticles. However, the choice of an appropriate method depends upon various factors, which will be known to the person of ordinary skill in the art.

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 HSD17B13 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. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze drying that yields a powder of delivery system comprising the one or more isolated oligonucleotides (e.g., dsRNA and siRNA) targeting HSD17B13 disclosed herein, plus any additional desired ingredient from a previously sterile filtered solution thereof.

Methods of Use

The present disclosure also provides a method of inhibiting or downregulating the expression or level of HSD17B13 in a subject in need thereof, wherein the method comprises administering to the subject an effective amount an isolated oligonucleotide disclosed herein, a vector of the 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 present disclosure also provides a method of treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 or a disease or disorder where HSD17B13 plays a role in a subject in need thereof, wherein the method comprises administering to the subject an effective amount an isolated oligonucleotide disclosed herein, a vector of the 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 present disclosure also provides an isolated oligonucleotide disclosed herein, a vector of the 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, for use in treatment or prevention of a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 or a disease or disorder where HSD17B13 plays a role, in a subject in need thereof.

The present disclosure also provides use of an isolated oligonucleotide disclosed herein, a vector of the 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, in the manufacture of a medicament for treatment or prevention of a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 or a disease or disorder where HSD17B13 plays a role in a subject in need thereof.

Provided herein are methods of inhibiting or downregulating HSD17B13 expression or activity in a cell, comprising contacting the cell with the one or more oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 as described herein. The one or more oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 as described herein can reduce or inhibit HSD17B13 activity through the RNAi pathway. The cell can be in vitro, in vivo or ex vivo. For example, the cell can be from a cell line, or in vivo in a subject in need thereof.

In some embodiments, the one or more oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 as described herein are capable of inducing RNAi-mediated degradation of an HSD17B13 mRNA in a cell of a subject.

As used herein, the terms “contacting,” “introducing” and “administering” are used interchangeably, and refer to a process by which 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.

The term “inhibit” 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%).

In contrast, the term “increase” or grammatical variations thereof as used herein refers to an increase or elevation 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. Increases in activity can be described in terms of fold change. For example, activity can be increased 1.2×, 1.5×, 2×, 3×, 5×, 6×, 7×, 8×, 9×, 10× or more compared to a baseline level of activity.

As used herein, the term “IC50” or “IC₅₀value” refers to the concentration of an agent where cell viability is reduced by half. The IC₅₀is thus a measure of the effectiveness of an agent in inhibiting a biological process. In an exemplary model, cell lines are cultured using standard techniques, treated with any of the one or more oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 as described herein, and the IC₅₀value of the oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 is calculated after 24, 48 and/or 72 hours to determine its effectiveness in downregulating or inhibiting the level of HSD17B13 mRNA or protein to 50%, as compared to the level of HSD17B13 mRNA or protein in an untreated cell or in the same cell before initiation of treatment with the isolated oligonucleotide.

Methods of monitoring of HSD17B13 mRNA and/or protein expression can be used to characterize gene silencing, and to determine the effectiveness of the compositions described herein. Expression of HSD17B13 may be evaluated by any known technique. Examples thereof include immunoprecipitations methods, utilizing HSD17B13 antibodies in assays such as ELISAs, Western Blot, or immunohistochemistry to visualize HSD17B13 protein expression in cells, or flow cytometry. Additional methods include various hybridization methods utilizing a nucleic acid that specifically hybridizes with a nucleic acid encoding HSD17B13 or a unique fragment thereof, or a transcription product (e.g., mRNA) or splicing product of said nucleic acid, Northern Blot methods, Southern blot methods, and various PCR-based methods such as RT-PCR, qPCR or digital droplet PCR. HSD17B13 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 HSD17B13 include transfecting representative cell lines with isolated oligonucleotides, and measuring viability. For example, cells from representative cell lines can be transfected using methods known in the art, such as the Lipofectamine RNAiMAX (Invitrogen-13778-150, Carlsbad, CA), and cultured using any suitable technique known in the art. Optionally additional therapeutic agents as described herein can be added at variable concentrations to cell culture media following transfection. Following a suitable incubation period, such as 24-96 hours, cell viability can be measured using methods such as Cell Titer Glo 2.0 (Promega, CA) to determine cell viability, and/or HSD17B13 mRNA and protein levels can be assessed using the methods described herein.

In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, wherein the isolated oligonucleotide, the vector, the delivery system, or the pharmaceutical composition is administered parenterally.

In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, wherein the parenteral administration is intravenous, subcutaneous, intraperitoneal, or intramuscular.

In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, the subject is a human. In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, the subject has nonalcoholic fatty liver disease (NAFLD) and related liver diseases like non-alcoholic steatohepatitis (NASH). In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, the subject has nonalcoholic fatty liver disease (NAFLD), fatty liver disease, liver injury, inflammation, fibrosis, cirrhosis, or carcinoma. In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, the method comprises administering the isolated oligonucleotide, the vector, the delivery system, or the pharmaceutical composition, in combination with at least a second therapeutic agent.

In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, the second therapeutic agent is an antibody, a small molecule drug, a peptide, a nucleotide molecule, or a combination thereof.

In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, the second therapeutic agent is an isolated oligonucleotide of the present disclosure.

In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, wherein the first and at least second oligonucleotides are administered simultaneously.

In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, the subject is a human. In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, the subject has nonalcoholic fatty liver disease (NAFLD) and related liver diseases like non-alcoholic steatohepatitis (NASH). In some embodiments of the methods of inhibiting or downregulating HSD17B13 expression or activity in a cell of the present disclosure, the subject has nonalcoholic fatty liver disease (NAFLD), fatty liver disease, liver injury, inflammation, fibrosis, cirrhosis, or carcinoma. In some embodiments of the method of treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 or a disease or disorder where HSD17B13 plays a role of the present disclosure, the subject is a human. In some embodiments of the method of treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 or a disease or disorder where HSD17B13 plays a role of the present disclosure, the disease or disorder is nonalcoholic fatty liver disease (NAFLD) and related liver diseases like non-alcoholic steatohepatitis (NASH).

In some embodiments of the use for treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 or a disease or disorder where HSD17B13 plays a role of the present disclosure, the subject is a human. In some embodiments of the use for treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 or a disease or disorder where HSD17B13 plays a role of the present disclosure, the disease or disorder is nonalcoholic fatty liver disease (NAFLD) and related liver diseases like non-alcoholic steatohepatitis (NASH). In some embodiments of the use for treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 or a disease or disorder where HSD17B13 plays a role of the present disclosure, the subject has nonalcoholic fatty liver disease (NAFLD), fatty liver disease, liver injury, inflammation, fibrosis, cirrhosis, or carcinoma. In some embodiments of the use in the manufacture of a medicament for treatment or prevention of a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 of the present disclosure, the subject is a human. In some embodiments of the use in the manufacture of a medicament for treatment or prevention of a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 of the present disclosure of the present disclosure, the disease or disorder is nonalcoholic fatty liver disease (NAFLD) and related liver diseases like non-alcoholic steatohepatitis (NASH). The treatment or prevention of a disease or disorder is associated with aberrant or increased expression or activity of HSD17B13.

Routes of Administration

Nanoparticles comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 mRNA of the present disclosure can be administered to a subject by many of the well-known methods currently used for therapeutic treatment. For example, for treatment of mammalian diseases associated with expression or activity of HSD17B13, a compositions comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 mRNA of the present disclosure may be injected directly into cells, injected into the blood stream or body cavities or 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.

The compositions comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 mRNA of the present disclosure can be administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperintoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and parenterally. In some embodiments, the parenteral administration comprises intramuscular, intraperitoneal, subcutaneous or intravenous administration. One skilled in the art will recognize the advantages of certain routes of 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. For intravenous parenteral administration, compositions comprising nanoparticles may be administered by injection or by infusion. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.

Dosages

In therapeutic applications, 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. Generally, 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 HSD17B13. Dosages may vary depending on the age and size of the subject and the type and severity of the disease or disorder associated with HSD17B13 expression.

The term “effective amount” or “therapeutically effective amount”, as used interchangeably herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, inhibit, downregulate or control the expression of HSD17B13 or symptoms associated with aberrant or abnormal expression of HSD17B13 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.

For any of the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 mRNA of the present disclosure, 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. In some embodiments, 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 HSD17B13 mRNA of the present disclosure. Such information can then be used to determine useful doses and routes for administration in humans. 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 HSD17B13 mRNA of the present disclosure, required 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). 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) may increase the efficiency of delivery, particularly for oral delivery.

A therapeutically effective dose of the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 mRNA of the present disclosure, can optionally be combined with approved amounts of therapeutic agents, and described herein.

Kits and Articles of Manufacture

The present disclosure also provides a kit comprising an 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 kits are for use in the treatment of diseases related to abnormal or aberrant expression of HSD17B13, in a mammal. The kits are for use in downregulating or inhibiting expression of HSD17B13 partially or completely, in a mammal. In some embodiments, 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. In some embodiments, the mammal is a human

Nanoparticles comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HSD17B13 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.

In some embodiments of the kits of the disclosure, 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 HSD17B13 (dsRNA or siRNA), and instructions for use of the same. In some embodiments, the kit further comprises at least one additional therapeutic agents, as described herein.

Articles of manufacture include, but are not limited to, instructions for use of the kit in treating diseases related to abnormal or aberrant expression of HSD17B13 or diseases related to expression of HSD17B13.

In some embodiments, the kits further comprise instructions for administering the isolated oligonucleotides, the vector, the delivery systems and the pharmaceutical compositions of the disclosure.

All percentages and ratios used herein, unless otherwise indicated, are by weight. Other features and advantages of the present disclosure are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present disclosure. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present disclosure

TABLE 2

Exemplary Sequences of the Present Application and Their in vitro Potency in

Silencing Human HSD17B13 mRNA

Passenger

% gene
% gene

Guide

Guide strand

strand

remaining at
remaining at

Strand
Guide
Guide
sequence
SEQ ID
sequence
SEQ ID
0.05 nM
0.5 nM

position
start
end
(5′-3′)
NO
(5′-3′)
NO
Mean
SEM
Mean
SEM

28
7
27
UCUCUUCCUUCU
61
ACUGAACCAGA
249
94.2
4.21
98.80
5.21

GGUUCAGUCC

AGGAAGAGA

78
57
77

UGAUCAGAAGC

62

AAUCCUUCUG

250
90.17
2.96
39.67
2.42

AGAAGGAUUUC

CUUCUGAUCA

83
62
82

UAUGGUGAUCA

63

UUCUGCUUCU

251
90.63
2.61
46.03
1.07

GAAGCAGAAGG

GAUCACCAUA

89
68
88

UUAGAUGAUGG

64

UUCUGAUCAC

252
65.43
1.36
18.53
0.55

UGAUCAGAAGC

CAUCAUCUAA

90
69
89

UGUAGAUGAUG

65

UCUGAUCACC

253
102.13
6.19
75.00
2.58

GUGAUCAGAAG

AUCAUCUACA

92
71
91

UGAGUAGAUGA

66

UGAUCACCAU

254
95.50
3.08
61.53
1.77

UGGUGAUCAGA

CAUCUACUCA

96
75
95
UGUAGGAGUAG
67
CACCAUCAUCU
255
109.40
8.45
98.20
12.11

AUGAUGGUGAU

ACUCCUACA

99
78
98
UCAAGUAGGAG
68
CAUCAUCUACU
256
108.93
2.23
95.07
5.49

UAGAUGAUGGU

CCUACUUGA

102
81
101
UCUCCAAGUAG
69
CAUCUACUCCU
257
102.50
7.95
105.60
5.15

GAGUAGAUGAU

ACUUGGAGA

250
229
249

UUAUUAAUAUC

29

GUUCUGUGGG

58
52.57
3.10
14.83
1.45

CCACAGAACCA

AUAUUAAUAA

251
230
250

UUUAUUAAUAU

70

UUCUGUGGGA

258
70.97
6.74
16.03
1.48

CCCACAGAACC

UAUUAAUAAA

252
231
251

UCUUAUUAAUA

71

UCUGUGGGAU

259
89.07
3.58
53.60
2.89

UCCCACAGAAC

AUUAAUAAGA

281
260
280

UCACUCAGCUG

72

AGGAAACUGC

260
103.80
3.45
74.50
0.55

CAGUUUCCUCC

AGCUGAGUGA

288
267
287

UUUUUCGGCAC

73

UGCAGCUGAG

261
80.50
2.86
39.60
0.76

UCAGCUGCAGU

UGCCGAAAAA

289
268
288

UGUUUUCGGCA

74

GCAGCUGAGU

262
101.77
2.35
72.47
5.40

CUCAGCUGCAG

GCCGAAAACA

291
270
290

UUAGUUUUCGG

75

AGCUGAGUGC

263
76.50
7.07
38.97
1.27

CACUCAGCUGC

CGAAAACUAA

292
271
291

UCUAGUUUUCG

76

GCUGAGUGCC

264
86.77
5.25
58.23
2.63

GCACUCAGCUG

GAAAACUAGA

300
279
299
UAGUGACGCCU
77
CCGAAAACUAG
265
111.83
6.74
88.57
7.83

AGUUUUCGGCA

GCGUCACUA

301
280
300

UCAGUGACGCC

78

CGAAAACUAG

266
105.93
8.78
80.00
6.26

UAGUUUUCGGC

GCGUCACUGA

311
290
310

UUACGCAUGCG

79

GCGUCACUGC

267
95.83
1.72
54.63
4.11

CAGUGACGCCU

GCAUGCGUAA

360
339
359

UCUGAUUUAGA

80

CUAUCGCUCU

268
68.77
3.28
22.90
1.70

GAGCGAUAGAU

CUAAAUCAGA

365
344
364

UUUCACCUGAU

10

GCUCUCUAAA

39
48.63
8.06
18.57
0.90

UUAGAGAGCGA

UCAGGUGAAA

366
345
365

UCUUCACCUGA

81

CUCUCUAAAUC

269
62.43
1.07
30.47
1.78

UUUAGAGAGCG

AGGUGAAGA

369
348
368

UUUUCUUCACC

82

UCUAAAUCAG

270
75.20
4.76
32.63
1.66

UGAUUUAGAGA

GUGAAGAAAA

415
394
414

UAUACUGUCCC

83

AAUAAUGCUG

271
96.33
7.08
71.70
0.17

AGCAUDAUUCA

GGACAGUAUA

417
396
416

UAUAUACUGUC

84

UAAUGCUGGG

272
85.93
2.50
43.93
2.05

CCAGCAUUAUU

ACAGUAUAUA

418
397
417

UGAUAUACUGU

85

AAUGCUGGGA

273
100.90
5.75
65.40
5.27

CCCAGCAUUAU

CAGUAUAUCA

427
406
426

UGAUCGGCUGG

86

ACAGUAUAUC

274
98.73
3.89
73.13
1.89

AUAUACUGUCC

CAGCCGAUCA

429
408
428

UAAGAUCGGCU

87

AGUAUAUCCA

275
81.60
4.31
38.93
2.70

GGAUAUACUGU

GCCGAUCUUA

453
432
452

UAAUCUCUUCA

88

CACCAAGGAU

276
89.13
4.31
31.13
3.42

UCCUUGGUGCU

GAAGAGAUUA

454
433
453

UUAAUCUCUUC

89

ACCAAGGAUG

277
68.53
1.74
20.73
0.38

AUCCUUGGUGC

AAGAGAUUAA

455
434
454

UGUAAUCUCUU

90

CCAAGGAUGA

278
76.63
5.06
33.83
1.04

CAUCCUUGGUG

AGAGAUUACA

480
459
479

UUAGGAUGUUG

91

AUUUGAGGUC

279
90.40
5.79
49.77
2.57

ACCUCAAAUGU

AACAUCCUAA

484
463
483

UGUCCUAGGAU

92

GAGGUCAACA

280
98.77
8.00
54.93
4.68

GUUGACCUCAA

UCCUAGGACA

486
465
485

UAUGUCCUAGG

93

GGUCAACAUC

281
100.50
5.61
80.13
8.88

AUGUUGACCUC

CUAGGACAUA

487
466
486

UAAUGUCCUAG

94

GUCAACAUCC

282
98.50
1.19
33.03
1.78

GAUGUUGACCU

UAGGACAUUA

488
467
487

UAAAUGUCCUA

95

UCAACAUCCUA

283
106.27
11.23
51.03
7.18

GGAUGUUGACC

GGACAUUUA

489
468
488

UAAAAUGUCCU

96

CAACAUCCUA

284
110.20
12.39
58.90
1.53

AGGAUGUUGAC

GGACAUUUUA

490
469
489

UAAAAAUGUCC

97

AACAUCCUAG

285
125.23
4.44
71.40
6.81

UAGGAUGUUGA

GACAUUUUUA

497
476
496

UGUGAUCCAAA

11

UAGGACAUUU

40
64.03
13.72
17.23
3.18

AAUGUCCUAGG

UUGGAUCACA

499
478
498

UUUGUGAUCCA

98

GGACAUUUUU

286
76.77
2.86
29.57
3.73

AAAAUGUCCUA

GGAUCACAAA

500
479
499

UUUUGUGAUCC

99

GACAUUUUUG

287
86.73
4.49
37.73
4.39

AAAAAUGUCCU

GAUCACAAAA

501
480
500

UUUUUGUGAUC

100

ACAUUUUUGG

288
110.67
11.50
59.40
7.45

CAAAAAUGUCC

AUCACAAAAA

502
481
501
UCUUUUGUGAU
101
CAUUUUUGGAU
289
110.30
2.46
95.00
3.13

CCAAAAAUGUC

CACAAAAGA

511
490
510

UGAAGAAGUGC

102

AUCACAAAAG

290
79.67
5.81
49.07
1.05

UUUUGUGAUCC

CACUUCUUCA

514
493
513

UAUGGAAGAAG

103

ACAAAAGCAC

291
86.33
4.25
54.07
1.33

UGCUUUUGUGA

UUCUUCCAUA

518
497
517

UAUCGAUGGAA

104

AAGCACUUCU

292
94.80
2.34
48.27
1.66

GAAGUGCUUUU

UCCAUCGAUA

521
500
520

UAUCAUCGAUG

105

CACUUCUUCCA

293
98.97
11.57
74.47
7.57

GAAGAAGUGCU

UCGAUGAUA

544
523
543

UCGAUGUGGCC

106

AGAAAUCAUG

294
103.80
1.69
77.57
7.42

AUGAUUUCUCU

GCCACAUCGA

546
525
545

UGACGAUGUGG

107

AAAUCAUGGC

295
88.30
8.55
51.87
3.79

CCAUGAUUUCU

CACAUCGUCA

643
622
642

UAUGUCAGACC

108

UUUCACAGAG

296
96.10
3.48
71.20
4.92

UCUGUGAAAGC

GUCUGACAUA

646
625
645

UCUGAUGUCAG

109

CACAGAGGUC

297
91.60
1.57
50.53
6.43

ACCUCUGUGAA

UGACAUCAGA

649
628
648

UGUUCUGAUGU

110

AGAGGUCUGA

298
99.17
6.79
74.40
2.52

CAGACCUCUGU

CAUCAGAACA

651
630
650

UAAGUUCUGAU

111

AGGUCUGACA

299
79.57
3.56
51.23
2.86

GUCAGACCUCU

UCAGAACUUA

679
658
678

UUUUUGAUACC

112

GGAAAAACUG

300
79.27
4.93
42.50
2.13

AGUUUUUCCCA

GUAUCAAAAA

680
659
679

UGUUUUGAUAC

113

GAAAAACUGG

301
79.40
1.04
44.07
2.95

CAGUUUUUCCC

UAUCAAAACA

690
669
689

UGAGACAUGAG

12

UAUCAAAACCU

41
58.23
1.54
19.03
0.80

GUUUUGAUACC

CAUGUCUCA

699
678
698
UAACUGGGCAG
114
CUCAUGUCUCU
302
94.53
3.21
89.33
4.10

AGACAUGAGGU

GCCCAGUUA

700
679
699
UAAACUGGGCA
115
UCAUGUCUCUG
303
97.37
2.66
83.67
2.78

GAGACAUGAGG

CCCAGUUUA

701
680
700
UAAAACUGGGC
116
CAUGUCUCUGC
304
95.97
2.46
89.27
5.40

AGAGACAUGAG

CCAGUUUUA

702
681
701
UAAAAACUGGG
117
AUGUCUCUGCC
305
110.00
1.80
90.03
9.67

CAGAGACAUGA

CAGUUUUUA

703
682
702

UCAAAAACUGG

118

UGUCUCUGCC

306
92.83
3.65
79.67
1.62

GCAGAGACAUG

CAGUUUUUGA

705
684
704
UCACAAAAACU
119
UCUCUGCCCAG
307
96.10
5.11
93.33
5.23

GGGCAGAGACA

UUUUUGUGA

707
686
706

UUUCACAAAAA

120

UCUGCCCAGU

308
105.13
6.48
75.53
3.85

CUGGGCAGAGA

UUUUGUGAAA

708
687
707

UAUUCACAAAA

121

CUGCCCAGUU

309
97.10
1.91
66.10
2.83

ACUGGGCAGAG

UUUGUGAAUA

709
688
708

UUAUUCACAAA

122

UGCCCAGUUU

310
93.87
3.70
52.37
1.66

AACUGGGCAGA

UUGUGAAUAA

710
689
709

UGUAUUCACAA

123

GCCCAGUUUU

311
110.97
4.49
74.77
5.23

AAACUGGGCAG

UGUGAAUACA

711
690
710
UAGUAUUCACA
124
CCCAGUUUUUG
312
104.53
3.86
86.37
2.59

AAAACUGGGCA

UGAAUACUA

712
691
711
UCAGUAUUCAC
125
CCAGUUUUUGU
313
103.33
11.14
86.90
2.63

AAAAACUGGGC

GAAUACUGA

722
701
721

UUUGGUGAACC

126

UGAAUACUGG

314
85.03
6.34
40.27
3.47

CAGUAUUCACA

GUUCACCAAA

723
702
722

UUUUGGUGAAC

127

GAAUACUGGG

315
86.10
7.15
46.10
1.15

CCAGUAUUCAC

UUCACCAAAA

724
703
723

UUUUUGGUGAA

128

AAUACUGGGU

316
88.57
10.42
43.73
4.74

CCCAGUAUUCA

UCACCAAAAA

725
704
724

UUUUUUGGUGA

129

AUACUGGGUU

317
98.20
4.35
73.60
1.53

ACCCAGUAVUC

CACCAAAAAA

727
706
726

UGAUUUUUGGU

130

ACUGGGUUCA

318
83.10
3.96
29.90
0.65

GAACCCAGUAU

CCAAAAAUCA

731
710
730

UCUUGGAUUUU

131

GGUUCACCAA

319
85.47
3.14
35.17
3.39

UGGUGAACCCA

AAAUCCAAGA

736
715
735

UUUGUGCUUGG

132

ACCAAAAAUCC

320
77.10
2.08
29.07
2.77

AUUUUUGGUGA

AAGCACAAA

737
716
736

UCUUGUGCUUG

133

CCAAAAAUCCA

321
90.57
5.94
37.67
3.58

GAUUUUUGGUG

AGCACAAGA

739
718
738

UAUCUUGUGCU

134

AAAAAUCCAA

322
81.37
2.42
35.00
3.62

UGGAUUUUUGG

GCACAAGAUA

742
721
741

UAUAAUCUUGU

13

AAUCCAAGCA

42
47.43
0.94
17.87
1.13

GCUUGGAUUUU

CAAGAUUAUA

750
729
749

UUACAGGCCAU

135

CACAAGAUUA

323
76.60
5.50
25.43
3.19

AAUCUUGUGCU

UGGCCUGUAA

751
730
750

UAUACAGGCCA

136

ACAAGAUUAU

324
90.43
2.45
31.90
2.86

UAAUCUUGUGC

GGCCUGUAUA

752
731
751

UAAUACAGGCC

137

CAAGAUUAUG

325
101.47
5.20
53.47
2.22

AUAAUCUUGUG

GCCUGUAUUA

753
732
752

UCAAUACAGGC

138

AAGAUUAUGG

326
93.70
3.57
36.37
1.62

CAUAAUCUUGU

CCUGUAUUGA

756
735
755

UCUCCAAUACA

139

AUUAUGGCCU

327
100.60
5.95
59.37
4.79

GGCCAUAAUCU

GUAUUGGAGA

762
741
761

UAUCUGUCUCC

140

GCCUGUAUUG

328
111.37
7.41
66.93
2.22

AAUACAGGCCA

GAGACAGAUA

763
742
762

UCAUCUGUCUC

141

CCUGUAUUGG

329
103.90
6.90
68.73
4.77

CAAUACAGGCC

AGACAGAUGA

765
744
764

UUUCAUCUGUC

142

UGUAUUGGAG

330
73.03
6.39
32.13
1.79

UCCAAUACAGG

ACAGAUGAAA

766
745
765

UCUUCAUCUGU

143

GUAUUGGAGA

331
72.53
1.77
29.30
1.50

CUCCAAUACAG

CAGAUGAAGA

787
766
786

UCAUCUAUCAG

144

GUAAGAAGUC

332
66.33
3.22
24.83
1.48

ACUUCUUACGA

UGAUAGAUGA

792
771
791

UUAUUCCAUCU

145

AAGUCUGAUA

333
95.10
2.14
48.03
0.73

AUCAGACUUCU

GAUGGAAUAA

795
774
794

UAAGUAUUCCA

146

UCUGAUAGAU

334
67.53
1.64
23.33
2.36

UCUAUCAGACU

GGAAUACUUA

803
782
802

UUUAUUGGUAA

147

AUGGAAUACU

335
66.30
1.21
18.13
1.19

GUAUUCCAUCU

UACCAAUAAA

804
783
803

UCUUAUUGGUA

148

UGGAAUACUU

336
92.60
2.98
39.23
2.50

AGUAUUCCAUC

ACCAAUAAGA

806
785
805

UUUCUUAUUGG

149

GAAUACUUAC

337
67.97
0.73
23.43
2.08

UAAGUAUUCCA

CAAUAAGAAA

807
786
806

UUUUCUUAUUG

150

AAUACUUACCA

338
82.47
3.94
|28.27
0.97

GUAAGUAUUCC

AUAAGAAAA

812
791
811

UAUCAUUUUCU

151

UUACCAAUAA

339
80.67
5.19
32.17
1.95

UAUUGGUAAGU

GAAAAUGAUA

902
881
901

UAUAUUCUGCA

152

UAAAUCGUAU

340
72.00
2.15
23.30
0.82

UACGAUUUAAA

GCAGAAUAUA

903
882
902

UAAUAUUCUGC

14

AAAUCGUAUG

43
63.00
0.80
19.03
0.47

AUACGAUUUAA

CAGAAUAUUA

909
888
908

UAAAUUGAAUA

15

UAUGCAGAAU

44
34.90
1.38
9.37
0.18

UUCUGCAUACG

AUUCAAUUUA

912
891
911

UUUCAAAUUGA

16

GCAGAAUAUU

45
50.70
2.95
17.57
1.66

AUAUUCUGCAU

CAAUUUGAAA

913
892
912

UCUUCAAAUUG

153

CAGAAUAUUC

341
60.50
2.25
23.63
1.98

AAUAUUCUGCA

AAUUUGAAGA

916
895
915

UCUGCUUCAAA

17

AAUAUUCAAUU

46
60.80
2.35
17.97
1.20

UUGAAUAUUCU

UGAAGCAGA

948
927
947

UUAUUCAUUUC

2

AAUCAAAAUG

31
55.80
2.65
13.53
0.59

AUUUUGAUUUU

AAAUGAAUAA

1020
999
1019

UAUAAAGCUUU

18

CAAUGCUGCA

47
44.93
3.09
20.80
2.06

GCAGCAUUGAU

AAGCUUUAUA

1021
1000
1020

UAAUAAAGCUU

19

AAUGCUGCAA

48
36.73
2.10
13.43
0.65

UGCAGCAUUGA

AGCUUUAUUA

1025
1004
1024

UGUGAAAUAAA

20

CUGCAAAGCU

49
28.17
2.32
13.53
1.59

GCUUUGCAGCA

UUAUUUCACA

1028
1007
1027

UAAUGUGAAAU

3

CAAAGCUUUA

32
33.17
1.41
14.03
0.59

AAAGCUUUGCA

UUUCACAUUA

1029
1008
1028

UAAAUGUGAAA

21

AAAGCUUUAU

50
32.23
1.13
14.30
0.96

UAAAGCUUUGC

UUCACAUUUA

1030
1009
1029

UAAAAUGUGAA

22

AAGCUUUAUU

51
41.53
2.58
15.10
0.15

AUAAAGCUUUG

UCACAUUUUA

1031
1010
1030

UAAAAAUGUGA

23

AGCUUUAUUU

52
51.80
1.65
19.93
0.65

AAUAAAGCUUU

CACAUUUUUA

1033
1012
1032

UGAAAAAAUGU

4

CUUUAUUUCAC

33
38.60
3.85
14.67
0.78

GAAAUAAAGCU

AUUUUUUCA

1115
1094
1114

UGAGAAACAGG

154

ACCUGUCUUC

342
57.63
3.72
23.63
1.32

AAGACAGGUAA

CUGUUUCUCA

1117
1096
1116

UUUGAGAAACA

155

CUGUCUUCCU

343
91.80
2.94
59.30
1.68

GGAAGACAGGU

GUUUCUCAAA

1118
1097
1117

UCUUGAGAAAC

156

UGUCUUCCUG

344
63.30
4.28
20.13
1.72

AGGAAGACAGG

UUUCUCAAGA

1120
1099
1119

UUUCUUGAGAA

157

UCUUCCUGUU

345
87.67
7.75
30.10
0.76

ACAGGAAGACA

UCUCAAGAAA

1121
1100
1120

UAUUCUUGAGA

158

CUUCCUGUUU

346
88.07
2.71
32.20
0.86

AACAGGAAGAC

CUCAAGAAUA

1122
1101
1121

UUAUUCUUGAG

24

UUCCUGUUUC

53
42.23
4.49
17.63
1.54

AAACAGGAAGA

UCAAGAAUAA

1124
1103
1123

UAAUAUUCUUG

159

CCUGUUUCUC

347
66.10
2.69
21.03
1.92

AGAAACAGGAA

AAGAAUAUUA

1159
1138
1158

UAUGAAAGGAA

160

GGUCUGUUUU

348
68.80
3.60
23.53
1.13

AAACAGACCUA

UCCUUUCAUA

1160
1139
1159

UCAUGAAAGGA

161

GUCUGUUUUU

349
81.67
3.43
33.37
1.45

AAAACAGACCU

CCUUUCAUGA

1171
1150
1170

UUUUUUAAGAG

162

CUUUCAUGCC

350
67.00
0.76
28.17
1.54

GCAUGAAAGGA

UCUUAAAAAA

1172
1151
1171

UGUUUUUAAGA

163

UUUCAUGCCU

351
86.30
6.63
41.87
2.82

GGCAUGAAAGG

CUUAAAAACA

1215
1194
1214

UAUCUUAAAGA

5

AAAAGGUUUU

34
40.17
1.88
18.30
1.89

AAACCUUUUAA

CUUUAAGAUA

1217
1196
1216

UAUAUCUUAAA

6

AAGGUUUUCU

35
39.73
3.80
15.60
1.10

GAAAACCUUUU

UUAAGAUAUA

1219
1198
1218

UAAAUAUCUUA

164

GGUUUUCUUU

352
70.70
3.67
31.37
2.42

AAGAAAACCUU

AAGAUAUUUA

1225
1204
1224

UAAAAUAAAAU

165

CUUUAAGAUA

353
95.30
3.36
54.60
2.65

AUCUUAAAGAA

UUUUAUUUUA

1246
1225
1245

UUUUUGUCCAC

166

CAUUUAAAGG

354
70.77
4.96
24.87
0.59

CUUUAAAUGGA

UGGACAAAAA

1318
1297
1317

UAUGCUACUUG

25

AAGACUGUUC

54
57.73
3.22
18.63
1.04

AACAGUCUUAA

AAGUAGCAUA

1323
1302
1322

UUUGGAAUGCU

26

UGUUCAAGUA

55
61.17
2.11
18.40
0.51

ACUUGAACAGU

GCAUUCCAAA

1325
1304
1324

UGAUUGGAAUG

167

UUCAAGUAGC

355
78.07
4.45
29.80
1.85

CUACUUGAACA

AUUCCAAUCA

1327
1306
1326

UCAGAUUGGAA

27

CAAGUAGCAU

56
54.17
1.88
20.03
0.78

UGCUACUUGAA

UCCAAUCUGA

1366
1345
1365

UCUCAUUCUGU

168

AACAAGAACA

356
65.43
3.56
26.30
2.16

GUUCUUGUUGA

CAGAAUGAGA

1377
1356
1376

UUUAGCUGUGC

169

AGAAUGAGUG

357
68.73
3.64
34.13
1.60

ACUCAUUCUGU

CACAGCUAAA

1378
1357
1377

UCUUAGCUGUG

170

GAAUGAGUGC

358
86.33
3.05
37.27
3.02

CACUCAUUCUG

ACAGCUAAGA

1442
1421
1441

UUUUCAAAUGC

7

UAAGAUUCAG

36
37.40
4.95
18.57
1.44

UGAAUCUUAAA

CAUUUGAAAA

1443
1422
1442

UCUUUCAAAUG

171

AAGAUUCAGC

359
65.40
4.62
22.67
0.52

CUGAAUCUUAA

AUUUGAAAGA

1445
1424
1444

UAUCUUUCAAA

8

GAUUCAGCAU

37
50.87
1.91
20.17
2.38

UGCUGAAUCUU

UUGAAAGAUA

1446
1425
1445

UAAUCUUUCAA

9

AUUCAGCAUU

38
53.30
3.15
20.70
0.67

AUGCUGAAUCU

UGAAAGAUUA

1500
1479
1499

UGUCCAGAAUA

172

GUGCAACUCU

360
96.13
6.89
52.10
3.50

GAGUUGCACCG

AUUCUGGACA

1503
1482
1502

UAAAGUCCAGA

173

CAACUCUAUUC

361
81.93
7.28
32.90
0.74

AUAGAGUUGCA

UGGACUUUA

1504
1483
1503

UUAAAGUCCAG

174

AACUCUAUUCU

362
62.30
3.82
30.27
2.43

AAUAGAGUUGC

GGACUUUAA

1506
1485
1505

UAAUAAAGUCC

175

CUCUAUUCUG

363
88.33
1.72
43.87
1.04

AGAAUAGAGUU

GACUUUAUUA

1507
1486
1506

UUAAUAAAGUC

176

UCUAUUCUGG

364
70.93
3.95
32.43
2.36

CAGAAUAGAGU

ACUUUAUUAA

1508
1487
1507

UGUAAUAAAGU

28

CUAUUCUGGA

57
48.43
2.98
22.90
2.10

CCAGAAUAGAG

CUUUAUUACA

1559
1538
1558

UUAGAGGGUCC

177

ACCAAAAGUG

365
88.43
3.56
65.30
3.37

ACUUUUGGUGG

GACCCUCUAA

1560
1539
1559

UAUAGAGGGUC

178

CCAAAAGUGG

366
85.10
6.42
42.37
1.53

CACUUUUGGUG

ACCCUCUAUA

1561
1540
1560

UUAUAGAGGGU

179

CAAAAGUGGA

367
82.17
5.11
39.27
2.31

CCACUUUUGGU

CCCUCUAUAA

1562
1541
1561

UAUAUAGAGGG

180

AAAAGUGGAC

368
95.10
5.95
51.73
1.96

UCCACUUUUGG

CCUCUAUAUA

1563
1542
1562

UAAUAUAGAGG

181

AAAGUGGACC

369
91.83
4.90
41.97
1.52

GUCCACUUUUG

CUCUAUAUUA

1564
1543
1563

UAAAUAUAGAG

182

AAGUGGACCC

370
90.47
11.50
44.87
4.44

GGUCCACUUUU

UCUAUAUUUA

1565
1544
1564

UGAAAUAUAGA

183

AGUGGACCCU

371
98.57
5.33
48.77
0.78

GGGUCCACUUU

CUAUAUUUCA

1572
1551
1571

UAAGGGAGGAA

184

CCUCUAUAUUU

372
78.37
2.62
39.47
0.79

AUAUAGAGGGU

CCUCCCUUA

1573
1552
1572

UAAAGGGAGGA

185

CUCUAUAUUUC

373
74.33
2.02
29.47
1.91

AAUAUAGAGGG

CUCCCUUUA

1574
1553
1573

UAAAAGGGAGG

186

OCUADAUUUCC

374
64.93
3.88
28.93
1.69

AAAUAUAGAGG

UCCCUUUUA

1576
1555
1575

UUAAAAAGGGA

187

UAUAUUUCCUC

375
58.43
4.49
30.50
2.80

GGAAAUAUAGA

CCUUUUUAA

1577
1556
1576

UAUAAAAAGGG

188

AUAUUUCCUCC

376
81.80
5.99
38.37
2.02

AGGAAAUAUAG

CUUUUUAUA

1578
1557
1577

UUAUAAAAAGG

189

UAUUUCCUCCC

377
66.00
3.69
24.10
0.72

GAGGAAAUAUA

UUUUUAUAA

1700
1679
1699
UCUCUGGGACC
190
UAUAUCCUUGG
378
104.13
9.56
101.37
8.64

AAGGAUAUAUG

UCCCAGAGA

1722
1701
1721
UGAGCCUAAAA
191
UUUAGACAAUU
379
108.13
9.84
87.53
5.96

UUGUCUAAACA

UUAGGCUCA

1726
1705
1725
UUUUUGAGCCU
192
GACAAUUUUAG
380
111.20
7.13
100.33
7.53

AAAAUUGUCUA

GCUCAAAAA

1727
1706
1726
UUUUUUGAGCC
193
ACAAUUUUAGG
381
116.47
5.24
113.50
3.72

UAAAAUUGUCU

CUCAAAAAA

1728
1707
1727
UAUUUUUGAGC
194
CAAUUUUAGGC
382
111.73
3.15
96.43
3.31

CUAAAAUUGUC

UCAAAAAUA

1729
1708
1728
UAAUUUUUGAG
195
AAUUUUAGGCU
383
107.27
8.71
91.57
5.43

CCUAAAAUUGU

CAAAAAUUA

1731
1710
1730
UUUAAUUUUUG
196
UUUUAGGCUCA
384
105.97
2.26
108.80
2.52

AGCCUAAAAUU

AAAAUUAAA

1732
1711
1731
UUUUAAUUUUU
197
UUUAGGCUCAA
385
112.63
3.03
103.60
1.99

GAGCCUAAAAU

AAAUUAAAA

1733
1712
1732
UCUUUAAUUUU
198
UUAGGCUCAAA
386
96.73
5.77
95.73
6.94

UGAGCCUAAAA

AAUUAAAGA

1738
1717
1737
UGUUAGCUUUA
199
CUCAAAAAUUA
387
105.03
7.98
101.13
2.75

AUUUUUGAGCC

AAGCUAACA

1739
1718
1738
UUGUUAGCUUU
200
UCAAAAAUUAA
388
102.47
5.19
110.8
8.53

AAUUUUUGAGC

AGCUAACAA

1748
1727
1747
UCUUUUCCUGU
201
AAAGCUAACAC
389
116.20
11.36
110.23
5.33

GUUAGCUUUAA

AGGAAAAGA

1757
1736
1756
UGUACAGUUCC
202
ACAGGAAAAGG
390
111.27
6.94
102.90
5.35

UUUUCCUGUGU

AACUGUACA

1759
1738
1758
UCAGUACAGUU
203
AGGAAAAGGAA
391
119.80
9.44
104.57
1.62

CCUUUUCCUGU

CUGUACUGA

1766
1745
1765
UUAAUAGCCAG
204
GGAACUGUACU
392
108.93
6.94
113.03
9.70

UACAGUUCCUU

GGCUAUUAA

1767
1746
1766
UGUAAUAGCCA
205
GAACUGUACUG
393
102.17
4.40
112.2
2.58

GUACAGUUCCU

GCUAUUACA

2185
2164
2184
UUAGUCUUGAU
206
UCCCACUACAU
394
107.33
5.50
100.00
10.00

GUAGUGGGAGU

CAAGACUAA

2187
2166
2186
UAUUAGUCUUG
207
CCACUACAUCA
395
101.70
6.69
104.13
3.91

AUGUAGUGGGA

AGACUAAUA

2188
2167
2187
UGAUUAGUCUU
208
CACUACAUCAA
396
96.73
1.89
88.60
7.95

GAUGUAGUGGG

GACUAAUCA

2191
2170
2190
UCAAGAUUAGU
209
UACAUCAAGAC
397
84.80
2.24
96.60
10.83

CUUGAUGUAGU

UAAUCUUGA

2215
2194
2214
UAAUACAUGUG
210
UGUGUUUUUCA
398
115.10
3.63
98.30
6.80

AAAAACACACA

CAUGUAUUA

2217
2196
2216
UAUAAUACAUG
211
UGUUUUUCACA
399
123.80
12.11
99.17
3.17

UGAAAAACACA

UGUAUUAUA

2219
2198
2218
UCUAUAAUACA
212
UUUUUCACAUG
400
102.37
3.24
104.17
5.27

UGUGAAAAACA

UAUUAUAGA

2222
2201
2221
UAUUCUAUAAU
213
UUCACAUGUAU
401
106.20
8.71 7
106.63
8.91

ACAUGUGAAAA

UAUAGAAUA

2227
2206
2226
UAAAGCAUUCU
214
AUGUAUUAUAG
402
111.90
5.61
102.67
5.93

AUAAUACAUGU

AAUGCUUUA

2238
2217
2237
UUAGUCCAUGC
215
AAUGCUUUUGC
403
110.70
8.23
99.07
6.89

AAAAGCAUUCU

AUGGACUAA

2243
2222
2242
UGAGGAUAGUC
216
UUUUGCAUGGA
404
99.63
4.60
92.17
6.77

CAUGCAAAAGC

CUAUCCUCA

2248
2227
2247
UAACAAGAGGA
217
CAUGGACUAUC
405
108.33
5.35
98.90
2.39

UAGUCCAUGCA

CUCUUGUUA

2249
2228
2248
UAAACAAGAGG
218
AUGGACUAUCC
406
94.87
3.87
94.40
7.83

AUAGUCCAUGC

UCUUGUUUA

2252
2231
2251
UUAAAAACAAG
219
GACUAUCCUCU
407
102.60
9.02
91.20
9.71

AGGAUAGUCCA

UGUUUUUAA

2253
2232
2252
UAUAAAAACAA
220
ACUAUCCUCUU
408
97.90
8.52
87.57
4.98

GAGGAUAGUCC

GUUUUUAUA

2254
2233
2253
UAAUAAAAACA
221
CUAUCCUCUUG
409
102.43
15.43
111.30
3.52

AGAGGAUAGUC

UUUUUAUUA

2255
2234
2254
UUAAUAAAAAC
222
UAUCCUCUUGU
410
103.63
6.55
105.30
3.37

AAGAGGAUAGU

UUUUAUUAA

2256
2235
2255
UUUAAUAAAAA
223
AUCCUCUUGUU
411
109.50
9.14
96.43
5.18

CAAGAGGAUAG

UUUAUUAAA

2258
2237
2257
UUUUUAAUAAA
224
CCUCUUGUUUU
412
113.93
5.43
109.37
9.09

AACAAGAGGAU

UAUUAAAAA

2259
2238
2258
UUUUUUAAUAA
225
CUCUUGUUUUU
413
110.93
7.05
105.03
5.25

AAACAAGAGGA

AUUAAAAAA

2305
2284
2304
UUUUAUUUUUA
226
ACAAUUCACUA
414
120.27
5.42
97.93
3.82

GUGAAUUGUUU

AAAAUAAAA

2306
2285
2305
UAUUUAUUUUU
227
CAAUUCACUAA
415
109.70
2.31
102.23
7.35

AGUGAAUUGUU

AAAUAAAUA

2349
2328
2348
UUUUUAUAACU
228
ACCUCUUGUAG
416
92.70
5.45
94.80
5.60

ACAAGAGGUUA

UUAUAAAAA

2350
2329
2349
UAUUUUAUAAC
229
CCUCUUGUAGU
417
101.53
3.93
99.63
9.25

UACAAGAGGUU

UAUAAAAUA

2351
2330
2350
UUAUUUUAUAA
230
CUCUUGUAGUU
418
102.63
4.43
96.87
0.55

CUACAAGAGGU

AUAAAAUAA

2354
2333
2353
UUUUUAUUUUA
231
UUGUAGUUAUA
419
92.03
3.33
96.73
3.24

UAACUACAAGA

AAAUAAAAA

2062
2041
2061
UAGAUCGAGAC
232
GCCAAGAUGGU
420
95.20
5.19
88.37
9.80

CAUCUUGGCUA

CUCGAUQUA

1878
1857
1877
UGGCGAAAGAG
233
GGAGUCUCACU
421
93.67
5.03
97.17
10.86

UGAGACUCCAU

CUUUCGOuA

2063
2042
2062
UGAGAUCGAGA
234
CCAAGAUGGUC
422
91.80
1.39
109.47
9.00

CCAUCUUGGCU

UCGAUuUuA

1942
1921
1941
UAUGGCGUGAA
235
CCUCCCGGGUU
423
101.67
16.39
88.37
3.52

CCCGGGAGGUG

CACGCuAUA

1985
1964
1984
UGGGCGCCUGU
236
GCUGGGACUAC
424
107.17
4.89
92.40
8.34

AGUCCCAGCUA

AGGCGuuuA

2004
1983
2003
UAUUAGCCGGG
237
GCCACCACACC
425
100.87
8.61
95.03
13.48

UGUGGUGGCGG

CGGCUAAUA

1977
1956
1976
UGUAGUCCCAG
238
CCUGAGUAGCU
426
93.00
9.96
99.83
4.32

CUACUCAGGAG

GGGACUAuA

2046
2025
2045
UGGCUAACAUG
239
GGGUUUCACCA
427
104.63
4.50
91.57
3.08

GUGAAACCCCG

UGUUAGUuA

1899
1878
1898
UAUACCACUGC
240
GGCUGGAGUGC
428
98.50
2.80
86.30
3.59

ACUCCAGCCUG

AGUGGUAUA

1936
1915
1935
UUGAACCCGGG
241
GCUCCACCUCC
429
83.47
9.58
96.43
9.20

AGGUGGAGCUU

CGGGUUuAA

2081
2060
2080
UGGUGGAUCAC
242
CCUGACCUCGU
430
96.77
8.76
90.30
4.91

GAGGUCAGGAG

GAUCCAuuA

2005
1984
2004
UAAUUAGCCGG
243
CCACCACACCC
431
109.40
3.66
97.60
2.54

GUGUGGUGGCG

GGCUAAUUA

2047
2026
2046
UUGGCUAACAU
244
GGUUUCACCAU
432
109.23
2.44
96.20
8.01

GGUGAAACCCC

GUUAGuuAA

1900
1879
1899
UuAUACCACUGC
245
GCUGGAGUGCA
433
106.43
2.21
86.23
3.42

ACUCCAGCCU

GUGGUAUGA

1903
1882
1902
UGAUCAUACCA
246
GGAGUGCAGUG
434
127.17
11.14
88.57
14.23

CUGCACUCCAG

GUAUGAUuA

1967
1946
1966
UuUACUCAGGAG
247
GCCUCAGCCUC
435
105.90
8.94
100.37
10.83

GCUGAGGCAG

CUGAGUAGA

1945
1924
1944
UAGAAUGGCGU
248
CCCGGGUUCAC
436
124.13
11.41
100.27
2.74

GAACCCGGGAG

GCCAUUuUA

495
474
494

UGAUCCAAAAA

30

CCUAGGACAU

59
74.67
8.78
19.47
0.50

UGUCCUAGGAU

UUUUGGAUCA

495
474
494

UGAUCCAAAAA

30

CCUAGGACAU

60
64.37
5.61
23.43
3.11

UGUCCUAGGAU

UUUUGIAUCA

TABLE 3

Exemplary Sequences of the Present Application and Their in

vivo Potency in Silencing Cynomolgus HSD17B13 mRNA in Liver Samples

Guide
Passenger
% mRNA
% mRNA
% mRNA
% mRNA
% mRNA
% mRNA

strand
strand
remaining
remaining at
remaining at
remaining at
remaining at
remaining at

SEQ ID
SEQ ID
at Day −7
Day 15
Day 29
Day 57
Day 85
Day 113

NO:
NO:
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD

440
441
100
0
8.84
2.83
10.60
3.64
15.57
3.19
16.95
8.56
57.23
25.97

442
443
100
0
14.39
6.34
9.06
3.88
12.81
12.29
20.29
19.64
58.05
54.37

444
445
100
0
19.18
6.56
14.05
5.84
20.00
11.56
27.95
14.12
81.54
72.19

TABLE 4

Exemplary Sequences of the Present Application

G(5′-3′)
[MeEPmUS][fAs][fA][mU][fG][mU][fG][[mA][mA][fA][mU][mA][mA][fA][mG][fC][mU][mU]
SEQ ID NO: 440

[mU][mGs][mCs][mA]

P(5′-3′)
[mCs][mAs][mA][mA][mG][fC][mU][fU][fU][fA][fU][mU][mU][mC][mA][mC][mA][mUs][mUs]
SEQ ID NO: 441

[mA][Glb][Glb][Glb]

G(5′-3′)
[MeEPmUs][fGs][fA][mG][fA][mC][fA][mU][mG][fA][mG][mG][mU][fU][mU][fU][mG][mA]
SEQ ID NO: 442

[mU][mAs][mCs][mC]

P(5′-3′)
[mUs][mAs][mU][mC][mA][fA][mA][fA][fC][fC][fU][mC][mA][mU][mG][mU][mC][mUs][mCs]
SEQ ID NO: 443

[mA][Glb][Glb][Glb]

G(5′-3′)
[MeEPmUs][fUs][fA][mU][fU][mA][fA][mU][mA][fU][mC][mC][mC][fA][mC][fA][mG][mA]
SEQ ID NO: 444

[mA][mCs][mCs][mA]

P(5′-3′)
[mGs][mUs][mU][mC][mU][fG][mU][fG][fG][fG][fA][mU][mA][mU][mU][mA][mA][mUs][mAs]
SEQ ID NO: 445

[mA][Glb][Glb][Glb]

G: guide strand, P: passenger strand

″m″ indicates 2′-O-methyl modification, ″f″ indicates a 2′-F modification, ″McEPmU″ is a mono methyl protected phosphate mimic linked to a 5′-terminal uracil (shown below), ″s″ is a phosphorothioate internucleotide linkage, and ″Glb″ is a GalNAc Glb moiety (shown below).

embedded image

TABLE 5

Exemplary Sequences of the Present Application and Their in vivo Potency

in Silencing Human HSD17B13 mRNA in HDI mouse liver

Guide

Guide strand

Passenger strand

% gene

Strand
Guide
Guide
sequence
SEQ ID
sequence
SEQ ID
remaining

position
start
end
(5′-3′)
NO
(5′-3′)
NO
Mean
SD

365
344
364
UUUCACCUGAUUUA
10
GCUCUCUAAAUCAG
39
22.20
7.98

GAGAGCGA

GUGAAA

497
476
496
UGUGAUCCAAAAA
11
UAGGACAUUUUUG
40
17.20
3.96

UGUCCUAGG

GAUCACA

690
669
689
UGAGACAUGAGGU
12
UAUCAAAACCUCAU
41
11.00
3.67

UUUGAUACC

GUCUCA

742
721
741
UAUAAUCUUGUGC
13
AAUCCAAGCACAAG
42
16.40
2.30

UUGGAUUUU

AUUAUA

903
882
902
UAAUAUUCUGCAU
14
AAAUCGUAUGCAGA
43
19.00
1.58

ACGAUUUAA

AUAUUA

909
888
908
UAAAUUGAAUAUU
15
UAUGCAGAAUAUUC
44
23.20
6.91

CUGCAUACG

AAUUUA

912
891
911
UUUCAAAUUGAAU
16
GCAGAAUAUUCAAU
45
19.40
3.36

AUUCUGCAU

UUGAAA

916
895
915
UCUGCUUCAAAUUG
17
AAUAUUCAAUUUG
46
34.20
8.53

AAUAUUCU

AAGCAGA

1020
999
1019
UAUAAAGCUUUGC
18
CAAUGCUGCAAAGC
47
21.00
6.67

AGCAUUGAU

UUUAUA

1021
1000
1020
UAAUAAAGCUUUG
19
AAUGCUGCAAAGCU
48
18.20
1.64

CAGCAUUGA

UUAUUA

1029
1008
1028
UAAAUGUGAAAUA
21
AAAGCUUUAUUUCA
50
24.00
7.65

AAGCUUUGC

CAUUUA

1030
1009
1029
UAAAAUGUGAAAU
22
AAGCUUUAUUUCAC
51
23.20
6.98

AAAGCUUUG

AUUUUA

1031
1010
1030
UAAAAAUGUGAAA
23
AGCUUUAUUUCACA
52
32.20
11.01

UAAAGCUUU

UUUUUA

1033
1012
1032
UGAAAAAAUGUGA
4
CUUUAUUUCACAUU
33
15.40
2.41

AAUAAAGCU

UUUUCA

1122
1101
1121
UUAUUCUUGAGAA
24
UUCCUGUUUCUCAA
53
27.00
4.12

ACAGGAAGA

GAAUAA

1215
1194
1214
UAUCUUAAAGAAA
5
AAAAGGUUUUCUU
34
33.80
9.88

ACCUUUUAA

UAAGAUA

1217
1196
1216
UAUAUCUUAAAGA
6
AAGGUUUUCUUUA
35
26.60
7.23

AAACCUUUU

AGAUAUA

1318
1297
1317
UAUGCUACUUGAAC
25
AAGACUGUUCAAGU
54
28.00
8.46

AGUCUUAA

AGCAUA

1323
1302
1322
UUUGGAAUGCUAC
26
UGUUCAAGUAGCAU
55
25.40
10.97

UUGAACAGU

UCCAAA

1327
1306
1326
UCAGAUUGGAAUG
27
CAAGUAGCAUUCCA
56
28.20
7.43

CUACUUGAA

AUCUGA

1442
1421
1441
UUUUCAAAUGCUG
7
UAAGAUUCAGCAUU
36
25.40
4.88

AAUCUUAAA

UGAAAA

1445
1424
1444
UAUCUUUCAAAUGC
8
GAUUCAGCAUUUGA
37
27.60
7.96

UGAAUCUU

AAGAUA

1446
1425
1445
UAAUCUUUCAAAU
19
AUUCAGCAUUUGAA
38
21.40
3.13

GCUGAAUCU

AGAUUA

1508
1487
1507
UGUAAUAAAGUCC
28
CUAUUCUGGACUUU
57
29.80
7.26

AGAAUAGAG

AUUACA

250
229
249
UUAUUAAUAUCCCA
29
GUUCUGUGGGAUA
58
18.60
7.86

CAGAACCA

UUAAUAA

948
927
947
UUAUUCAUUUCAU
2
AAUCAAAAUGAAA
31
28.20
7.86

UUUGAUUUU

UGAAUAA

1025
1004
1024
UGUGAAAUAAAGC
20
CUGCAAAGCUUUAU
49
25.60
4.16

UUUGCAGCA

UUCACA

1028
1007
1027
UAAUGUGAAAUAA
3
CAAAGCUUUAUUUC
32
17.20
2.59

AGCUUUGCA

ACAUUA

EXAMPLES
Example 1: Design and Testing of siRNA Compounds Against HSD17B13 mRNA

The example described herein determined the potency of the siRNA compounds against HSD17B13 mRNA, including compounds described in Tables 1-4.

Materials and Methods

A set of 218 siRNA compounds against human HSD17B13 transcript (Accession No: NM_178135.5) were designed (Table 1 and Table 4). Due to the low expression levels of HSD17B13 in the in vitro cell lines, a Dual-Glo Lucierase assay was performed to evaluate the compound potency in silencing human HSD17B13 mRNA. Huh-7 cells were first transfected with psiCHECK2-HSD17B13 plasmids with Fugene-HD reagents on day 0. On day 1, the siRNA compounds were diluted into the desired concentration with PBS and transfected into the psiCHECK2-HSD17B13-plasmid transfected Huh-7 cells, at two concentrations of 0.05 nM and 0.5 nM, with Lipofectamine RNAiMAX reagents. At 24 hours post-siRNA transfection (day 2), Firefly (transfection control) and Renilla (fused to HSD17B13 transcript sequence) luciferase activities were measured using the Dual-Glo Luciferase reagent kit. Data is represented as Mean+/−SEM.

Compound Synthesis

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.

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.

RT-qPCR

Liver mRNA samples were prepared with RNeasy Plus mini kit. mRNAs were reverse transcribed into cDNAs using High-Capacity cDNA Reverse transcription kits with RNase Inhibitors. TaqMan multiplex qPCR assays were performed to determine the relative HSD17B13 mRNA levels over time.

Western Blotting

10 mg of liver samples were homogenized in RIPA lysis buffer. Total protein concentrations were determined with BCA assays. 10 jig of total protein samples were loaded for electrophoresis. Cyno HSD17B13 proteins were blotted with rabbit anti-HSD17B13 (N-terminus) polyclonal antibody (Absin, Abs110383, 1:1000 dilution). GAPDH proteins were blotted with rabbit anti-GAPDH polyclonal antibody (Absin, Abs132004, 1:5000 dilution). Goat anti-rabbit IgG-HRP secondary antibodies (Absin, Abs20002, 1:20000) were used to detect the proteins.

Ex Vivo Potency Evaluation in Primary Human Hepatocytes (PHH)

Compounds with GalNAc conjugations were tested through free-uptake in PHH. The compounds were directly added to the cultured primary hepatocytes at 2 doses (10 nM and 100 nM). 48 hrs later, the cells were harvested for mRNA analysis through RT-qPCR. Data shown as Mean+/−SD (FIG. 2)

In vivo potency and duration evaluation in Macaca fascicularis

To determine HSD17B13 knockdown in non-human primates, a nonterminal study was conducted in cynomolgus macaques. A liver biopsy was taken from cyno monkeys in the study to determine the baseline mRNA expression level one week before dosing (Day −7). Animals were dosed with a single dose of 3 mg/kg of compounds listed in Table 4 a week after the liver biopsy (Day 0). A biopsy of the liver and blood was taken at 15, 29, 57, 85 and 113 days post-dose. Liver samples were used for mRNA and protein remaining analysis by RT-qPCR and Western blot, respectively.

In Vivo Compound Screening in HDI Mouse Liver

Compounds with GalNAc conjugations were formulated in 1×PBS, and dosed on day 1 through subcutaneous dosing to BALB/c female animals (6-8 weeks old). Animals then received 10 μg of pcDNA3. 1-hsHSD17B13 plasmids on day 4. Liver biopsies were taken on day 5 for mRNA remaining analysis through RT-qPCR (FIG. 3A and Table 5).

For the dose response study shown in FIG. 3B, 6-8 weeks old female BALB/c mice were dosed subcutaneously at 0.25 mg/kg, 0.5 mg/kg or 1 mg/kg. The control animals were dosed with PBS. Animals were sacrificed 4 days post-dose and liver samples were collected for RNA extraction and HSD171313 mRNA expression analysis by RT-gPCR (FIG. 3B).

Results and Observations

The percentage of human HSD17B13 mRNA remaining in cells relative to mock transfection were normalized to Firefly Luciferase levels, was determined for each compound at a concentration of either 0.05 nM or 0.5 nM. The results identified several compounds that were able to reduce the level of human HSD17B13 mRNA in transfected Huh-7 cells by 20% to 50% or more than 50% at a concentration of 0.05 nM, as shown in Table 2. Also, several compounds were able to reduce the level of human HSD17B13 mRNA in transfected cells by between 50% to 75%, more than 75%, 80%, or 85% at a concentration of 0.5 nM (Table 2).

The percentage of remaining cynomolgus HSD17B13 mRNA in Macaca fascicularis after a 3 mg/kg single subcutaneous dosing of compounds listed in Table 4 is shown in Table 3. All three compounds reduced mRNA levels in the liver by greater than 80%, and one compound was able to reduce mRNA levels greater than 90% at Day 15 post-dosing. As shown in Table 3, the mRNA levels remained about 20% or greater for some compounds out to day 85 post-dosing.

As shown in FIGS. 1A-1B, all three compounds listed in Table 4 were able to reduce cynomolgus HSD17B13 protein in Macaca fascicularis liver samples after a 3 mg/kg single subcutaneous dosing. The HSD17B13 protein levels remained reduced relative to pre-dose levels (Day −7, D-7) out to 85 days post-dosing (Day 85, D85), as shown in FIG. 1A. As shown in FIG. 1B, by Day 15, all compounds had reduced HSD17B13 protein by at least 40% relative to pre-dosing levels (Day −7). The maximum reduction in protein for all compounds was seen at Day 29 post-dosing, as shown in FIG. 1B.

As shown in FIG. 2, all compounds were able to reduce the level of HSD17B13 mRNA in primary human hepatocytes ex vivo greater than 80% at both doses (10 nM and 100 nM) tested.

These results demonstrate several siRNA compounds disclosed herein are effective at reducing human HSD17B13 mRNA in vitro in multiple cells including human primary hepatocytes ex vivo. The compounds were also able to reduce cynomolgus HSD17B13 mRNA and protein in vivo following a single subcutaneous dose.

Additional embodiments of the disclosure include the following:

Embodiment 1. An isolated oligonucleotide comprising a sense strand and an anti-sense 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) 229 to 249;
  - b) 344 to 364;
  - c) 474 to 496;
  - d) 669 to 741;
  - e) 882 to 947;
  - f) 999 to 1032;
  - g) 1101 to 1216;
  - h) 1297 to 1326; and
  - i) 1421 to 1507,
- from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) mRNA sequence according to SEQ ID NO: 1,
  - and the anti-sense strand is substantially complementary to the sense strand such that the sense strand and the anti-sense strand together form a double stranded region.

Embodiment 2. The isolated oligonucleotide of Embodiment 1, 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) 229 to 249;
- b) 344 to 364;
- c) 474 to 496;
- d) 669 to 741;
- e) 882 to 947;
- f) 999 to 1032;
- g) 1101 to 1216;
- h) 1297 to 1326; and

i) 1421 to 1507,

- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 3. The isolated oligonucleotide of Embodiment 1, 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) 229 to 249;
- b) 344 to 364;
- c) 474 to 496;
- d) 669 to 741;
- e) 882 to 947;
- f) 999 to 1032;
- g) 1101 to 1216;
- h) 1297 to 1326; and
- i) 1421 to 1507,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 4. The isolated oligonucleotide of any one of Embodiments 1-3, 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) 927 to 947;
- b) 1007 to 1032;
- c) 1194 to 1216; and
- d) 1421 to 1445,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 5. The isolated oligonucleotide of Embodiment 4, 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) 927 to 947;
- b) 1007 to 1032;
- c) 1194 to 1216; and
- d) 1421 to 1445,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 6. The isolated oligonucleotide of Embodiment 4, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from:

- a) 927 to 947;
- b) 1007 to 1032;
- c) 1194 to 1216; and
- d) 1421 to 1445,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 7. The isolated oligonucleotide of any one of Embodiments 1-3, 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) 344 to 364;
- b) 476 to 496;
- c) 669 to 741;
- d) 882 to 915;
- e) 999 to 1030;
- f) 1101 to 1121;
- g) 1297 to 1326; and
- h) 1487 to 1507,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 8. The isolated oligonucleotide of Embodiment 7, 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) 344 to 364;
- b) 476 to 496;
- c) 669 to 741;
- d) 882 to 915;
- e) 999 to 1030;
- f) 1101 to 1121;
- g) 1297 to 1326; and
- h) 1487 to 1507,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 9. The isolated oligonucleotide of Embodiment 7, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from:

- a) 344 to 364;
- b) 476 to 496;
- c) 669 to 741;
- d) 882 to 915;
- e) 999 to 1030;
- f) 1101 to 1121;
- g) 1297 to 1326; and
- h) 1487 to 1507,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 10. The isolated oligonucleotide of any one of Embodiments 1-3, 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) 229 to 249; and
- b) 474 to 494,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 11. The isolated oligonucleotide of Embodiment 10, 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) 229 to 249; and
- b) 474 to 494,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 12. The isolated oligonucleotide of Embodiment 10, wherein 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) 229 to 249; and
- b) 474 to 494,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 13. The isolated oligonucleotide of any one of Embodiments 1-12, wherein the isolated oligonucleotide is capable of inducing degradation of the HSD17B13 mRNA.

Embodiment 14. The isolated oligonucleotide of any one of Embodiments 1-13, wherein the sense strand is a single stranded RNA molecule.

Embodiment 15. The isolated oligonucleotide of any one of Embodiments 1-13, wherein the anti-sense strand is a single stranded RNA molecule.

Embodiment 16. The isolated oligonucleotide of any one of Embodiments 1-13, wherein both the sense strand and the anti-sense strand are single stranded RNA molecules.

Embodiment 17. The isolated oligonucleotide of Embodiment 15 or 16, wherein the anti-sense strand comprises a 3′ overhang.

Embodiment 18. The isolated oligonucleotide of Embodiment 17, wherein the 3′ overhang comprise at least one nucleotide.

Embodiment 19. The isolated oligonucleotide of Embodiment 18, wherein the 3′ overhang comprise two nucleotides.

Embodiment 20. The isolated oligonucleotide of Embodiment 19, wherein the 3′ overhang comprises any one of thymidine-thymidine (dTdT), Adenine-Adenine (AA), Cysteine-Cysteine (CC), Guanine-Guanine (GG) or Uracil-Uracil (UU).

Embodiment 21. The isolated oligonucleotide of any one of Embodiments 1-20, wherein the sense strand comprises an RNA sequence of at least 20 nucleotides in length.

Embodiment 22. The isolated oligonucleotide of Embodiment 21, wherein the sense strand comprises an RNA sequence of 20 nucleotides in length.

Embodiment 23. The isolated oligonucleotide of any one of Embodiments 1-22, wherein the anti-sense strand comprises an RNA sequence of at least 22 nucleotides in length.

Embodiment 24. The isolated oligonucleotide of Embodiment 23, wherein the anti-sense strand comprises an RNA sequence of 22 nucleotides in length.

Embodiment 25. The isolated oligonucleotide of any one of Embodiments 1-24, wherein the double stranded region is between 19 and 21 nucleotides in length.

Embodiment 26. The isolated oligonucleotide of Embodiment 25, wherein the double stranded region is 20 nucleotides in length.

Embodiment 27. The isolated oligonucleotide of any one of Embodiments 1-26, wherein the anti-sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 2-30.

Embodiment 28. The isolated oligonucleotide of any one of Embodiments 1-27, wherein the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 31-60.

Embodiment 29. The isolated oligonucleotide of Embodiment 6, wherein the double stranded region comprises:

- i) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UUAUUCAUUUCAUUUUGAUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ AAUCAAAAUGAAAUGAAUAA 3′);
- ii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UAAUGUGAAAUAAAGCUUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CAAAGCUUUAUUUCACAUUA 3′);
- iii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UGAAAAAAUGUGAAAUAAAGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUUUAUUUCACAUUUUUUCA 3′);
- iv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UAUCUUAAAGAAAACCUUUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ AAAAGGUUUUCUUUAAGAUA 3′);
- v) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UAUAUCUUAAAGAAAACCUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ AAGGUUUUCUUUAAGAUAUA 3′);
- vi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UUUUCAAAUGCUGAAUCUUAAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ UAAGAUUCAGCAUUUGAAAA 3′);
- vii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUCUUUCAAAUGCUGAAUCUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GAUUCAGCAUUUGAAAGAUA 3′); or
- viii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAAUCUUUCAAAUGCUGAAUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ AUUCAGCAUUUGAAAGAUUA 3′).

Embodiment 30. The isolated oligonucleotide of Embodiment 9, wherein the double stranded region comprises:

- i) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UUUCACCUGAUUUAGAGAGCGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ GCUCUCUAAAUCAGGUGAAA 3′);
- ii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UGUGAUCCAAAAAUGUCCUAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ UAGGACAUUUUUGGAUCACA 3′);
- iii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UGAGACAUGAGGUUUUGAUACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ UAUCAAAACCUCAUGUCUCA 3′);
- iv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UAUAAUCUUGUGCUUGGAUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ AAUCCAAGCACAAGAUUAUA 3′);
- v) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UAAUAUUCUGCAUACGAUUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ AAAUCGUAUGCAGAAUAUUA 3′);
- vi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAAAUUGAAUAUUCUGCAUACG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ UAUGCAGAAUAUUCAAUUUA 3′);
- vii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UUUCAAAUUGAAUAUUCUGCAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ GCAGAAUAUUCAAUUUGAAA 3′);
- viii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UCUGCUUCAAAUUGAAUAUUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ AAUAUUCAAUUUGAAGCAGA 3′);
- ix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAUAAAGCUUUGCAGCAUUGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CAAUGCUGCAAAGCUUUAUA 3′);
- x) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UAAUAAAGCUUUGCAGCAUUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ AAUGCUGCAAAGCUUUAUUA 3′);
- xi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UGUGAAAUAAAGCUUUGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ CUGCAAAGCUUUAUUUCACA 3′);
- xii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UAAAUGUGAAAUAAAGCUUUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ AAAGCUUUAUUUCACAUUUA 3′);
- xiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UAAAAUGUGAAAUAAAGCUUUG3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ AAGCUUUAUUUCACAUUUUA 3′);
- xiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAAAAAUGUGAAAUAAAGCUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 52 (5′ AGCUUUAUUUCACAUUUUUA 3′);
- xv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UUAUUCUUGAGAAACAGGAAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUCCUGUUUCUCAAGAAUAA 3′);
- xvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAUGCUACUUGAACAGUCUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ AAGACUGUUCAAGUAGCAUA 3′);
- xviii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUUGGAAUGCUACUUGAACAGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 55 (5′ UGUUCAAGUAGCAUUCCAAA3′);
- xix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ UCAGAUUGGAAUGCUACUUGAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ CAAGUAGCAUUCCAAUCUGA 3′); or
- xx) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ UGUAAUAAAGUCCAGAAUAGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ CUAUUCUGGACUUUAUUACA 3′).

Embodiment 31. The isolated oligonucleotide of Embodiment 12, wherein the double stranded region comprises:

- i) an anti-sense strand of nucleic acid sequence according to SEQ CD NO: 29 (5′ UUAUUAAUAUCCCACAGAACCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ GUUCUGUGGGAUAUUAAUAA 3′);
- ii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ UGAUCCAAAAAUGUCCUAGGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 59 (5′ CCUAGGACAUUUUUGGAUCA 3′); or
- iii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ UGAUCCAAAAAUGUCCUAGGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO:60 (5′ CCUAGGACAUUUUUGIAUCA 3′).

Embodiment 32. The isolated oligonucleotide of any one of Embodiments 1-31, wherein the isolated oligonucleotide attenuates expression of the HSD17B13 mRNA by 20% to 50% at a dose of 0.05 nM.

Embodiment 33. The isolated oligonucleotide of Embodiment 32, wherein the double stranded region comprises:

- i) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UUAUUCAUUUCAUUUUGAUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ AAUCAAAAUGAAAUGAAUAA 3′);
- ii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUCUUUCAAAUGCUGAAUCUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GAUUCAGCAUUUGAAAGAUA 3′);
- iii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAAUCUUUCAAAUGCUGAAUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ AUUCAGCAUUUGAAAGAUUA 3′);
- iv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UGUGAUCCAAAAAUGUCCUAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ UAGGACAUUUUUGGAUCACA 3′);
- v) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UGAGACAUGAGGUUUUGAUACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ UAUCAAAACCUCAUGUCUCA 3′);
- vi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UAAUAUUCUGCAUACGAUUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ AAAUCGUAUGCAGAAUAUUA 3′);
- vii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UUUCAAAUUGAAUAUUCUGCAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ GCAGAAUAUUCAAUUUGAAA 3′);
- viii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UCUGCUUCAAAUUGAAUAUUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ AAUAUUCAAUUUGAAGCAGA 3′);
- ix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAAAAAUGUGAAAUAAAGCUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 52 (5′ AGCUUUAUUUCACAUUUUUA 3′);
- x) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAUGCUACUUGAACAGUCUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ AAGACUGUUCAAGUAGCAUA 3′);
- xi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUUGGAAUGCUACUUGAACAGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 55 (5′ UGUUCAAGUAGCAUUCCAAA3′);
- xii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ UCAGAUUGGAAUGCUACUUGAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ CAAGUAGCAUUCCAAUCUGA 3′);
- xiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ UUAUUAAUAUCCCACAGAACCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ GUUCUGUGGGAUAUUAAUAA 3′);
- xiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ UGAUCCAAAAAUGUCCUAGGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 59 (5′ CCUAGGACAUUUUUGGAUCA 3′); or
- xv) an anti-sense strand of nucleic acid sequence according to SEQ CD NO: 30 (5′ UGAUCCAAAAAUGUCCUAGGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO:60 (5′ CCUAGGACAUUUUUGIAUCA 3′).

Embodiment 34. The isolated oligonucleotide of any one of Embodiments 1-31, wherein the isolated oligonucleotide attenuates expression of the HSD17B13 mRNA by at least 50% at a dose of 0.05 nM.

Embodiment 35. The isolated oligonucleotide of Embodiment 34, wherein the double stranded region comprises:

- i) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UAAUGUGAAAUAAAGCUUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CAAAGCUUUAUUUCACAUUA 3′);
- ii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UGAAAAAAUGUGAAAUAAAGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUUUAUUUCACAUUUUUUCA 3′);
- iii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UAUCUUAAAGAAAACCUUUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ AAAAGGUUUUCUUUAAGAUA 3′);
- iv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UAUAUCUUAAAGAAAACCUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ AAGGUUUUCUUUAAGAUAUA 3′);
- v) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UUUUCAAAUGCUGAAUCUUAAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ UAAGAUUCAGCAUUUGAAAA 3′);
- vi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UUUCACCUGAUUUAGAGAGCGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ GCUCUCUAAAUCAGGUGAAA 3′);
- vii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UAUAAUCUUGUGCUUGGAUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ AAUCCAAGCACAAGAUUAUA 3′);
- viii) an anti-sense strand of nucleic acid sequence according to SEQ CD NO: 15 (5′ UAAAUUGAAUAUUCUGCAUACG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ UAUGCAGAAUAUUCAAUUUA 3′);
- ix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAUAAAGCUUUGCAGCAUUGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CAAUGCUGCAAAGCUUUAUA 3′);
- x) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UAAUAAAGCUUUGCAGCAUUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ AAUGCUGCAAAGCUUUAUUA 3′);
- xi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UGUGAAAUAAAGCUUUGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ CUGCAAAGCUUUAUUUCACA 3′);
- xii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UAAAUGUGAAAUAAAGCUUUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ AAAGCUUUAUUUCACAUUUA 3′);
- xiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UAAAAUGUGAAAUAAAGCUUUG3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ AAGCUUUAUUUCACAUUUUA 3′);
- xiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UUAUUCUUGAGAAACAGGAAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUCCUGUUUCUCAAGAAUAA 3′); or
- xv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ UGUAAUAAAGUCCAGAAUAGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ CUAUUCUGGACUUUAUUACA 3′).

Embodiment 36. The isolated oligonucleotide of any one of Embodiments 1-31, wherein the isolated oligonucleotide attenuates expression of the HSD17B13 mRNA by at least 50% at a dose of 0.5 nM.

Embodiment 37. The isolated oligonucleotide of Embodiment 36, wherein the double stranded region comprises:

- i) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UUAUUCAUUUCAUUUUGAUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ AAUCAAAAUGAAAUGAAUAA 3′);
- ii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UAAUGUGAAAUAAAGCUUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CAAAGCUUUAUUUCACAUUA 3′);
- iii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UGAAAAAAUGUGAAAUAAAGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUUUAUUUCACAUUUUUUCA 3′);
- iv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UAUCUUAAAGAAAACCUUUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ AAAAGGUUUUCUUUAAGAUA 3′);
- v) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UAUAUCUUAAAGAAAACCUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ AAGGUUUUCUUUAAGAUAUA 3′);
- vi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UUUUCAAAUGCUGAAUCUUAAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ UAAGAUUCAGCAUUUGAAAA 3′);
- vii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUCUUUCAAAUGCUGAAUCUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GAUUCAGCAUUUGAAAGAUA 3′);
- viii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAAUCUUUCAAAUGCUGAAUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ AUUCAGCAUUUGAAAGAUUA 3′);
- ix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UUUCACCUGAUUUAGAGAGCGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ GCUCUCUAAAUCAGGUGAAA 3′);
- x) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UGUGAUCCAAAAAUGUCCUAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ UAGGACAUUUUUGGAUCACA 3′);
- xi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UGAGACAUGAGGUUUUGAUACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ UAUCAAAACCUCAUGUCUCA 3′);
- xii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UAUAAUCUUGUGCUUGGAUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ AAUCCAAGCACAAGAUUAUA 3′);
- xiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UAAUAUUCUGCAUACGAUUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ AAAUCGUAUGCAGAAUAUUA 3′);
- xiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAAAUUGAAUAUUCUGCAUACG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ UAUGCAGAAUAUUCAAUUUA 3′);
- xv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UUUCAAAUUGAAUAUUCUGCAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ GCAGAAUAUUCAAUUUGAAA 3′);
- xvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UCUGCUUCAAAUUGAAUAUUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ AAUAUUCAAUUUGAAGCAGA 3′);
- xvii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAUAAAGCUUUGCAGCAUUGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CAAUGCUGCAAAGCUUUAUA 3′);
- xviii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UAAUAAAGCUUUGCAGCAUUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ AAUGCUGCAAAGCUUUAUUA 3′);
- xix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UGUGAAAUAAAGCUUUGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ CUGCAAAGCUUUAUUUCACA 3′);
- xx) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UAAAUGUGAAAUAAAGCUUUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ AAAGCUUUAUUUCACAUUUA 3′);
- xxi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UAAAAUGUGAAAUAAAGCUUUG3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ AAGCUUUAUUUCACAUUUUA 3′);
- xxii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAAAAAUGUGAAAUAAAGCUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 52 (5′ AGCUUUAUUUCACAUUUUUA 3′);
- xxiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UUAUUCUUGAGAAACAGGAAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUCCUGUUUCUCAAGAAUAA 3′);
- xxiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAUGCUACUUGAACAGUCUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ AAGACUGUUCAAGUAGCAUA 3′);
- xxv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUUGGAAUGCUACUUGAACAGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 55 (5′ UGUUCAAGUAGCAUUCCAAA3′);
- xxvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ UCAGAUUGGAAUGCUACUUGAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ CAAGUAGCAUUCCAAUCUGA 3′);
- xxvii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ UGUAAUAAAGUCCAGAAUAGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ CUAUUCUGGACUUUAUUACA 3′);
- xxviii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ UUAUUAAUAUCCCACAGAACCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ GUUCUGUGGGAUAUUAAUAA 3′);
- xxix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ UGAUCCAAAAAUGUCCUAGGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 59 (5′ CCUAGGACAUUUUUGGAUCA 3′); or
- xxx) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ UGAUCCAAAAAUGUCCUAGGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO:60 (5′ CCUAGGACAUUUUUGIAUCA 3′).

Embodiment 38. An isolated oligonucleotide comprising a sense strand and an anti-sense 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) 57 to 91;
  - b) 230 to 368;
  - c) 394 to 428;
  - d) 432 to 520;
  - e) 523 to 765;
  - f) 766 to 811;
  - g) 881 to 912;
  - h) 1094 to 1123;
  - i) 1138 to 1171;
  - j) 1198 to 1245;
  - k) 1304 to 1324;
  - j) 1345 to 1377;
  - 1) 1422 to 1442;
  - m) 1479 to 1506; and
  - n) 1538 to 1577,
- from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) mRNA sequence according to SEQ ID NO: 1, and the anti-sense strand is substantially complementary to the sense strand such that the sense strand and the anti-sense strand together form a double stranded region.

Embodiment 39. The isolated oligonucleotide of Embodiment 38, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from:

- a) 57 to 88;
- b) 230 to 250;
- c) 267 to 290;
- d) 339 to 368;
- e) 396 to 428;
- f) 432 to 454;
- g) 459 to 517;
- h) 630 to 679;
- i) 701 to 765;
- j) 766-912;
- k) 1094 to 1123;
- l) 1138 to 1171;
- m) 1198 to 1218;
- n) 1225-1245;
- o) 1304 to 1324;
- p) 1345 to 1377;
- q) 1422 to 1442;
- r) 1479 to 1506; and
- s) 1538 to 1577,
- from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) mRNA sequence according to SEQ ID NO: 1,
- and the anti-sense strand is substantially complementary to the sense strand such that the sense strand and the anti-sense strand together form a double stranded region.

Embodiment 40. The isolated oligonucleotide of Embodiment 39, wherein the isolated oligonucleotide attenuates expression of the HSD17B13 mRNA by 20% to 50% at a dose of 0.05 nM.

Embodiment 41. The isolated oligonucleotide of any one of Embodiments 39-40, wherein the isolated oligonucleotide attenuates expression of the HSD17B13 mRNA by at least 50% at a dose of 0.5 nM.

Embodiment 42. The isolated oligonucleotide of any one of Embodiments 40-41, wherein the double stranded region comprises:

- i) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 64 (5′ UUAGAUGAUGGUGAUCAGAAGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 252 (5′ UUCUGAUCACCAUCAUCUAA 3′);
- ii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUUAUUAAUAUCCCACAGAACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 258 (5′ UUCUGUGGGAUAUUAAUAAA 3′);
- iii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 75 (5′ UUAGUUUUCGGCACUCAGCUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 263 (5′ AGCUGAGUGCCGAAAACUAA 3′);
- iv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 80 (5′ UCUGAUUUAGAGAGCGAUAGAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 268 (5′ CUAUCGCUCUCUAAAUCAGA 3′);
- v) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 81 (5′ UCUUCACCUGAUUUAGAGAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 269 (5′ CUCUCUAAAUCAGGUGAAGA 3′);
- vi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 82 (5′ UUUUCUUCACCUGAUUUAGAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 270 (5′ UCUAAAUCAGGUGAAGAAAA 3′);
- vii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 89 (5′ UUAAUCUCUUCAUCCUUGGUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 277 (5′ ACCAAGGAUGAAGAGAUUAA 3′);
- viii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 90 (5′ UGUAAUCUCUUCAUCCUUGGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 278 (5′ CCAAGGAUGAAGAGAUUACA 3′);
- ix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 98 (5′ UUUGUGAUCCAAAAAUGUCCUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 286 (5′ GGACAUUUUUGGAUCACAAA 3′);
- x) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 102 (5′ UGAAGAAGUGCUUUUGUGAUCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 290 (5′ AUCACAAAAGCACUUCUUCA 3′);
- xi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 111 (5′ UAAGUUCUGAUGUCAGACCUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 299 (5′ AGGUCUGACAUCAGAACUUA 3′);
- xii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 112 (5′ UUUUUGAUACCAGUUUUUCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 300 (5′ GGAAAAACUGGUAUCAAAAA 3′);
- xiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 113 (5′ UGUUUUGAUACCAGUUUUUCCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 301 (5′ GAAAAACUGGUAUCAAAACA 3′);
- xiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 132 (5′ UUUGUGCUUGGAUUUUUGGUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 320 (5′ ACCAAAAAUCCAAGCACAAA 3′);
- xv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 135 (5′ UUACAGGCCAUAAUCUUGUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 323 (5′ CACAAGAUUAUGGCCUGUAA 3′);
- xvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 142 (5′ UUUCAUCUGUCUCCAAUACAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 330 (5′ UGUAUUGGAGACAGAUGAAA 3′);
- xvii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 143 (5′ UCUUCAUCUGUCUCCAAUACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 331 (5′ GUAUUGGAGACAGAUGAAGA 3′);
- xviii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 144 (5′ UCAUCUAUCAGACUUCUUACGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 332 (5′ GUAAGAAGUCUGAUAGAUGA 3′);
- xix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 146 (5′ UAAGUAUUCCAUCUAUCAGACU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 334 (5′ UCUGAUAGAUGGAAUACUUA 3′);
- xx) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 147 (5′ UUUAUUGGUAAGUAUUCCAUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 335 (5′ AUGGAAUACUUACCAAUAAA 3′);
- xxi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 149 (5′ UUUCUUAUUGGUAAGUAUUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 337 (5′ GAAUACUUACCAAUAAGAAA 3′);
- xxii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 149 (5′ UUUCUUAUUGGUAAGUAUUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 337 (5′ GAAUACUUACCAAUAAGAAA 3′);
- xxiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 152 (5′ UAUAUUCUGCAUACGAUUUAAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 340 (5′ UAAAUCGUAUGCAGAAUAUA 3′);
- xxiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 153 (5′ UCUUCAAAUUGAAUAUUCUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 341 (5′ CAGAAUAUUCAAUUUGAAGA 3′);
- xxv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 154 (5′ UGAGAAACAGGAAGACAGGUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 342 (5′ ACCUGUCUUCCUGUUUCUCA 3′);
- xxvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 156 (5′ UCUUGAGAAACAGGAAGACAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 344 (5′ UGUCUUCCUGUUUCUCAAGA 3′);
- xxvii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 159 (5′ UAAUAUUCUUGAGAAACAGGAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 347 (5′ CCUGUUUCUCAAGAAUAUUA 3′);
- xxviii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 160 (5′ UAUGAAAGGAAAAACAGACCUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 348 (5′ GGUCUGUUUUUCCUUUCAUA 3′);
- xxix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 162 (5′ UUUUUUAAGAGGCAUGAAAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 350 (5′ CUUUCAUGCCUCUUAAAAAA 3′);
- xxx) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 164 (5′ UAAAUAUCUUAAAGAAAACCUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 352 (5′ GGUUUUCUUUAAGAUAUUUA 3′);
- xxxi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 166 (5′ UUUUUGUCCACCUUUAAAUGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 354 (5′ CAUUUAAAGGUGGACAAAAA 3′);
- xxxii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 167 (5′ UGAUUGGAAUGCUACUUGAACA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 355 (5′ UUCAAGUAGCAUUCCAAUCA 3′);
- xxxiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 168 (5′ UCUCAUUCUGUGUUCUUGUUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 356 (5′ AACAAGAACACAGAAUGAGA 3′);
- xxxiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 169 (5′ UUUAGCUGUGCACUCAUUCUGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 357 (5′ AGAAUGAGUGCACAGCUAAA 3′);
- xxxv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 171 (5′ UCUUUCAAAUGCUGAAUCUUAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 359 (5′ AAGAUUCAGCAUUUGAAAGA 3′);
- xxxvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 174 (5′ UUAAAGUCCAGAAUAGAGUUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 362 (5′ AACUCUAUUCUGGACUUUAA 3′);
- xxxvii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 176 (5′ UUAAUAAAGUCCAGAAUAGAGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 364 (5′ UCUAUUCUGGACUUUAUUAA 3′);
- xxxviii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 184 (5′ UAAGGGAGGAAAUAUAGAGGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 372 (5′ CCUCUAUAUUUCCUCCCUUA 3′);
- xxxix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 185 (5′ UAAAGGGAGGAAAUAUAGAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 373 (5′ CUCUAUAUUUCCUCCCUUUA 3′);
- xL) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 186 (5′ UAAAAGGGAGGAAAUAUAGAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 374 (5′ UCUAUAUUUCCUCCCUUUUA 3′);
- xLi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 187 (5′ UUAAAAAGGGAGGAAAUAUAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 375 (5′ UAUAUUUCCUCCCUUUUUAA 3′); or
- xLii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 189 (5′ UUAUAAAAAGGGAGGAAAUAUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 377 (5′ UAUUUCCUCCCUUUUUAUAA 3′).

Embodiment 43. The isolated oligonucleotide of Embodiment 39, wherein the isolated oligonucleotide attenuates expression of the HSD17B13 mRNA by at least 50% at a dose of 0.5 nM.

Embodiment 44. The isolated oligonucleotide of Embodiment 43, wherein the double stranded region comprises:

- (i) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 62 (5′ UGAUCAGAAGCAGAAGGAUUUC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 250 (5′ AAUCCUUCUGCUUCUGAUCA 3′);
- (ii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 63 (5′ UAUGGUGAUCAGAAGCAGAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 251 (5′ UUCUGCUUCUGAUCACCAUA 3′);
- (iii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 73 (5′ UUUUUCGGCACUCAGCUGCAGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 261 (5′ UGCAGCUGAGUGCCGAAAAA 3′);
- (iv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 84 (5′ UAUAUACUGUCCCAGCAUUAUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 272 (5′ UAAUGCUGGGACAGUAUAUA 3′);
- (v) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 87 (5′ UAAGAUCGGCUGGAUAUACUGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 275 (5′ AGUAUAUCCAGCCGAUCUUA 3′);
- (vi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 88 (5′ UAAUCUCUUCAUCCUUGGUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 276 (5′ CACCAAGGAUGAAGAGAUUA 3′);
- (vii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 91 (5′ UUAGGAUGUUGACCUCAAAUGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 279 (5′ AUUUGAGGUCAACAUCCUAA 3′);
- (viii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 94 (5′ UAAUGUCCUAGGAUGUUGACCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 282 (5′ GUCAACAUCCUAGGACAUUA 3′);
- (ix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 99 (5′ UUUUGUGAUCCAAAAAUGUCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 287 (5′ GACAUUUUUGGAUCACAAAA 3′);
- (x) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 104 (5′ UAUCGAUGGAAGAAGUGCUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 292 (5′ AAGCACUUCUUCCAUCGAUA 3′);
- (xi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 126 (5′ UUUGGUGAACCCAGUAUUCACA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 314 (5′ UGAAUACUGGGUUCACCAAA 3′);
- (xii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 127 (5′ UUUUGGUGAACCCAGUAUUCAC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 315 (5′ GAAUACUGGGUUCACCAAAA 3′);
- (xiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 128 (5′ UUUUUGGUGAACCCAGUAUUCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 316 (5′ AAUACUGGGUUCACCAAAAA 3′);
- (xiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 130 (5′ UGAUUUUUGGUGAACCCAGUAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 318 (5′ ACUGGGUUCACCAAAAAUCA 3′);
- (xv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 131 (5′ UCUUGGAUUUUUGGUGAACCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 319 (5′ GGUUCACCAAAAAUCCAAGA 3′);
- (xvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 133 (5′ UCUUGUGCUUGGAUUUUUGGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 321 (5′ CCAAAAAUCCAAGCACAAGA 3′);
- (xvii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 134 (5′ UAUCUUGUGCUUGGAUUUUUGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 322 (5′ AAAAAUCCAAGCACAAGAUA 3′);
- (xviii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 136 (5′ UAUACAGGCCAUAAUCUUGUGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 324 (5′ ACAAGAUUAUGGCCUGUAUA 3′);
- (xix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 138 (5′ UCAAUACAGGCCAUAAUCUUGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 326 (5′ AAGAUUAUGGCCUGUAUUGA 3′);
- (xx) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 145 (5′ UUAUUCCAUCUAUCAGACUUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 333 (5′ AAGUCUGAUAGAUGGAAUAA 3′);
- (xxi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 148 (5′ UCUUAUUGGUAAGUAUUCCAUC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 336 (5′ UGGAAUACUUACCAAUAAGA 3′);
- (xxii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 150 (5′ UUUUCUUAUUGGUAAGUAUUCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 338 (5′ AAUACUUACCAAUAAGAAAA 3′);
- (xxiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 151 (5′ UAUCAUUUUCUUAUUGGUAAGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 339 (5′ UUACCAAUAAGAAAAUGAUA 3′);
- (xxiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 157 (5′ UUUCUUGAGAAACAGGAAGACA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 345 (5′ UCUUCCUGUUUCUCAAGAAA 3′);
- (xxv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 158 (5′ UAUUCUUGAGAAACAGGAAGAC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 346 (5′ CUUCCUGUUUCUCAAGAAUA 3′);
- (xxvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 161 (5′ UCAUGAAAGGAAAAACAGACCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 349 (5′ GUCUGUUUUUCCUUUCAUGA 3′);
- (xxvii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 163 (5′ UGUUUUUAAGAGGCAUGAAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 351 (5′ UUUCAUGCCUCUUAAAAACA 3′);
- (xxviii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 170 (5′ UCUUAGCUGUGCACUCAUUCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 358 (5′ GAAUGAGUGCACAGCUAAGA 3′);
- (xxix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 173 (5′ UAAAGUCCAGAAUAGAGUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 361 (5′ CAACUCUAUUCUGGACUUUA 3′);
- (xxx) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 175 (5′ UAAUAAAGUCCAGAAUAGAGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 363 (5′ CUCUAUUCUGGACUUUAUUA 3′);
- (xxxi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 178 (5′ UAUAGAGGGUCCACUUUUGGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 366 (5′ CCAAAAGUGGACCCUCUAUA 3′);
- (xxxii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 179 (5′ UUAUAGAGGGUCCACUUUUGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 367 (5′ CAAAAGUGGACCCUCUAUAA 3′);
- (xxxiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 181 (5′ UAAUAUAGAGGGUCCACUUUUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 369 (5′ AAAGUGGACCCUCUAUAUUA 3′);
- (xxxiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 182 (5′ UAAAUAUAGAGGGUCCACUUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 370 (5′ AAGUGGACCCUCUAUAUUUA 3′);
- (xxxv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 183 (5′ UGAAAUAUAGAGGGUCCACUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 371 (5′ AGUGGACCCUCUAUAUUUCA 3′);
- (xxxvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 183 (5′ UGAAAUAUAGAGGGUCCACUUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 371 (5′ AGUGGACCCUCUAUAUUUCA 3′); or
- (xxxvii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 188 (5′ UAUAAAAAGGGAGGAAAUAUAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 376 (5′ AUAUUUCCUCCCUUUUUAUA 3′).

Embodiment 45. The isolated oligonucleotide of Embodiment 38, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from.

- a) 69 to 91;
- b) 231 to 310;
- c) 394 to 426;
- d) 463 to 648;
- e) 682 to 724;
- f) 731 to 762;
- g) 1096 to 1116;
- h) 1204 to 1224;
- i) 1479 to 1499; and
- j) 1538 to 1561,
- from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) mRNA sequence according to SEQ ID NO: 1,
- and the anti-sense strand is substantially complementary to the sense strand such that the sense strand and the anti-sense strand together form a double stranded region.

Embodiment 46. The isolated oligonucleotide of Embodiment 45, wherein the isolated oligonucleotide attenuates expression of the HSD17B13 mRNA by 20% to 50% at a dose of 0.5 nM.

Embodiment 47. The isolated oligonucleotide of Embodiment 46, wherein the double stranded region comprises:

- (i) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 65 (5′ UGUAGAUGAUGGUGAUCAGAAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 253 (5′ UCUGAUCACCAUCAUCUACA 3′);
- (ii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 66 (5′ UGAGUAGAUGAUGGUGAUCAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 254 (5′ UGAUCACCAUCAUCUACUCA 3′);
- (iii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 71 (5′ UCUUAUUAAUAUCCCACAGAAC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 259 (5′ UCUGUGGGAUAUUAAUAAGA 3′);
- (iv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 72 (5′ UCACUCAGCUGCAGUUUCCUCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 260 (5′ AGGAAACUGCAGCUGAGUGA 3′);
- (v) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 74 (5′ UGUUUUCGGCACUCAGCUGCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 262 (5′ GCAGCUGAGUGCCGAAAACA 3′);
- (vi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 76 (5′ UCUAGUUUUCGGCACUCAGCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 264 (5′ GCUGAGUGCCGAAAACUAGA 3′);
- (vii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 78 (5′ UCAGUGACGCCUAGUUUUCGGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 266 (5′ CGAAAACUAGGCGUCACUGA 3′);
- (viii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 79 (5′ UUACGCAUGCGCAGUGACGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 267 (5′ GCGUCACUGCGCAUGCGUAA 3′);
- (ix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 83 (5′ UAUACUGUCCCAGCAUUAUUCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 271 (5′ AAUAAUGCUGGGACAGUAUA 3′);
- (x) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 85 (5′ UGAUAUACUGUCCCAGCAUUAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 273 (5′ AAUGCUGGGACAGUAUAUCA 3′);
- (xi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 86 (5′ UGAUCGGCUGGAUAUACUGUCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 274 (5′ ACAGUAUAUCCAGCCGAUCA 3′);
- (xii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 92 (5′ UGUCCUAGGAUGUUGACCUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 280 (5′ GAGGUCAACAUCCUAGGACA 3′);
- (xiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 93 (5′ UAUGUCCUAGGAUGUUGACCUC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 281 (5′ GGUCAACAUCCUAGGACAUA 3′);
- (xiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 95 (5′ UAAAUGUCCUAGGAUGUUGACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 283 (5′ UCAACAUCCUAGGACAUUUA 3′);
- (xv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 96 (5′ UAAAAUGUCCUAGGAUGUUGAC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 284 (5′ CAACAUCCUAGGACAUUUUA 3′);
- (xvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 97 (5′ UAAAAAUGUCCUAGGAUGUUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 285 (5′ AACAUCCUAGGACAUUUUUA 3′);
- (xvii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 100 (5′ UUUUUGUGAUCCAAAAAUGUCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 288 (5′ ACAUUUUUGGAUCACAAAAA 3′);
- (xviii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 103 (5′ UAUGGAAGAAGUGCUUUUGUGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 291 (5′ ACAAAAGCACUUCUUCCAUA 3′);
- (xix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 105 (5′ UAUCAUCGAUGGAAGAAGUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 293 (5′ CACUUCUUCCAUCGAUGAUA 3′);
- (xx) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 106 (5′ UCGAUGUGGCCAUGAUUUCUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 294 (5′ AGAAAUCAUGGCCACAUCGA 3′);
- (xxi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 107 (5′ UGACGAUGUGGCCAUGAUUUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 295 (5′ AAAUCAUGGCCACAUCGUCA 3′);
- (xxii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 108 (5′ UAUGUCAGACCUCUGUGAAAGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 296 (5′ UUUCACAGAGGUCUGACAUA 3′);
- (xxiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 109 (5′ UCUGAUGUCAGACCUCUGUGAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 297 (5′ CACAGAGGUCUGACAUCAGA 3′);
- (xxiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 110 (5′ UGUUCUGAUGUCAGACCUCUGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 298 (5′ AGAGGUCUGACAUCAGAACA 3′);
- (xxiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 118 (5′ UCAAAAACUGGGCAGAGACAUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 306 (5′ UGUCUCUGCCCAGUUUUUGA 3′);
- (xxv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 120 (5′ UUUCACAAAAACUGGGCAGAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 308 (5′ UCUGCCCAGUUUUUGUGAAA 3′);
- (xxvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 121 (5′ UAUUCACAAAAACUGGGCAGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 309 (5′ CUGCCCAGUUUUUGUGAAUA 3′);
- (xxvii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 122 (5′ UUAUUCACAAAAACUGGGCAGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 310 (5′ UGCCCAGUUUUUGUGAAUAA 3′);
- (xxviii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 123 (5′ UGUAUUCACAAAAACUGGGCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 311 (5′ GCCCAGUUUUUGUGAAUACA 3′);
- (xxix) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 129 (5′ UUUUUUGGUGAACCCAGUAUUC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 317 (5′ AUACUGGGUUCACCAAAAAA 3′);
- (xxx) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 137 (5′ UAAUACAGGCCAUAAUCUUGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 325 (5′ CAAGAUUAUGGCCUGUAUUA 3′);
- (xxx) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 139 (5′ UCUCCAAUACAGGCCAUAAUCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 327 (5′ AUUAUGGCCUGUAUUGGAGA 3′);
- (xxxi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 140 (5′ UAUCUGUCUCCAAUACAGGCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 328 (5′ GCCUGUAUUGGAGACAGAUA 3′);
- (xxxi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 141 (5′ UCAUCUGUCUCCAAUACAGGCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 329 (5′ CCUGUAUUGGAGACAGAUGA 3′);
- (xxxii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 155 (5′ UUUGAGAAACAGGAAGACAGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 343 (5′ CUGUCUUCCUGUUUCUCAAA 3′);
- (xxxiii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 165 (5′ UAAAAUAAAAUAUCUUAAAGAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 353 (5′ CUUUAAGAUAUUUUAUUUUA 3′);
- (xxxiv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 172 (5′ UGUCCAGAAUAGAGUUGCACCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 360 (5′ GUGCAACUCUAUUCUGGACA 3′);
- (xxxv) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 177 (5′ UUAGAGGGUCCACUUUUGGUGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 365 (5′ ACCAAAAGUGGACCCUCUAA 3′);
- (xxxvi) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 178 (5′ UAUAGAGGGUCCACUUUUGGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 366 (5′ CCAAAAGUGGACCCUCUAUA 3′); or
- (xxxvii) an anti-sense strand of nucleic acid sequence according to SEQ ID NO: 180 (5′ UAUAUAGAGGGUCCACUUUUGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 368 (5′ AAAAGUGGACCCUCUAUAUA 3′).

Embodiment 48. The isolated oligonucleotide of any one of Embodiments 1-47, wherein the sense strand or the anti-sense strand or both comprise one or more modified nucleotide(s).

Embodiment 49. The isolated oligonucleotide of Embodiment 48, wherein the one or more modified nucleotide(s) increases the stability or potency or both of the isolated oligonucleotide.

Embodiment 50. A vector encoding the isolated oligonucleotide of any one of Embodiments 1-49.

Embodiment 51. The vector of Embodiment 50, wherein the vector is a plasmid.

Embodiment 52. A delivery system comprising the isolated oligonucleotide of any one of Embodiments 1-49 or the vector of any one of Embodiments 50-51.

Embodiment 53. The delivery system of Embodiment 52, wherein the delivery system is any one of a liposome, a nanoparticle, a polymer based delivery system, or a ligand-conjugate delivery system.

Embodiment 54. The delivery system of Embodiment 53, wherein the ligand-conjugate delivery system comprises one or more of an antibody, a peptide, a lipid, a sugar moiety or a combination thereof.

Embodiment 55. A pharmaceutical composition comprising the isolated oligonucleotide of any one of Embodiments 1-49, the vector of any one of Embodiments 50-51, the delivery system of any one of Embodiments 52-54, and a pharmaceutically acceptable carrier, diluent or excipient.

Embodiment 56. A kit comprising the isolated oligonucleotide of any one of Embodiments 1-49, the vector of any one of Embodiments 50-51, the delivery system of any one of Embodiments 52-54, or the pharmaceutical composition of Embodiment 55.

Embodiment 57. The kit of Embodiment 56, further comprising instructions for administrating the isolated oligonucleotide, the vector, the delivery system or the pharmaceutical composition to a subject.

Embodiment 58. A method of inhibiting or downregulating the expression or level of HSD17B13 in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the isolated oligonucleotide of any one of Embodiments 1-49, the vector of any one of Embodiments 50-51, the delivery system of any one of Embodiments 52-54, or the pharmaceutical composition of Embodiment 55.

Embodiment 59. The method of Embodiment 58, wherein the isolated oligonucleotide, the vector, the delivery system or the pharmaceutical composition is administered parenterally.

Embodiment 60. The method of Embodiment 59, wherein the parenteral administration is intravenous, subcutaneous, intraperitoneal or intramuscular.

Embodiment 61. The method of any one of Embodiments 58-60, wherein the subject is a human.

Embodiment 62. The method of any one of Embodiments 58-61, wherein the subject has nonalcoholic fatty liver disease (NAFLD), fatty liver disease, liver injury, inflammation, fibrosis, cirrhosis, or carcinoma.

Embodiment 63. The method of any one of Embodiments 58-62, wherein the method comprises administering the isolated oligonucleotide, the vector, the delivery system, or the pharmaceutical composition, in combination with at least a second therapeutic agent.

Embodiment 64. The method of Embodiment 62, wherein the second therapeutic agent is an antibody, a small molecule drug, a peptide, a nucleic acid molecule or a combination thereof. Embodiment 65. The method of Embodiment 63, wherein the second therapeutic agent is an isolated oligonucleotide of any one of Embodiments 1-49.

Embodiment 66. A method of inhibiting or downregulating the expression or level of HSD17B13 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 of any one of Embodiments 1-49, wherein the first and at least second oligonucleotides comprise different sequences.

Embodiment 67. The method of Embodiment 66, wherein the first and at least second oligonucleotides are administered simultaneously.

Embodiment 68. The method of Embodiment 66, wherein the first and at least second oligonucleotides are administered sequentially.

Embodiment 69. A method of treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 or a disease or disorder where HSD17B13 plays a role in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the isolated oligonucleotide of any one of Embodiments 1-49, the vector of any one of Embodiments 50-51, the delivery system of any one of Embodiments 52-54, or the pharmaceutical composition of Embodiment 55.

Embodiment 70. The method of Embodiment 69, wherein the subject is a human.

Embodiment 71. 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 between any one of the nucleotide positions selected from:
- a) 229 to 249;
- b) 669 to 689; and
- c) 1007 to 1027,
- from the 5′ end of a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) 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.

Embodiment 72. The isolated oligonucleotide of Embodiment 71, 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) 229 to 249;
- b) 669 to 689; and
- c) 1007 to 1027,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 73. The isolated oligonucleotide of Embodiment 71, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from:

- a) 229 to 249;
- b) 669 to 689; and
- c) 1007 to 1027,
- from the 5′ end of a HSD17B13 mRNA sequence according to SEQ ID NO: 1.

Embodiment 74. The isolated oligonucleotide of any one of Embodiments 71-73, wherein the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 3, 12 and 29.

Embodiment 75. The isolated oligonucleotide of any one of Embodiments 71-74, wherein the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 32, 41 and 58.

Embodiment 76. The isolated oligonucleotide of Embodiment 73, wherein the double stranded region comprises an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UAAUGUGAAAUAAAGCUUUGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CAAAGCUUUAUUUCACAUUA 3′).

Embodiment 77. The isolated oligonucleotide of Embodiment 73, wherein the double stranded region comprises an anti sense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UGAGACAUGAGGUUUUGAUACC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ UAUCAAAACCUCAUGUCUCA 3′).

Embodiment 78. The isolated oligonucleotide of Embodiment 73, wherein the double stranded region comprises an antisense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ UUAUUAAUAUCCCACAGAACCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ GUUCUGUGGGAUAUUAAUAA 3′).

Embodiment 79. The isolated oligonucleotide of any one of Embodiments 71-78, wherein the sense strand or the antisense strand or both comprise one or more modified nucleotide(s).

Embodiment 80. The isolated oligonucleotide of Embodiment 79, wherein the antisense strand comprises a mono methyl protected phosphate mimic (5′-MeEP).

Embodiment 81. The isolated oligonucleotide of any one of Embodiments 71-80, wherein in the sense strand or the antisense strand or both, a terminal or internal nucleotide is linked to a targeting ligand.

Embodiment 82. The isolated oligonucleotide of Embodiment 81, wherein the targeting ligand is attached to the 3′ end (e.g., 3′ terminal position) of the sense strand.

Embodiment 83. The isolated oligonucleotide of any one of Embodiments 81-82, wherein the targeting ligand comprises a GalNAc.

Embodiment 84. The isolated oligonucleotide of any one of Embodiments 81-83, wherein the targeting ligand comprises at least one GalNAc G1b moiety.

Embodiment 85. The isolated oligonucleotide of any one of Embodiments 71-84, wherein the antisense strand comprises nucleotides modified with 2′-F modification, and nucleotides modified with 2′-O-methyl modification, according to the formula:

3′(M)₀(F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁5′.

Embodiment 86. The isolated oligonucleotide of any one of Embodiments 71-85, wherein the sense strand comprises nucleotides modified with 2′-F modification, and nucleotides modified with 2′-O-methyl modification, according to the formula:

5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉3′.

Embodiment 87. The isolated oligonucleotide of any one of Embodiments 71-86, wherein the antisense strand comprises any one of:

- i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 440 (5′ [MeEPmUs][fAs][fA][mU][f 3][mU][fG][mA][mA][fA][mU][mA][mA][fA][mG][fC][mU][mU][mU][mGs][mCs][mA] 3′);
- ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 442 (5′ [McEPmUs][fGs][fA][mG][fA][mC][fA][mU][mG][fA][mG][mG][mU][fU][mU][fU][mG][mA][mU][mAs][mCs][mC] 3′); or
- iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 444 (5′ [MeEPmUs][fUs][fA][mU][fU][mA][fA][mU][mA][fU][mC][mC][mC][fA][mC][fA][mG][mA][mA][mCs][mCs][mA] 3′),
- wherein “m” is a 2′-O-methyl modified nucleotide, “f” is a 2′-F modified nucleotide, “s” is a phosphorothioate internucleotide linkage, “MeEP” is a mono methyl protected phosphate mimic.

Embodiment 88. The isolated oligonucleotide of any one of Embodiments 71-87, wherein the sense strand comprises any one of:

- i) a sense strand of nucleic acid sequence according to SEQ ID NO: 441 (5′ [mCs][mAs][mA][mA][mG][fC][mU][fU][fU][fA][fU][mU][mU][mC][mA][mC][mA][mUs][m Us][mA][G1b][G1b][G1b] 3′);
- ii) a sense strand of nucleic acid sequence according to SEQ ID NO: 443 (5′ [mUs][mAs][mU][mC][mA][fA][mA][fA][fC][fC][fU][mC][mA][mU][mG][mU][mC][mUs][m Cs][mA][G1b][G1b][G1b] 3′); or
- iii) a sense strand of nucleic acid sequence according to SEQ ID NO: 445 (5′ [mGs][mUs][mU][mC][mU][fG][mU][fG][fG][fG][fA][mU][mA][mU][mU][mA][mA][mUs][m As][mA][G1b][G1b][G1b] 3′),
- wherein “m” is a 2′-O-methyl modified nucleotide, “f” is a 2′-F modified nucleotide, “s” is a phosphorothioate internucleotide linkage, and “G1b” is a GalNac G1b moiety.

Embodiment 89. A vector encoding the isolated oligonucleotide of any one of Embodiments 71-88.

Embodiment 90. The vector of Embodiment 79, wherein the vector is a plasmid.

Embodiment 91. A delivery system comprising the isolated oligonucleotide of any one of Embodiments 71-88 or the vector of any one of Embodiments 89-90.

Embodiment 92. A pharmaceutical composition comprising the isolated oligonucleotide of any one of Embodiments 71-88, the vector of any one of Embodiments 89-90, the delivery system of Embodiment 91, and a pharmaceutically acceptable carrier, diluent or excipient.

Embodiment 93. A kit comprising the isolated oligonucleotide of any one of Embodiments 71-88, the vector of any one of Embodiments 89-90, the delivery system of Embodiment 91, or the pharmaceutical composition of Embodiment 92.

Embodiment 94. A method of inhibiting or downregulating the expression or level of HSD17B13 in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the isolated oligonucleotide of any one of Embodiments 71-88, the vector of any one of Embodiments 89-90, the delivery system of Embodiment 91, or the pharmaceutical composition of Embodiment 92.

Embodiment 95. The method of Embodiment 94, wherein the subject has nonalcoholic fatty liver disease (NAFLD), fatty liver disease, liver injury, inflammation, fibrosis, cirrhosis, or carcinoma.

Embodiment 96. The method of any one of Embodiments 94-95, wherein the method comprises administering the isolated oligonucleotide, the vector, the delivery system, or the pharmaceutical composition, in combination with at least a second therapeutic agent.

Embodiment 97. A method of inhibiting or downregulating the expression or level of HSD17B13 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 of any one of Embodiments 71-88, wherein the first and at least second oligonucleotides comprise different sequences.

Embodiment 98. A method of treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HSD17B13 or a disease or disorder where HSD17B13 plays a role in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the isolated oligonucleotide of any one of Embodiments 71-88, the vector of any one of Embodiments 89-90, the delivery system of Embodiment 91, or the pharmaceutical composition of Embodiment 92.

DOUBLE STRANDED RNA TARGETING 17-BETA HYDROXYSTEROIDDEHYDROGENASE 13 (HSD17B13) AND METHODS OF USE THEREOF

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