SMALL INTERFERING RNA TARGETING HBV AND USES THEREOF

Abstract

This disclosure relates to isolated oligonucleotides comprising duplex regions targeting hepatitis B virus (HBV) mRNA, and delivery systems, kits and compositions comprising the same, and methods of using the same for inhibiting or downregulating HBV gene and surface antigen expression.

Description

BACKGROUND

Hepatitis B is a life-threatening liver infection caused by the hepatitis B virus (HBV). HBV infection can cause both acute and chronic disease. Acute infection with HBV can cause symptoms such as fatigue, poor appetite, stomach pain, inflammation, nausea, vomiting and jaundice. In some instances, HBV infection can become chronic and can lead to serious, life-threatening health issues such as liver failure, cirrhosis, or liver cancer.

Despite there being a vaccine to prevent hepatitis B, HBV continues to pose a global health problem. The World Health Organization estimates that 296 million people were living with chronic hepatitis B infection in 2019, with 1.5 million new infections each year. There is currently no specific treatment for acute hepatitis B and care is largely limited to replacing patient fluids and maintaining patient comfort. Accordingly, there is a need for therapies for subjects having diseases, disorders and symptoms associated with elevated HBV expression levels, such as for example hepatitis B, cirrhosis of the liver and hepatocellular carcinoma. The present disclosure provides compositions targeting HBV and methods of reducing HBV mRNA and surface antigen (HBsAg) expression for treatment of subjects having an HBV-associated disease, disorder or symptom.

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) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a hepatitis B virus (HBV) 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) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a HBV 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 comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a HBV 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) 158 to 240; h) 389 to 439; c) 621 to 686; and d) 775 to 823, from the 5′ end of a HBV 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) 158 to 240; b) 389 to 439; c) 621 to 686; and d) 775 to 823, from the 5′ end of a HBV 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) 158 to 240; b) 389 to 439; c) 621 to 686; and d) 775 to 823, from the 5′ end of a HBV 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) 206 to 226; b) 558 to 578; and c) 720 to 807, from the 5′ end of a HBV 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 as 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) 206 to 226; b) 558 to 578; and c) 720 to 807, from the 5′ end of a. BV 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) 206 to 226; b) 558 to 578, and c) 720 to 807, from the 5′ end of a HBV 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 HBV mRNA.

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

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

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 (BTU).

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises an RN 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 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 Table 1, as described in herein.

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: 2-26.

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: 27-51.

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: 2-26; and the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 27-51, 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 strands.

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) 158 to 240; h) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% (e.g., 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% or 45% to 50%), at a dose of 0.5 nM.

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) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by at least 50% (e.g., 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95% or 95% to 99%, 99% to 100%4 at a dose of 0.5 nM.

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) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 62.1 to 823, from the 5′ end of a. HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% (e.g., 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% or 45% to 50%), at a dose of 0.05 nM.

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 substantially identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 157 to 177; b) 196 to 410; c) 578 to 598; and d) 762 to 782, from the 5′ end of a HBV 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 comprising a sense strand and an antisense strand, 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) 157 to 177; b) 196 to 410, c) 578 to 598; and d) 762 to 782, from the 5′ end of a HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.5 nM.

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, 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) 157 to 177; b) 196 to 410; c) 578 to 598; and d) 762 to 782, from the 5′ end of a HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.05 nM.

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 substantially identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 158 to 276; b) 389 to 578; and c) 666 to 823, from the 5′ end of a HBV 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 comprising a sense strand and an antisense strand, the sense strand comprises a nucleotide sequence that is substantially identical to a region comprising 19-25 nucleoli des between any one of the nucleotide positions selected from: a) 158 to 276; b) 389 to 578; and c) 666 to 823, from the 5′ end of a HBV 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 the isolated oligonucleotide reduces levels of Hepatitis B surface antigen (HBsAg) by at least 50% at a dose of 0.05 nM.

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, 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) 158 to 276; h) 389 to 578; and c) 666 to 823, from the 5′ end of a HBV 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 the isolated oligonucleotide reduces levels of HBsAg by at least 20% at a dose of 0.01 nM.

The present disclosure also provides an isolated oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand or the antisense strand or both comprise one or more modified nucleotide(s). In some embodiments, the antisense strand comprises a mono methyl protected phosphate mimic (5′-MeEP). In some embodiments, in the sense strand or the antisense strand or both, a terminal or internal nucleotide is linked to a targeting ligand. In some embodiments, the targeting ligand comprises at least one GalNAc G1b moiety.

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, the antisense strand comprises nucleotides modified with 2′-F modification, and nucleotides modified With 2′-O-methyl modification, according to the formula: 3′ (M)₀F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁ (M)₂F)₁(M)₁(F)₁(M)₁(F)₂(M)₁ 5′

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, 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)₉ 3′

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, the antisense strand comprises any one of i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 232 (5′ [mUs][fAs][fU][mC][fC][mU][fG][mA][mU][fG][mU][mG][mA][fU][mG][fU][mU][mC][m U][mCs][mCs][mA] 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID: 233 (5′ [mUs][fUs][fA][mG][fG][mA][fA][mU][mC][fC][mU][mG][mA][fU][mG][fU][mG][mA][mU][mGs][mUs][mU] 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID: 234 (5′ [mUs][fU][fC][mA][fA][mC][fA][mA][mG][fA][mA][mA][mA][fA][mC][fC][mC][mC][m G][mCs][mCs][mU] 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID: 235 (5′ [mUs][fUs][fU][mG][fU][mC][fA][mA][mC][fA][mA][mG][mA][fA][mA][fA][mA][mC][m C][mCs][mCs][mG] 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID: 236 (5′ [mUs][fUs][fA][mU][fU][mG][fU][mG][mA][fG][mG][mA][mU][fU][mC][fU][mU][mG][mU][mCs][mAs][mA] 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID: 237 (5′ [mUs][fUs][fA][mG][fC][mA][fA][mU][mU][fU][mU][mC][mC][fC][mA][fA][mA][mG][mC][mCs][mCs][mA] 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID: 238 (5′ [mUs][fAs][fU][mA][fA][mC][fU][mG][mA][fA][mA][mG][mC][fC][mA][fA][mA][mC][m A][mGs][[mUs][mG] 3′); viii) an antisense strand of nucleic acid sequence according to SEQ ID: 239 (5′ [mUs][fAs][fG][mA][fA][mA][fA][mU][mU][fG][mG][mU][mA][fA][mC]][fA][mG][mC][mG][mGs][mUs][mA] 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID: 240 (5′ [mUs][fAs][fA][mA][fA][mG][fA][mA][mA][fA][mU][mU][mG][fG][mU][fA][mA][mC][mA][mGs][mCs][mG] 3′); or x) an antisense strand of nucleic acid sequence according to SEQ ID: 241 (5′ [mUs][fAs][fC][mA][fA][mA][fA][mG][mA][fA][mA][mA][mU][fU][mG][fG][mU][mA][mA][mCs][mAs][mG] 3′), wherein “m” is a 2′-O-methyl modified nucleotide, “f” is a 2′-F modified nucleotide, “s” is a phosphorothioate internucleotide linkage.

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprises any one of: i) a sense strand of nucleic acid sequence according to SEQ ID NO: 242 (5′ [mGs][mAs][mG][mA][mA][f C][mA][fU][fC][fA][fC][mA][mU][mC][mA][mG][mG][mAs][mUs][mA][G1b][G1b][G1b] 3′); ii) a sense strand of nucleic acid sequence according to SEQ ID NO: 243 (5′ [mCs][mAs][mU][mC][mA][fC][mA][fU][fC][fA][fG][mG][mA][mU][mU][mC][mC][mUs][mAs][mA][G1b][G1b][G1b] 3′); iii) a sense strand of nucleic acid sequence according to SEQ ID NO: 244 (5′ [mGs][mCs][mG][mU][mU][fG][mU][fU][fU][fU][fU][fU][mC][mU][mU][mG][mU][mU][mGs][mAs][mA][G1b][G1b][G1b] 3′); iv) a sense strand of nucleic acid sequence according to SEQ ID NO: 245 (5′ [mGs][mGs][mG][mU][mU][fU][mU][fU][fC][fU][fU][mG][mU][mU][mG][mA][mC][mAs][mAs][mA][G1b][G1b][G1b] 3′); v) a sense strand of nucleic acid sequence according to SEQ ID NO: 246 (5′ [mGs][mAs][mC][mA][mA][fG][mA][fA][U][fC][fC][mU][mC][mA][mC][mA][mA][mUs][mAs][mA][G1b][G1b][G1b] 3′); vi) a sense strand of nucleic acid sequence according to SEQ ID NO: 247 (5′ [mGs][mGs][mC][mU][mU][fU][mC][fG][fA][fA][mA][mA][mU][mU][mC][mC][mUs][mAs][mA][G1b][G1b][G1b] 3′); vii) a sense strand of nucleic acid sequence according to SEQ ID NO: 248 (5′ [mCs][mUs][mC][mU][mU][fU][mC][fG][fC][fU][fU][fU][mU][mC][mA][mG][mU][mU][mAs][mUs][mA][G1b][G1b][G1b] 3); viii) a sense strand of nucleic acid sequence according to SEQ ID NO: 249 (5′ [mCs][mCs][mG][mC][mU][fG][mU][fU][fA][fC][fC][mA][mA][mU][mU][mU][mU][mCs][mUs][mA][G1b][G1b][G1b] 3′) ix) a sense strand of nucleic acid sequence according to SEQ ID NO: 250 (5′ [mCs][mUs][mG][mU][mU][mU][fA][mC][fC][fA][fA][fU][mU][mU][mU][mC][mU][mU][mUs][mUs][mA][G1b][G1b][G1b] 3′); or x) a sense strand of nucleic acid sequence according to SEQ ID NO: 251 (5′ [mGs][mUs][mU][mA][mC][fC][mA][fA][fU][fU][fU][mU][mC][mU][mU][mU][mU][mGs][mUs][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 comprising a sense strand and an antisense strand, the double stranded region comprises any one of: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 232 (5′ [mUs][fAs][fU][mC][fC][mU][fG][mA][mU][fG][mU][mG][mA][fU][mG][fU][mU][mC][m U][mCs][mCs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 242 (5′ [mGs][mAs][mG][mA][mA][fC][mA][fA][fC][fA][fC][mA][mU][mC][mA][mG][mG][mAs][mUs][mA][G1b][G1b][G1b] 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 233 (5′ [mUs][fUs][fA][mG][fG][mA][fA][mU][mC][fC][mU][mG][mA][fU][fU][mG][fU][mG][mA][mU][mGs][mUs][mU] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: NO: 243 (5′ [mCs][mAs][mU][mC][mA][fC][mA][fU][fC][fA][fG][mG][mA][mU][mU][mC][mC][mUs][mAs][mA][G1b][G1b][G1b] 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 234 (5′ [mUs][fUs][fC][mA][fA][mC][fA][mA][mG][mA][mA][mA][mA][fA][mC][fC][mC][mC][m G][mCs][mCs][mU] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 244 (5′ [mGs][mCs][mG][mG][mG][fG][mU][fU][fU][fU][fU][mC][mU][mU][mG][mU][mU][mGs][mAs][mA][G1b][G1b][G1b] 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 235 (5′ [mUs][fUs][fU][mG][fU][mC][fA][mA][mC][fA][mA][mG][mA][fA][mA][fA][mA][mC][mC][mCs][mCs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 245 (5′ [mCs][mCs][mG][mU][mU][fU][mU][fU][fC][fC][fU][fU][mG][mU][mU][mG][mA][mC][mAs][mAs][mA][G1b][G1b][G1b] 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 236 (5′ [mUs][fUs][fA][mU][fU][mG][fU][mG][mA][fU][mG][mA][mU][fU][mC][fU][mU][mG][mU][mCs][mAs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 246 (5′ [mGs][mAs][mC][mA][mA][fG][mA][fA][fU][fC][fC][mU][mC][mA][mC][mA][mA][mUs][mAs][mA][G1b][G1b][G1b] 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 237 (5′ [mUs][fUs][fA][mG][fG][mA][fA][mU][mU][fU][mU][mC][mC][fG][mA][fA][mA][mG][mC][mCs][mCs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 247 (5′[mGs][mGs][mC][mU][mU][fU][mC][fG][fG][fA][fA][mA][mA][mU][mU][mC][mC][m Us][mAs][mA][G1b][G1b][G1b] 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 238 (5′ [mUs][fAs][fU][mA][fA][mC][fU][mG][mA][fA][mA][mC][mC][fC][mA][fA][mA][mC][m A][mGs][mUs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 248 (5′ [mCs][mUs][mG][mU][mU][fU][mG][fG][fG][fC][fU][fU][mU][mC][mA][mG][mU][mU][mAs][mUs][mA][G1b][G1b][G1b] 3); viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 239 (5′ [mUs][fAs][fG][mA][fA][mA][fA][mU][mU][fG][mG][mU][mA][fA][mC][fA][mC][mC][mG][mGs][mUs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 249 (5′ [mCs][mCs][mG][mC][mU][fG][mU][fU][fA][fC][fC][mA][mA][mU][mU][mU][mU][mCs][mUs][mA][G1b][G1b][G1b] 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 240 (5′ [mUs][fAs][fA][mA][fA][mG][fA][mA][mA][fA][mU][mU][mG][fG][mU][fA][mA][mC][mA][mGs][mCs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 250 (5′ [mCs][mUs][mG][mU][mU][fA][mC][fC][fA][fA][fU][mU][mU][mU][mC][mU][mU][mUs ][mUs][mA][G1b][G1b][G1b] 3′); or x) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 241 (5′ [mUs][fAs][fC][mA][fA][mA][fA][mG][mA][fA][mA][mA][mU][fU][mG][fG][mU][mA][mA][mCs][mAs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 251 (5′ [mGs][mUs][mU][mA][mC][fC][mA][fA][fU][fU][fU][mU][mC][mU][mU][mU][mU][mGs][mUs][mA][10b][G1b][G11b] 3′).

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 HBV in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of at least one isolated oligonucleotide, vector, delivery system or pharmaceutical composition disclosed herein.

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

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the efficacy of siRNA compounds listed in Table 2 in silencing hepatitis B HBsAg expression in cultured HepG2.2.15 cells. The compounds were transfected into cells at 0.05 nM concentration. Data is presented as % of HBsAg remaining in culture media detected by ELISA relative to mock transfection (Mean, +/− SEM). Each bar represents a single compound tested.

FIG. 2 is a graph showing the efficacy of siRNA compounds listed in Table 2 in silencing hepatitis B HBsAg expression in cultured HepG2.2.15 cells. The compounds were transfected into cells at 0.01 nM concentration. Data is presented as % of HBsAg remaining in culture media detected by ELISA relative to mock transfection (Mean, +/− SEM), Each bar represents a single compound tested.

FIG. 3 is a graph showing the efficacy of a subset of siRNA compounds listed in Table 2 in silencing hepatitis B 2.1 kb+2.4 kb+3.5 kb mRNA in cultured HepG2.2.15 cells. The compounds were transfected into cells at 0.5 nM concentration, Data is presented as % of HBV 2.1 kb+2.4 kb+3.5 kb mRNA remaining relative to mock transfection when normalized to Gapdh mRNA levels (Mean, +/− SEM). Each bar represents a single compound tested.

FIG. 4 is a graph showing the efficacy of siRNA compounds listed in Table 2 in silencing hepatitis B 2.1 kb+2.4 kb+3.5 kb mRNA in cultured HepG2.2.15 cells. The compounds were transfected into cells at 0.05 nM concentration. Data is presented as % of HBV 2.1 kb+2.4 kb+3.5 kb mRNA remaining relative to mock transfection when normalized to Gapdh mRNA levels (Mean, +/− SEM). Each bar represents a single compound tested,

FIG. 5 is a graph showing the efficacy of siRNA compounds listed in Table 2 in silencing plasma HBsAg in HDI mouse model on day-S post dosing (1 mg/Kg). Data is presented as Log HBsAg remaining in HBV-genotype D HDI mice (Mean, +/− SEM).

FIG. 6 is a graph showing the efficacy of siRNA compounds listed in Table 2 in lowering HBV pregenomic RNA (pgRNA) in HDI mouse liver on day-5 post dosing (1 mg/kg). Data is presented as % of liver pgRNA remaining relative to PBS injected HDT mice when normalized to Gapdh miRNA levels (Mean, +/− SEM).

FIG. 7 is a graph showing the efficacy of siRNA compounds listed in Table 2 in lowering HBV 2.1 kb+2.4 kb+pgRNA in 1-DI mouse liver on day-5 post dosing (1 mg/kg). Data is presented as % of liver 2.1 kb+2.4 kb+pg RNA remaining relative to PBS injected HDI mice when normalized to Gapdh mRNA levels (Mean, +/− SEM).

FIG. 8 is a graph showing the efficacy and dose response of siRNA compounds selected from Table 2 in lowering plasma HBsAg in HD mouse model on day-5 post dosing at 1 mg/kg (black squares), 3 mg/kg (grey squares), or 6 mg/kg (empty squares). Data is presented as Log HBsAg remaining in HDI mice (Mean, +/− SEM).

FIG. 9 is a graph showing the efficacy and dose response of siRNA compounds selected from Table 2 in silencing liver pgRNA in HDI mouse model on day-5 post dosing at 1 mg/kg (black squares), 3 mg/kg (grey squares), or 6 mg/kg (empty squares). Data is presented as liver pgRNA remaining relative to PBS injected HDI mice when normalized to Gapdh mRNA levels (Mean, +/− SEM).

FIG. 10 is a graph showing the efficacy and duration of a subset of siRNA compounds listed in Table 4 (at 3 mg/kg) in lowering plasma HBsAg in AAV-HBV-genotype D mice. Data is presented as Log HBsAg remaining HBV-AAV mice (Mean, +/− SEM).

FIG. 11 is a graph showing the efficacy and duration of a subset of siRNA compounds listed in Table 4 (at 3 mg/kg) in lowering plasma Hepatitis B e antigen (HBeAg) in AAV-HBV-genotype D mice. Data is presented as Log HBeAg remaining in HBV-AAV mice (Mean, +/− SEM).

FIG. 12 is a graph showing the efficacy and duration of siRNA compounds listed in Table 4 (at 3 mg/kg) in lowering plasma HBV DNA in AAV-HBV-genotype D mice. Data is presented as Log HBV DNA remaining in HBV-AAV mice plasma (Mean, +/− SEM).

FIG. 13A-C is a graph showing the efficacy of siRNA compounds (at 3 mg/kg) listed in Table 4 in lowering liver HBV total RNA (FIG. 13A), HBV 3.5 kB RNA (FIG. 13B), and HBV DNA copy number (FIG. 13C) in HBV-genotype-D AAV mice at day 77 post dosing.

FIG. 14 is a graph showing the plasma ALT and AST levels in AAV-HBV mice on day 7- and day 28-post dosing of compounds selected from Table 4.

FIG. 15 is a graph showing the efficacy and dose response (1 nM, 0.5 nM, 0.20 nM, 0.01 nM) of siRNA compounds listed in Table 4 in silencing hepatitis B HBsAg expression in HepG2.2.15 cells that were transfected with HBV genotype B. Data is presented as % of HBsAg inhibition relative to Mock transfection (Mean, +/− SEM).

FIG. 16 is a graph showing the efficacy and dose response (1 nM and 0.01 nM) of siRNA compounds listed in Table 4 in silencing hepatitis B HBsAg expression in HepG2.2.15 cells that were transfected with HBV genotype C. Data is presented as % of HBsAg remaining relative to Mock transfection (Mean, +/− SEM).

FIG. 17 is a graph showing the efficacy and eight concentration dose responses of siRNA compounds listed in Table 4 in silencing hepatitis B HBsAg expression in HepG2.2.15 cells that were transfected with HBV genotype C. Data is presented as % of HBsAg remaining relative to Mock transfection (Mean, +/− SEM).

DETAILED DESCRIPTION

The present disclosure provides isolated oligonucleotides that form a double stranded region, preferably small interfering RNAs (siRNAs), that can decrease hepatitis B virus (HBV) mRNA expression, in turn leading to a decrease in the degree of HBV surface antigen (HBsAg) protein expression in target cells. The oligonucleotides disclosed herein can have therapeutic application in regulating the expression of HBV transcript, for treatment of HBV disease or an HBV-associated disease such as, but not limited to, chronic hepatitis B infection, hepatitis D virus (HDV) infection, hepatitis infection-induced inflamatory liver disease, liver cirrhosis and HBV-associated cancer such as for example, hepatocellular carcinoma (HCC), or a combination thereof.

In some aspects, the present invention provides compositions and methods of treating a subject having a disorder that would benefit from the reduction in HBV mRNA or HBsAg expression. In some aspects, the methods disclosed herein prevent at least one symptom in a subject having a disease or disorder that would benefit from reduction in HBV mRNA or HBsAg expression.

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

The hepatitis B virus (HBV) mRNA sequence described herein, is an mRNA sequence encoded by a HBV gene according to GenBank Accession No. U95551.1:

(SEQ ID NO: 1)
AATTCCACAACCTTTCACCAAACTCTGCAAGATCCCAGAGTGAGAG
GCCTGTATTTCCCTGCTGGTGGCTCCAGTTCAGGAGCAGTAAACC
CTGTTCCGACTACTGCCTCTCCCTTATCGTCAATCTTCTCGAGGA
TTGGGGACCCTGCGCTGAACATGGAGAACATCACATCAGGATTCC
TAGGACCCCTTCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAA
GAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTC
TCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGC
AGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTT
GTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCC
TCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGG
ACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCCT
CAACCACCAGCACGGGACCATGCCGAACCTGCATGACTACTGCTC
AAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGG
ACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCG
GAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCA
GTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCA
CTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAA
GTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATTT
TCTTTTGTCTTTGGGTATACATTTAAACCCTAACAAAACAAAGAG
ATGGGGTTACTCTCTGAATTTTATGGGTTATGTCATTGGAAGTTA
TGGGTCCTTGCCACAAGAACACATCATACAAAAAATCAAAGAATG
TTTTAGAAAACTTCCTATTAACAGGCCTATTGATTGGAAAGTATG
TCAACGAATTGTGGGTCTTTTGGGTTTTGCTGCCCCATTTACACA
ATGTGGTTATCCTGCGTTAATGCCCTTGTATGCATGTATTCAATC
TAAGCAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTGTG
TAAACAATACCTGAACCTTTACCCCGTTGCCCGGCAACGGCCAGG
TCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTT
GGTCATGGGCCATCAGCGCGTGCGTGGAACCTTTTCGGCTCCTCT
GCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAG
CAGGTCTGGAGCAAACATTATCGGGACTGATAACTCTGTTGTCCT
CTCCCGCAAATATACATCGTATCCATGGCTGCTAGGCTGTGCTGC
CAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGC
GCTGAATCCTGCGGACGACCCTTCTCGGGGTCGCTTGGGACTCTC
TCGTCCCCTTCTCCGTCTGCCGTTCCGACCGACCACGGGGCGCAC
CTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGA
CCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGACCACC
GTGAACGCCCACCGAATGTTGCCCAAGGTCTTACATAAGAGGACT
CTTGGACTCTCTGCAATGTCAACGACCGACCTTGAGGCATACTTC
AAAGACTGTTTGTTTAAAGACTGGGAGGAGTTGGGGGAGGAGATT
AGATTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTGGTC
TGCGCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCT
CTTGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGT
GGCTTTGGGGCATGGACATCGACCCTTATAAAGAATTTGGAGCTA
CTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTCAG
TACGAGATCTTCTAGATACCGCCTCAGCTCTGTATCGGGAAGCCT
TAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGC
AAGCAATTCTTTGCTGGGGGGAACTAATGACTCTAGCTACCTGGG
TGGGTGTTAATTTGGAAGATCCAGCATCTAGAGACCTAGTAGTCA
GTTATGTCAACACTAATATGGGCCTAAAGTTCAGGCAACTCTTGT
GGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACCGTTATAG
AGTATTTGGTGTCTTTCGGAGTGTGGATTCGCACTCCTCCAGCTT
ATAGACCACCAAATGCCCCTATCCTATCAACACTTCCGGAAACTA
CTGTTGTTAGACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCT
CGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCTC
AATCTCGGGAACCTCAATGTTAGTATTCCTTGGACTCATAAGGTG
GGGAACTTTACTGGTCTTTATTCTTCTACTGTACCTGTCTTTAAT
CCTCATTGGAAAACACCATCTTTTCCTAATATACATTTACACCAA
GACATTATCAAAAAATGTGAACAGTTTGTAGGCCCACTTACAGTT
AATGAGAAAAGAAGATTGCAATTGATTATGCCTGCTAGGTTTTAT
CCAAAGGTTACCAAATATTTACCATTGGATAAGGGTATTAAACCT
TATTATCCAGAACATCTAGTTAATCATTACTTCCAAACTAGACAC
TATTTACACACTCTATGGAAGGCGGGTATATTATATAAGAGAGAA
ACAACACATAGCGCCTCATTTTGTGGGTCACCATATTCTTGGGAA
CAAGATCTACAGCATGGGGCAGAATCTTTCCACCAGCAATCCTCT
GGGATTCTTTCCCGACCACCAGTTGGATCCAGCCTTCAGAGCAAA
CACAGCAAATCCAGATTGGGACTTCAATCCCAACAAGGACACCTG
GCCAGACGCCAACAAGGTAGGAGCTGGAGCATTCGGGCTGGGTTT
CACCCCACCGCACGGAGGCCTTTTGGGGTGGAGCCCTCAGGCTCA
GGGCATACTACAAACTTTGCCAGCAAATCCGCCTCCTGCCTCCAC
CAATCGCCAGACAGGAAGGCAGCCTACCCCGCTGTCTCCACCTTT
GAGAAACACTCATCCTCAGGCCATGCAGTGG.

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) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV 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) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV 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 comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV 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) 158 to 240; b) 389 to 439; c) 621 to 686; and d) 775 to 823, from the 5′ end of a human HBV 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) 158 to 240; b) 389 to 439; c) 621 to 686; and d) 775 to 823, from the 5′ end of a human HBV 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) 158 to 240; b) 389 to 439; c) 621 to 686, and d) 775 to 823, from the 5′ end of a human HBV 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) 206 to 226; b) 558 to 578; and c) 720 to 807, from the 5′ end of a human HBV 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) 206 to 226; b) 558 to 578; and c) 720 to 807, from the 5′ end of a human HBV 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) 206 to 226; b) 558 to 578; and c) 720 to 807, from the 5′ end of a human HBV miRNA sequence according to SEQ ID NO: 1.

The HBV mRNA sequence according to SEQ ID NO: 1, as described herein, is any heterologous mRNA sequence with sufficient identity to a HBV according to GenBank Accession No. U95551.1, as described herein, that allows binding to the antisense 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 HBV mRNA.

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, 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 HBV mRNA 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), piRNA (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 HBV mRNA sequence, as described herein, will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence of the HBV mRNA sequence and 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 thy mine (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.

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 (BLOSUM 62) 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 Math 48:1073 (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. Mot. 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 Hepatitis B Virus (HBV) mRNA

The disclosure provides isolated oligonucleotides comprising a double stranded RNAs (dsRNAs) duplex region which target a hepatitis B virus (HBV) 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 HBV mRNA sequence for degradation. In some embodiments, the siRNA targeting HBV is packaged in a delivery system described herein (e.g., nanoparticle).

The isolated oligonucleotides of the present disclosure targeting HBV for degradation can comprise a sense strand at least 70% identical to any fragment of a HBV mRNA, for example the HBV 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 HBV for degradation can comprise an antisense strand at least 70% identical to a sequence complementary to any fragment of a HBV mRNA, for example the HBV 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 HBV 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 oligonucleotide 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 HBV mRNA sequence. In some embodiments, the sense region is identical to a HBV mRNA sequence.

As used herein, the term “sense strand” or “sense region” refers to ar 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 HBV 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 ID 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 HBV has one or more mismatches between the sequence of the isolated oligonucleotides and the HBV 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 HBV sequence. In some embodiments, the HBV sequence is a HBV 3′ untranslated region sequence (3′ UTR). Without wishing to be bound by theory, it is thought that siRNAs targeting the 3′ UTR have elevated mismatch tolerance when compared to mismatches in the isolated oligonucleotides targeting coding regions of a gene. 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 HBV 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, both the sense strand and the antisense strand are single stranded RNA molecules. In some embodiments, the isolated oligonucleotide of the present disclosure is an siRNA targeting HBV 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. In some embodiments, the overhangs, which are non-base-paired, single strand regions, can be from one to eight nucleotides in length, or longer. In some embodiments, the overhang can be a 3′ overhang, wherein the 3′-end of a strand has a single strand region of from one to eight nucleotides. In some embodiments, the overhang can be a 5′ overhang, wherein the 5′-end of a strand has a single strand region of from one to eight nucleotides. In some embodiments, the overhangs of the isolated oligonucleotide are the same length. In some embodiments, the overhangs of the isolated oligonucleotide are 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, the 3′ overhang of the single stranded RNA molecule of the sense strand comprises at least one nucleotide. In some embodiments, the 3′ overhang of the single stranded RNA molecule of the sense strand comprises two nucleotides.

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, the 3′ overhang of the single stranded RNA molecule of the antisense strand comprises at least one nucleotide. In some embodiments, the 3′ overhang of the single stranded RNA molecule of the antisense strand comprises two nucleotides.

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

In some embodiments of the isolated oligonucleotide of the present disclosure, the overhang 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 of the isolated oligonucleotide of the present disclosure, 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 embodiments, 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 antisense strand and sense strand sequences in Table 1, as described below.

Hepatitis B Virus

As described in US 16/532,245, hepatitis B virus (HBV) is a member of the Hepadnavirus family and is divided into 4 major serotypes (adr, adw, ayr, ayw) based on antigenic epitopes. The virus is also classed into eight genotypes (A-H based on genomic sequence). Genotype A is common in the Americas, Africa, India and Western Europe. Genotype B and C are found in Asia and the US. Genotype D is most common in Southern Europe and India. Genotype E is found in Western and Southern Africa. Genotype F and H are commonly found in Central and Southern America. Genotype G is commonly found in Europe and the U.S.

The HBV genome consists of a circular strand of DNA that is partially double stranded. The genome is about 3.3 kb in length. It encodes 4 known genes (C, X, P and S). Viral polymerase, the central enzyme in genome replication, is encoded by the P (Pol) open reading frame (ORF). It has DNA polymerase (DNA Pol), reverse transcriptase (RT) and RNase H activities and also acts as the terminal protein (TP). The C (core) gene encodes the structural protein of the nucleocapsids as well as the ‘e’ antigen (HBeAg). The S (surface) region encodes 3 different envelope glycoproteins. The X region encodes the multifunctional X protein. The genome also contains regulatory elements (promoters, enhancers) and also encodes signals for poly adenylation and encapsidation. HBV is one of the few DNA viruses that utilize reverse transcriptase in the replication process. HBV replication involves multiples stages including entry, uncoating and transport of the virus genome to the nucleus. Initially, replication of the HBV genome involves the generation of an RNA intermediate that is then reverse transcribed to produce the DNA viral genome.

Transcription of the HBV genome produces four major mRNAs, including the 3.5 kb pregenomic RNA (pgPRNA)/preCore RNA and 2.4 kb, 2.1 kb, and 0.7 kb subgenomic RNA (Lin, Y. et al. 2020. Autophay). The pgRNA encodes both the polymerase and core protein and also serves as the template for HBV DNA replication, thus playing an essential role in HBV replication. The preCore RNA encodes the preCore protein, which is post-translationally processed to become the mature HBV e-antigen (HBeAg). The 2.4 kb RNA and 2.1 kb RNA encode HBV large surface protein (LHBs), middle surface protein (MHBs), and small surface protein (SHBs), respectively.

Coding of different virion proteins using the same ORE is done by exploiting more than one in-frame initiation codon (Datta, S. et al. 2012. J Clin Exp Hepatol). Due to this fact, three different surface molecules, with variable N-terminals, but a common C-terminal end, are synthesized from the preS/S ORF. The largest of these, the preS1 protein (or large HBsAg or LHBs) is the product of initiation at the first initiation codon of the ORF, while initiation at the second initiation codon produces preS2 middle hepatitis B surface antigen (or middle HBsAg or MHBs). Initiation at the third start codon produces the classical HBsAg small hepatitis B surface antigen (or small HBsAg or SHBs) that contains only the S domain and is commonly referred to as the surface antigen (HBsAg or Au antigen).

Several studies have attempted to use RNAi for the treatment of HBV and this approach has been comprehensively reviewed in RNAI for treating Hepatitis B Viral Infection, Chen, Y. et al., Pharmaceutical Research, 2008 Vol. 25. No. 1, pgs 72-86. Yet, as noted in the above reference transfection of a single siRNA often fails to provide adequate gene silencing. Thus, despite significant advances in the field of RNAi, there remains a need for agents that can effectively inhibit HBV gene expression and that can treat disease associated with HBV expression such as liver disease and cancer.

Since current therapies are limited due to their ineffectiveness, serious side effects or due to the generation of drug resistant variants, there is a clinical need for the development of new therapies to treat HBV infection.

Accordingly, the isolated oligonucleotides disclosed in the present disclosure are useful in treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HBV or a disease or disorder where HBV plays a role. Exemplary isolated oligonucleotides of the present disclosure are described in Table 1.

In some embodiments of the isolated oligonucleotide of the present disclosure, the sense strand comprises a sequence selected from any one of the group of sense strand/passenger strand sequences listed in Table 1, Table 2, Table 3, or Table 4. In some embodiments, the antisense strand comprises a sequence selected from any one of the group of antisense strand/guide strand sequences listed in Table 1, Table 2, Table 3, or Table 4. In some embodiments, the sense and antisense regions comprise complementary sequences selected from the group listed in Table 1, Table 2, Table 3, or Table 4.

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: 2-26, 52-68, and 86-158.

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: 27-51, 69-85, and 159-231.

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: 2-26, 52-68, and 86-158; and the sense strand comprises a nucleotide sequence according to any one of SEQ ID NOs: 27-51, 69-85, and 159-231, 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.

Sequences of the Present Disclosure

The present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV mRNA sequence according to SEQ ID NO: 1, and the antisense strand is substantially complementary to the sense strand such that the sense strand and the antisense strand together form a double stranded region.

The present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV mRNA sequence according to SEQ ID NO: 1.

The present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is identical to a region comprising 19-25 nucleotides between any one of the nucleotide positions selected from: a) 158 to 240; b) 389 to 439, c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV mRNA sequence according to SEQ ID NO: 1.

The present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 158 to 240; b) 389 to 439; c) 621 to 686; and d) 775 to 823, from the 5′ end of a human HBV miRNA sequence according to SEQ ID NO: 1.

The present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to a region between any one of the nucleotide positions selected from: a) 158 to 240; b) 389 to 439; c) 621 to 686; and d) 775 to 823, from the 5′ end of a human HIBV mRNA sequenc^e according to SEQ ID NO: 1.

The present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 158 to 240; b) 389 to 439; c) 621 to 686; and d) 775 to 823, from the 5′ end of a human HBV mRNA sequence according to SEQ ID NO: 1.

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) 158 to 240; b) 389 to 439; c) 621 to 686; and d) 775 to 823, from the 5′ end of a human HBV 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′ UAUCCUGAUGUGAUGUUCUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ GAGAACAUCACAUCAGGAUA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UUGUAACACGAGAAGGGGUCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ GACCCCUUCUCGUGUUACAA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′), v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UAGAGGAAGAUGAUAAAACGCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CGUUUUAUCAUCUUCCUCUA 3′) vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUAGCAGCAGGAUGAAGAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUCUUCAUCCUGCUGCUAUA 3′); viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAGAAGAUGAGGCAUAGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ CUGCUAUGCCUCAUCUUCUA 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′); x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UAGGAAUUUUCCGAAAGCCCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ GGGCUUUCGGAAAAUUCCUA 3′); xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GGCUUUCGGAAAAUUCCUAA 3′); xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCACGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ CCUGGCUCAGUUUACUAGUA 3′); xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUAA 3′); xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′); xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′); xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ ULAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′); xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′); xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUULUUCUUUUGUA 3′); or xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′).

The present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is substantially identical to a region between any one of the nucleotide positions selected from: a) 206 to 226; b) 558 to 578; and c) 720 to 807, from the 5′ end of a human HBV mRNA sequence according to SEQ ID NO: 1. The present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to a region between any one of the nucleotide positions selected from: a) 206 to 226; b) 558 to 578; and c) 720 to 807, from the 5′ end of a human HBV mRNA sequence according to SEQ ID NO: 1.

The present disclosure provides an isolated oligonucleotide comprising a sense and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is identical to a region between any one of the nucleotide positions selected from: a) 206 to 226; b) 558 to 578; and c) 720 to 807, from the 5′ end of a human HBV mRNA sequence according to SEQ ID NO: 1.

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) 206 to 226; b) 558 to 578; and c) 720 to 807, from the 5′ end of a human HBV 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: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ GGGUUUUCUUGUUGACAAA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CCUCCUOUUGCUGUACCAAA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ GUUAUAUGGAUGAUGUGGUA 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAAAAAGGGACUCAAGAUGCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ GCAUCUUGAGUCCCUUUUUA 3′); or vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUGGUAACAGCGGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′).

20%-50% Knockdown of HBV at 0.5 nM

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) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV 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, wherein the isolated oligonucleotide attenuates expression of the HBV % mRNA by 20% to 50% (e.g., 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% or 45% to 50%, at a dose of 0.5 nM.

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) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.5 nM, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UUGUAACACGAGAAGGGGUCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ GACCCCUUCUCGUGUUACAA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5 UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUAA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUAGCAGCAGGAUGAAGAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUCUUCAUCCUGCUGCUAUA 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′GGUUUUUCUUGUUGACAAA 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAAA 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UAGAGGAAGAUGAUAAAACGCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CGUUUUAUCAUCUUCCUCUA 3′); viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UACAAAAUUUGGAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUUACCAAUUUUCUA 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UAUCCUGAUGUGAUGUUCUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ GAGAACAUCACAUCAGGAUA 3′); x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′); xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′); xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CCUCCUGUUGCUGUACCAAA 3′); xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ GUUAUAUGGAUGAUGUGGUA 3′); xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUGGUAACAGCGCGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′); or xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ GGGUUUUCUUGUUGACAAA 3′).

At Least 50% Knockdown of HBV at 0.5 nM

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) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV 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, wherein the isolated oligonucleotide attenuates expression of the HBV mRNA by at least 50% (e.g., 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95% or 95% to 99%, 99% to 100%), at a dose of 0.5 nM.

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) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by at least 50%, at a dose of 0.5 nM, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GGCUUUCGGAAAAAUUCCUAA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAGAAGAUGAGGCAUAGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′CUGCUAUGCCUCAUCUUCUA 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ CCUGGCUCAGUUUACUAGUA 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAGAAAAUUCGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′); viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UAGGAAUUUUCCGAAAGCCCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ GGGCUUUCGGAAAAUUCCUA 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′); or x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′).

20% to 50% a Knockdown of HBV at 0.05 nM

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 comprising 19-25 nucleotides between any one of the nucleotide positions selected from a) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV 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, wherein the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% (e.g., 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% or 45% to 50%), at a dose of 0.05 nM.

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) 158 to 240; b) 389 to 439; c) 558 to 578; and d) 621 to 823, from the 5′ end of a human HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.05 nM, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUUA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CCUCCUGUUGCUGUACCAAA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ GUUAUAUGGAUGAUGUGGUA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GGCUUUCGGAAAAUUCCUAA 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′); viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAGAAGAUGAGGCAUAGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ CUGCUAUGCCUCAUCUUCUA 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ CCUGGCUCAGUUUACUAGUA 3′); x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UAGGAAUUUUUCCGAAAGCCCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ GGGCUUUCGGAAAAUUCCUA 3′); xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′); or xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′).

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) 157 to 177; b) 196 to 410; c) 578 to 598; and d) 762 to 782, from the 5′ end of a HBV 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.

20% to 50% Knockdown of HBV at 0.5 nM

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, 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) 157 to 177; b) 196 to 410; c) 578 to 598; and d) 762 to 782, from the 5′ end of a HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.5 nM.

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, 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) 157 to 177; b) 196 to 410; c) 578 to 598; and d) 762 to 782, from the 5′ end of a HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.5 nM, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ UACUGAGAGAAGUCCACCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 74 (5′ GUGGUGGACUUCUCUCAGUA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 66 (5′ UAAGAGGAAGAUGAUAAAACGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 83 (5′ GUUUUAUCAUCUUCCUCUUA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ UCUGAGAGAAGUCCACCACGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 73 (5′ CGUGGTGGACUUCUCUCAGA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 63 (5′ UUUUUGGCCAAGACACACGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 80 (5′ CCGUGUGUCUUGGCCAAAAA 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 65 (5′ UAGGACAAGUUGGAGGACAGGA 3′), and sense strand of nucleic acid sequence according to SEQ ID NO: 82 (5′ CUGUCCUCCAACUUGUCCUA 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 68 (5′ UAAGAUGCUGUACAGACUUGGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 85 (5′ CAAGUCUGUACAGCAUCUUA 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 52 (5′ UUCCUGAUGUGAUGUUCUCCAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 69 (5′ GGAGAACAUCACAUCAGGAA 3′); viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 59 (5′ UUUGAGAGAAGUCCACCACGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 76 (5′ CGUGGUGGACUUCUCUCAAA 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 55 (5′ UUGUCAACAAGAAAAACCCCGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 72 (5′ GGGGUUUUUCUUGUUGACAA 3′); x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ UUAUUGUGAGGAUUUUUGUCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 71 (5′ GACAAAAAUCCUCACAAUAA 3′); xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 67 (5′ UUGCAAUUUCCGUCCGAAGGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 84 (5′ CCUUCGGACGGAAAUUGCAA 3′); or xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGGAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′).

20% to 50% Knockdown of HBV at 0.05 nM

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, 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) 157 to 177; b) 196 to 410; c) 578 to 598; and d) 762 to 782, from the 5′ end of a HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.05 nM.

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, 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) 157 to 177; b) 196 to 410; c) 578 to 598; and d) 762 to 782, from the 5′ end of a HBV 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 the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.05 nM, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGOAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′); or ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 67 (5′ UUGCAAUUUCCGUCCGAAGGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 84 (5′ CCUUCGGACGGAAAUUGCAA 3′).

Knockdown of Hepatitis B Virus Surface Antigen (HBsAg)

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) 158 to 276; b) 389 to 578; and c) 666 to 823, from the 5′ end of a HBV 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.

At Least 50% Knockdown of HBsAg at 0.05 nM

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, 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) 158 to 276; b) 389 to 578; and c) 666 to 823, from the 5′ end of a HBV 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 the isolated oligonucleotide reduces levels of hepatitis B virus surface antigen (HBsAg) by at least 50% at a dose of 0.05 nM.

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, 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) 158 to 276; b) 389 to 578; and c) 666 to 823, from the 5′ end of a HBV 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 the isolated oligonucleotide reduces levels of HBsAg by at least 50% at a dose of 0.05 nM, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAGAAAAUUGGUUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ UUAUUGUGAGGAUUUUUGUCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 71 (5′ GACAAAAAUCCUCACAAUAA 3′); viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ UCUGAGAGAAGUCCACCACGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 73 (5′ CGUGGUGGACUUCUCUCAGA 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUGGUAACAGCGGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′); x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAACAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ GGGUUUUUCUUGUUGACAAA 3′); xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 3′); xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGGAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′), xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ CCUGGCUCAGUUUACUAGUA 3′); xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′); xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UAUCCUGAUGUGAUGUUCUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ GAGAACAUCACAUCAGGAUA 3′); xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UAGAGGAAGAUGAUAAAACGCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CGUUUUAUCAUCUUCCUCUA 3′); xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUUAA 3′); xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUAGCAGCAGGAUGAAGAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUCUUCAUCCUGCUGCUAUA 3′); xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ UACUGAGAGAAGUCCACCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 74 (5′ GUGGUGGACUUCUCUCAGUA 3′); xx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UUGUAACACGAGAAGGGGUCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ GACCCCUUCUCGUGUUACAA 3′); xxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′); xxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAAAAAGGGACUCAAGAUGCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5 GCAUCUUGAGUCCCUUUUA 3′) or xxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′).

At Least 20% Knockdown of HBsAg at 0.01 nM

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, 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) 158 to 276; b) 389 to 578; and c) 666 to 823, from the 5′ end of a HBV 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 the isolated oligonucleotide reduces levels of HBsAg by at least 20% at a dose of 0.01 nM.

In some embodiments of the isolated oligonucleotide comprising a sense strand and an antisense strand, 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) 158 to 276; b) 389 to 578; and c) 666 to 823, from the 5′ end of a HBV 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 the isolated oligonucleotide reduces levels of HBsAg by at least 20% at a dose of 0.01 nM, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ UUAUUGUGAGGAUUUUUGUCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 71 (5′ GACAAAAAUCCUCACAAUAA 3′); viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ UCUGAGAGAAGUCCACCACGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 73 (5′ CGUGGUGGACUUCUCUCAGA 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′UUGGUAACAGCGGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′); x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ GGGUUUUUCUUGUUGACAAA 3′), xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 3′); xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGGAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′); xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′); xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′); xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ GUUAUAUGGAGACUGGUA 3′); xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 126 (5′ UUACAUAGAGGUUCCUUGAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 199 (5′ CUCAAGGAACCUCUAUGUAA 3′), xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUAA 3′); xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 89 (5′ UUAGACUCUGCGGUAUUGUGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 162 (5′ CACAAUACCGCAGAGUCUAA 3′); xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ UACUGAGAGAAGUCCACCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 74 (5′ GUGGUGGACUUCUCUCAGUA 3′); xx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 92 (5′ UAUUGAGAGAAGUCCACCACGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 165 (5′ GUGGUGGACUUCUCUCAAUA 3′); xxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′); xxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAAAAAGGGACUCAAGAUGCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ GCAUCUUGAGUCCCUUUUUA 3′); xxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′); xxiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CCUCCUGUUGCUGUACCAAA 3′); xxv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 119 (5′ UUUGAGGAUCCUGGAAUUAGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 192 (5′ CUAAUUCCAGGAUCCUCAAA 3′); xxvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ UAAAACUGAGAGAAGUCCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 75 (5′ GUGGACUUCUCUCAGUUUUA 3′); or xxvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 90 (5′ UUCUAGACUCUGCGGUAUUGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 163 (5′ CAAUACCGCAGAGUCUAGAA 3′).

In some embodiments, the isolated oligonucleotide of the present disclosure can comprise a linker, sometimes referred to as a loop, siRNAs comprising a linker or loop are sometimes referred to as short hairpin RNAs (shRNAs). 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 antisense strand of the isolated oligonucleotide each comprise at least five modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least six modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least seven modified nucleotides. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least eight modified nucleotides. 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 acridinone 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 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 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-C6 alkyl, 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′-C N 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-C₆ alkyl, 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 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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 27, 28, 30, 46, 31, 37, 48, 40, 43, or 44.

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′)(M)_e(F)_d(M)_c(F)_b(M)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 27.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 27, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 2.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 28.

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)(F)(IM)_e(F)_a(M)_c(F)_b(M)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 28, 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)_c(F)_b(M)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 30.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 30, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 5.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₀ 3′, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 46.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 46, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 21.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3 the sense strand comprises a nucleotide sequence according to SEQ ID NO: 31.

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)_a 3′ wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 31, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 6.

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)_a 3′ wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 37.

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)_a 3′ wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 37, 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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 48.

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)_a 3, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 48, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 23.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3V the sense strand comprises a nucleotide sequence according to SEQ ID NO: 40.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3V and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 40, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 15.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₄(F)₁(M)₁(F)₄(M)₉ 3′, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 43.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)(F)₁(M)₁(F)₄(M)₉ 3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 43, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 18.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, L, c, d, e, f and g is any one of 0-16, and wherein the sense strand is 5′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₄(M)₉ 3′, the sense strand comprises a nucleotide sequence according to SEQ ID NO: 44.

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)_a 3′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, L, 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)(M)₁(F)₄(M)₉ 3′, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 44, the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 19.

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 158 to 178, from the 5′ end of a HBV 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: 2 (5′ UAUCCUGAUGUGAUGUUCUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ GAGAACAUCACAUCAGGAUA 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 the nucleotide positions 163 to 183, from the 5′ end of a HBV 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 UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 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 the nucleotide positions 203 to 223, from the 5′ end of a HBV 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: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′GCGGGGUUUUUCUUGUUGAA 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 the nucleotide positions 206 to 226, from the 5′ end of a HBV 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: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ GGGUUUUUCUUGUUGACAAA 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 the nucleotide positions 220 to 240, from the 5′ end of a HBV 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: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 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 the nucleotide positions 622 to 642, from the 5′ end of a HBV 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′ UUAGGAAUUUUCCGAAAGCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GGCUUUCGGAAAAUUCCUAA 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 the nucleotide positions 720 to 740, from the 5′ end of a HBV 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: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 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 the nucleotide positions 794 to 814, from the 5′ end of a HBV 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: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 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 the nucleotide positions 797 to 817, from the 5′ end of a HBV 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: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 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 the nucleotide positions 799 to 819, from the 5′ end of a HBV 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: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 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)_e(F)_f(M)_g(F)_h(M)_i(F)_j(M)_k(F)_l(M)_m(F)_n(M)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is 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)₁ 5′.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide. F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is 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)₁ 5′, the antisense strand comprises a nucleotide sequence according to any one of SEQ ID NOs: 2, 3, 5, 21, 6, 12, 23, 15, 18, or 19.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is 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)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 2.

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)_fIM)_g(F)_h(M)_i(F)_j(M)_k(F)_l(M)_m(F)_n(M)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is any one of 3′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 2, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 27.

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)(M)_m(F)_n(M)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is 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)₁ 5′, and 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 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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is 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)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 3, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 28.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is 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)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 5.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide. F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is 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)₁ 5, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 5, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 30.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is any one of 3′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 21.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is any one of: 3′(M)₀(F)₀(M)₆(F)₁(M)₁(F)(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 21, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 46.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is 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)₂(M)₁(F)₂(M)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 6.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is 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)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 6, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 31.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is 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)(F)₁(M)₁(F)₂(M)₁ 5′, and 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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is any one of 3′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 12, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 37.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is any one of: 3′(M)₀(F)₀(M)₆(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)_j(M)₁(F)₁(M)₁(F)₂(M)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 23.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is any one of: 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)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 23, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 48.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide. F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is any one of 3′(M)₀(F)₀(M)₆(F)₁(M)(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is any one of: 3′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 15, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 40.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide. F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is 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)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 18.

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)(M)_g(F)_h(M)_i(F)_j(M)_k(F)_l(M)_m(F)_n(M)_o 5′, wherein M is 2′-O-methyl modified nucleotide. F is 2′-F modified nucleotide, and a, b, c, d, e, f g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is any one of: 3′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)_j(M)₁(F)₁(M)₁(F)₂(M)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 18, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 43.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide, F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is any one of 3′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)₁ 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 19.

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)_o 5′, wherein M is 2′-O-methyl modified nucleotide. F is 2′-F modified nucleotide, and a, b, c, d, e, f, g, h, i, j, k, l, m, n and o is any one of 0-16, wherein the antisense strand is any one of: 3′(M)₀(F)₀(M)₅(F)₁(M)₁(F)₁(M)₃(F)₁(M)₂(F)₁(M)₁(F)₁(M)₁(F)₂(M)_i 5′, and the antisense strand comprises a nucleotide sequence according to SEQ ID NO: 19, and the sense strand comprises a nucleotide sequence according to SEQ ID NO: 44.

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 (ASCPR2).

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

In some embodiments, the ligand comprises three GalNAc moieties, or three derivatives thereof. In some embodiments, the ligand comprises three GalNAc G1b moieties. In some embodiments, wherein the ligand comprises three GalNAc G1b moeities, the GalNAc G1b moities 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 G1b moiety is linked to the second G1b moiety and the second G1b is linked to the third G1b 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 G1b moiety is linked to sense of the isolated oligonucleotide, the first GalNAc 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 G1b, or three GalNAc G1b 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.

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) 158 to 183; b) 203 to 240; c) 622 to 642; d) 720 to 740; and e) 794 to 819 from the 5′ end of a HBV 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, a targeting ligand is attached to the 3′ end of the sense strand. In some embodiments, the targeting ligand comprising three GalNAc G1b moieties.

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) 158 to 183; b) 203 to 240; c) 622 to 642; d) 720 to 740; and e) 794 to 819, from the 5′ end of HBV 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, wherein the targeting ligand comprises three GalNAc G1b moieties attached to the 3′ end of the sense strand, the sense strand comprises a nucleic acid sequence according to SEQ ID NO: 27 (5′ GAGAACAUCACAUCAGGAUA 3′); SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′); SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′); SEQ ID NO: 46 (5′ GGUUUUUCUUGUUGACAAA 3′); SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 3′); SEQ ID NO: 37 (5′ GGCUUUCGGAAAAUUCCUAA 3′); SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′); SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′); SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′); or SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′).

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, R¹ is H or C₁-C₆ alkyl, and R² is H or C₁-C₆ alkyl. 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-C₆ alkyl”, as used herein, is intended to include C₁, C₂, C₃, C₄, C₅ or C₆ straight chain (linear) saturated aliphatic hydrocarbon groups and C₃, C₄, C₅ or C₆ branched saturated aliphatic hydrocarbon groups. For example, C₁-C₆ alkyl is intended to include Cr, C₂, C₃, C₄, C₅ and C₆ alkyl 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-C₆ for straight chain, C₃-C₆ for 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:

wherein:

• • B is H or a nucleobase moiety;
  • X is O or S;
  • R¹ is H or C₁-C₆ alkyl;
  • R² is H or C₁-C₆ alkyl;
  • Y¹ is O or S;
  • Y² is O or S;
  • Z is H, halogen, or —OR^Z.
  • R^Z is H, C₁-C₆ alkyl, or —(C₁-C₆ alkyl)-(C₆-C₁₀ aryl), wherein the C₁-C₆ alkyl or —(C₁-C₆ alkyl)-(C₆-C₁₀ aryl) is optionally substituted with one or more R^Za
  • each R^Za independently is halogen, C₁-C₆ alkyl, or —O—(C₁-C₆ alkyl), wherein the C₁-C₆ alkyl or —O—(C₁-C₆ alkyl) is optionally substituted with one or more halogen; and

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:

wherein:

• • B is H or a nucleobase moiety;
  • X is O or S;
  • R¹ is H or C₁-C₆ alkyl;
  • R² is H or C₁-C₆ alkyl;
  • Y¹ is O or S;
  • Y² is O or S; and

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:

wherein:

• • B is H or a nucleobase moiety;
  • X is O or S;
  • R¹ is H or C₁-C₆ alkyl;
  • R² is H or C₁-C₆ alkyl; and

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, R¹ is H.

In some embodiments, R¹ is C₁-C₆ alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

In some embodiments, R¹ is methyl.

In some embodiments, R² is H.

In some embodiments, R² is C₁-C₆ alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, 1-butyl, pentyl, or hexyl).

In some embodiments, R² is methyl.

In some embodiments, Y¹ is O.

In some embodiments, Y¹ is S.

In some embodiments, Y² is O.

In some embodiments, Y² is 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-C₆ alkyl) (e.g., wherein the C₁-C₆ alkyl 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-C₆ alkyl)-O—(C₁-C₆ alkyl) (e.g., wherein the C₁-C₆ alkyl 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-C₆ alkyl)-(C₆-C₁₀ aryl) optionally substituted with one or more R^Za.

In some embodiments, Z is —O—(C₁-C₆ alkyl)-(C₆-C₁₀ aryl).

In some embodiments, Z is

In some embodiments, Z is

optionally substituted with one or more R^Za.

In some embodiments, Z is

optionally substituted with one or more halogen.

In some embodiments, Z is

optionally substituted with one or more C₁-C₆ alkyl or —O—(C₁-C₆ alkyl), wherein the C₁-C₆ alkyl or —O—(C₁-C₆ alkyl) is optionally substituted with one or more halogen.

In some embodiments, R^Z is H.

In some embodiments, R^Z is not H.

In some embodiments. R^Z is C₁-C₆ alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more R^Za.

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

In some embodiments, R^Z is C₁-C₆ alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

In some embodiments, R^Z is methyl, ethyl, or propyl.

In some embodiments, R^Z is methyl.

In some embodiments, R^Z is C₁-C₆ alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogen (e.g., F, Cl, Br, or I).

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

In some embodiments, R^Z is —(C₁-C₆ alkyl)-(C₆-C₁₀ aryl) optionally substituted with one or more R^Za.

In some embodiments, R^Z is —(C₁-C₆ alkyl)-(C₆-C₁₀ aryl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I), C₁-C₆ alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl), or —O—(C₁-C₆ alkyl) (e.g., wherein the C₁-C₆ alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl), wherein the C₁-C₆ alkyl or —O—(C₁-C₆ alkyl) is optionally substituted with one or more halogen.

In some embodiments, R^Z is —(C₁-C₆ alkyl)-(C₅-C₁₀ aryl).

In some embodiments, at least one R^Za is halogen (e.g., F, Cl, Br, or I).

In some embodiments, at least one R^Za is F or C₁.

In some embodiments, at least one R^Za is C₁-C₆ alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I).

In some embodiments, at least one R^Za is C₁-C₆ alkyl (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^Za is C₁-C₆ alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogen (e.g., F, Cl, Br, or I).

In some embodiments, at least one R^Za is —O—(C₁-C₆ alkyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I).

In some embodiments, at least one R^Za is —O—(C₁-C₆ alkyl).

In some embodiments, at least one R is —O—(C₁-C₆ alkyl) 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:

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;
  • R¹ is Cr alkyl;
  • R² is H; and

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 antisense strand of the isolated oligonucleotide having the following structure (5′-MeEPrnU.

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 antisense strand of the isolated oligonucleotide, having the following structure (5′-MeEPmUs).

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.

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

wherein “mU” is a 2′-O-methyl modified uridine nucleotide, “EP” is a 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 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) 158 to 183; b) 203 to 240; c) 622 to 642; d) 720 to 740; and e) 794 to 819, from the 5′ end of a HBV 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, the MeEP is linked to the 5′ end of the antisense strand (5′-MeEP).

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.

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) 158 to 183; b) 203 to 240; c) 622 to 642; d) 720 to 740; and e) 794 to 819, from the 5′ end of a HBV mRNA sequence according to SEQ ID NO: 1, and the antisense strand is substantially complementary to the sense strand such that the sense strand and the antisense strand together form a double stranded region, the antisense strand comprises a nucleic acid sequence according to SEQ ID NO: 2 (5′ UAUCCUGAUGUGAUGUUCUCCA 3′); SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′); SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′); SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′); SEQ ID NO: 6 (5′UUAUUGUGAGGAUUCUUGUCAA 3′); SEQ ID NO: 12 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′); SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′); SEQ ID NO: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′); SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′); or SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 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) 158 to 183; b) 203 to 240; c) 622 to 642; d) 720 to 740; and e) 794 to 819, from the 5′ end of a HBV mRNA sequence according to SEQ ID NO: 1, and the antisense strand is substantially complementary to the sense strand such that the sense strand and the antisense strand together form a double stranded region, the sense strand comprises a targeting ligand comprising three GalNAc G1b moieties attached to the 3′ end of the sense strand.

Modified Backbone Phosphate/Phosphodiester Bond

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. A phosphodiester bond comprises a linkage having the formula:

wherein

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

denotes attachment to a 5′ carbon of a second nucleotide in the isolated oligonucleotide of the present disclosure. In some embodiments, the phosphodiester bond is unmodified, wherein Z¹ is O and Z² is OH or O⁻. In some embodiments, the phosphodiester bond is modified, wherein Z¹ is O, S, NH, or N(C₁-C₆ alkyl) and Z² is OH, SH, NH₂, NH(C₁-C₆ alkyl), O⁻, S⁻, HN⁻, or (C₁-C₆ alkyl)N⁻, and wherein when Z¹ is O, Z² is not OH or O⁻.

In some embodiments, Z₁ is O.

In some embodiments, Z₁ is S.

In some embodiments, Z₁ is NH.

In some embodiments, Z₁ is N(C₁-C₆ alkyl).

In some embodiments, Z₂ is OH.

In some embodiments, Z₂ is SH.

In some embodiments, Z₂ is NH₂.

In some embodiments, Z₂ is NH(C₁-C₆ alkyl).

In some embodiments, Z₂ is SH, NH₂, or NH(C₁-C₆ alkyl).

In some embodiments, Z₂ is O⁻.

In some embodiments, Z₂ is S⁻.

In some embodiments, Z₂ is HN⁻.

In some embodiments, Z₂ is (C₁-C₆ alkyl)N⁻.

In some embodiments, Z₂ is S⁻, HN⁻, or (C₁-C₆ alkyl)N⁻.

In some embodiments, Z₁ is O and Z₂ is SH.

In some embodiments, Z₁ is O and Z₂ is NH₂.

In some embodiments, Z₁ is O and Z₂ is NH(C₁-C₆ alkyl).

In some embodiments, Z₁ is S and Z₂ is OH.

In some embodiments, Z₁ is S and Z₂ is SH.

In some embodiments, Z₁ is S and Z₂ is NH₂.

In some embodiments, Z₁ is S and Z₂ is NH(C₁-C₆ alkyl).

In some embodiments, Z₁ is NH and Z₂ is OH.

In some embodiments, Z₁ is NH and Z₂ is SH.

In some embodiments, Z₁ is NH and Z₂ is NH₂.

In some embodiments, Z₁ is NH and Z is NH(C₁-C₆ alkyl).

In some embodiments, Z₁ is N(C₁-C₆ alkyl) and Z₂ is OH.

In some embodiments, Z₁ is N(C₁-C₆ alkyl) and Z₂ is SH.

In some embodiments, Z₁ is N(C₁-C₆ alkyl) and Z₂ is NH₂.

In some embodiments, Z₁ is N(C₁-C₆ alkyl) and Z₂ is NH(C₁-C₆ alkyl).

In some embodiments, Z₁ is O and Z₂ is S⁻.

In some embodiments, Z₁ is O and Z₂ is HN⁻.

In some embodiments, Z₁ is O and Z₂ is (C₁-C₆ alkyl)N⁻.

In some embodiments, Z₁ is S and Z₂ is O⁻.

In some embodiments, Z₁ is S and Z₂ is S⁻.

In some embodiments, Z₁ is S and Z₂ is HN⁻.

In some embodiments, Z₁ is S and Z₂ is (C₁-C₆ alkyl)N.

In some embodiments, Z₁ is NH and Z₂ is O⁻.

In some embodiments, Z₁ is NH and Z₂ is S⁻.

In some embodiments, Z₁ is NH and Z₂ is HN⁻.

In some embodiments, Z₁ is NH and Z₂ is (C₁-C₆ alkyl)N⁻.

In some embodiments, Z₁ is N(C₁-C₆ alkyl) and Z₂ is O⁻.

In some embodiments, Z₁ is N(C₁-C₆ alkyl) and Z₂ is S⁻.

In some embodiments, Z₁ is N(C₁-C₆ alkyl) and Z₂ is HN⁻.

In some embodiments, Z₁ is N(C₁-C₆ alkyl) and Z₂ is (C₁-C₆ alkyl)N⁻.

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

In some embodiments, the modified phosphodiester bond comprises

wherein

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

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

In some embodiments, the modified phosphodiester bond comprises

wherein

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

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

In some embodiments, the modified phosphodiester bond comprises

wherein

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

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

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 antisense 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. In 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. In 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 antisense strand of the isolated oligonucleotide each comprise at least eleven phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least twelve phosphorothioate internucleotide linkages. 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 antisense strand of the isolated oligonucleotide each comprise at least sixteen phosphorothioate internucleotide linkages. In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least seventeen phosphorothioate internucleotide linkages.

In some embodiments, the sense strand and/or the antisense strand of the isolated oligonucleotide each comprise at least eighteen phosphorothioate internucleotide linkages. 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 antisense strand or both are not consecutively located.

Envisaged within the present disclosure is an isolated oligonucleotide, wherein any phosphodiester bond on the sense strand or antisense strand can be modified. In some embodiments, any phosphodiester bond on the antisense strand can be modified. In some embodiments, any phosphodiester bond on the antisense strand can be modified.

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 antisense strand comprises at least one, at least two, at least three, or at least four phosphorothioate internucleotide linkages, the phosphorothioate internucleotide linkages connect the nucleotides at position 1 and position 2 from the first nucleotide at the 5′-terminus of the antisense strand. 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 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 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 antisense strand comprises four phosphorothioate internucleotide linkages, the phosphorothioate internucleotide linkages are located between nucleotides at position 1 to 3 and nucleotides at position 20 to 22 from the first nucleotide at the 5′-terminus of the antisense strand.

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: 232 (5′ [mUs][fAs][fU][mC][fC][mU][fG][mA][mU][fG][mU][mG][mA][fU][mG][fU][mU][mC][mU][mCs][mCs][mA] 3′);

• • ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 233 (5′ [mUs][fUs][fA][mG][fG][mA][fA][mU][mC][fC][mU][mG][mA][fU][mG][fU][mG][mA][mU][mGs][mUs][mU] 3′);
  • iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 234 (5′ [mUs][fUs][fC][mA][fA][mC][fA][mA][mG][fA][mA][mA][mA][ ][fA][mC][fC][mC][mC][m G][mCs][mCs][mU] 3′);
  • iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 235 (5 [mUs][Us][fU][mC][fU][mC][fA,][mA][mC][fA][mA][mG][mA][fA][mA][fA][mA][mC][m C][mCs][mCs][mG] 3′);
  • v) an antisense strand of nucleic acid sequence according to SEQ ID: 236 (5′ [mUs][fUs][fA][mU][fU][mG][fU][mG][mA][fG][mC][mA][mU][fU][mC][fU][mU][mG][mU][mCs][mAs][mA] 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID: 237 (5, [mUs][fUs][fA][mG][fG][mA][fA][mU][mU][fU][mU][mC][mC][fG][mA][fA][mA][mG][mC][mCs][mCs][mA] 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID: 238 (5′ [mUs][fAs][fU][mA][fA][mC][fU][mG][mA][fA][mA][mG][mC][fC][mA][fA][mA][mC][m A][mGs][mUs][mG] 3′), viii) an antisense strand of nucleic acid sequence according to SEQ ID: 239 (5′ [mUs][fAs][fG][mA][fA][mA][fA][mU][mU][fG][mG][mU][mA][fA][mC][fA][mG][mC][mG][mGs][mUs][mA] 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID: 240 (5′ [mUs][fAs][fA][mA][fA][mC][fA][mA][mA][fA][mU][mU][mG][fC][mU][fA][mA][mC][mA][mGs][mCs][mG] 3′); or x) an antisense strand of nucleic acid sequence according to SEQ ID: 241 (5′ [mUs][fAs][fC][mA][fA][mA][fA][mG][mA][fA][mA][mA][mU][fU][mG][fC][mU][mA][mA][mCs][mAs][mG] 3′), wherein “m” is a 2′-O-methyl modified nucleotide, “f” is a 2′-F modified nucleotide, “s” is a phosphorothioate internucleotide linkage.

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: 242 (5′ [mGs][mAs][mG][mA][mA][fC][mA][fU][fC][fA][fC][mA][mU][mC][mA][mG][mG][mAs][mUs][mA][G1b][G1b][G1b] 3′);
  • ii) a sense strand of nucleic acid sequence according to SEQ ID NO: 243 (5′ [mCs][mAs][mU][mC][mA][fC][mA][fU][fC][fA][fG][mG][mA][mU][mU][mC][mC][mUs][mAs][mA][G1b][G1b][G1b] 3′);
  • iii) a sense strand of nucleic acid sequence according to SEQ ID NO: 244 (5′ [mGs][mCs][m G][mG][mG][fG][mU][fU][fU][fU][fU][mC][mU][mU][mG][mU][mU][mGs][mAs][mA][G1b][G1b][G1b] 3′);
  • iv) a sense strand of nucleic acid sequence according to SEQ ID NO: 245 (5′ [mGs][mGs][mG][mU][mU][fU][mU][fU][fC][fU][fU][mG][mU][mU][mG][mA][mC][mAs][mAs][mA][G1b][G1b][G1b] 3′);
  • v) a sense strand of nucleic acid sequence according to SEQ ID NO: 246 (5′ [mGs][mAs][mC][mA][mA][fG][mA][fA][fU][fC][fC][mU][mC][mA][mC][mA][mA][mUs][mAs][mA][G1b][G1b][G1b] 3′); vi) a sense strand of nucleic acid sequence according to SEQ ID NO: 247 (5′ [mGs][mGs][mC][mU][mU][fU][fG][fG][fA][fA][mA][mA][mU][mU][mC][mC][mUs][mAs][mA][G1b][G1b][G1b] 3′); vii) a sense strand of nucleic acid sequence according to SEQ ID NO: 248 (5′ [mCs][mUs][mG][mU][mU][fU][mG][fG][fC][fU][fU][mU][mC][mA][mG][mG][mU][mAs][mUs][mA][G1b][G1b][G1b] 3); viii) a sense strand of nucleic acid sequence according to SEQ ID NO: 249 (5′ [mCs][mCs][mG][mC][mU][fG][mU][fU][fA][fC][fC][mA][mA][mU][mU][mU][mU][mCs][mUs][mA][G1b][G1b][G1b] 3′); ix) a sense strand of nucleic acid sequence according to SEQ ID NO: 250 (5′ [mCs][mUs][mG][mU][mU][fA][mC][fC][fA][fA][fU][mU][mU][mU][mC][mU][mU][mUs][mUs][mA][G1b][G1b][G1b] 3′); or x) a sense strand of nucleic acid sequence according to SEQ ID NO: 251 (5′ [mGs][mUs][mU][mA][mC][fC][mA][fA][fU][fU][fU][mU][mC][mU][mU][mU][mU][mCs][mUs][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 nucleic acid sequence according to SEQ ID NO: 232 (5′ [mUs][fAs][fU][mC][fC][mU][fC][mA][mU][fG][mU][mC][mA][fU][mG][fU][mU][mC][m U][mCs][mCs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 242 (5′ [mGs][mAs][mG][mA][mA][fC][mA][fU][fC][fA][fC][mA][mU][mC][mA][mG][mG][mAs][mUs][mA][G1b][G1b][G1b] 3′); ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 233 (5′ [mUs][fUs][fA][mG][fG][mA][fA][mU][mC][fC][mU][mG][mA][fU][mG][fU][mG][mA][mU][mGs][mUs][mU] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: NO: 243 (5′ [mCs][mAs][mU][mC][mA][fC][mA][fU][fC][fA][fG][mC][mA][mU][mU][mC][mUs][mAs][mA][G1b][G1b][G1b] 3′); iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 234 (5′ [mUs][fUs][fC][mA][fA][mC][fA][mA][mG][fA][mA][mA][mA][fA][mC][fC][mC][mC][m G][mCs][mCs][mU] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 244 (5′ [mGs][mCs][mG][mG][mG][fG][mU][fU][fU][fU][fU][mC][mU][mU][mG][mU][mU][mGs ][mAs][mA][G1b][G1b][G1b] 3′); iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 235 (5′ [mUs][fU][fU][mG][fU][mC][fA][mA][mC][fA][mA][mG][mA][fA][mA][fA][mA][mC][m C][mCs][mCs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 245 (5′ [mGs][mGs][mG][mU][mU][fU][mU][fU][fC][fU][fU][mG][mU][mU][mG][mA][mC][mAs][mAs][mA][G1b][1 b][G b] 3′); v) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 236 (5′ [mUs][fUs][fA][mU][fU][mG][fU][mG][mA][fG][mG][mA][mU][fU][mC][fU][mU][mG][mU][mCs][mAs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 246 (5′ [mGs][mAs][mC][mA][mA][fG][mA][fA][fU][fC][fC][mU][mC][mA][mC][mA][mA][mUs][mAs][mA][G1b][G1b][G1b] 3′); vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 237 (5′ [mUs][fUs][fA][mG][fG][mA][fA][mU][mU][fU][mU][mC][mC][fG][mA][fA][mA][mG][mC][mCs][mCs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 247 (5′[mCs][mCs][mC][mU][mU][fU][mC][fG][fG][fA][fA][mA][mA][mU][mU][mC][mC][m Us][mAs][mA][G1b][G1b][G1b] 3′); vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 238 (5′ [mUs][fAs][fU][mA][fA][mC][fU][mG][mA][fA][mA][mG][mC][fC][mA][fA][mA][mC][m A][mGs][mUs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 248 (5′ [mCs][mUs][mG][mU][mU][fU][fG][fC][fU][fU][mU][mC][mA][mG][mU][mU][mAs][mUs][mA][G1b][G1b][G1b] 3); viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 239 (5′ [mUs][fAs][fG][mA][fA][mA][fA][mU][mU][fG][mG][mU][mA][fA][mC][fA][mG][mC][mG][mGs][mUs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 249 (5′ [mCs][mCs][mG][mC][mU][fG][mU][fU][fA][fC][fC][mA][mA][UI][mU][mU][mU][mCs][mUs][mA][G1b][G1b][G1b] 3′); ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 240 (5′ [mUs][fAs][fA][mA][fA][mG][fU][mA][mA][fA][mU][mU][mG][fG][mU][fA][mA][mC][mA][mGs][mCs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 250 (5′ [mCs][mUs][mG][mU][mU][fA][mC][fC][fA][fA][fU][mU][mU][mU][mC][mU][mU][mUs][mUs][mA][G1b][G1b][G1b] 3′); or x) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 241 (5′ [mUs][fAs][fC][mA][fA][mA][fA][mG][mA][fA][mA][mA][mU][fU][mG][fG][mU][mA][mA][mCs][mAs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 251 (5′ [mGs][mUs][mU][mA][mC][fC][mA][fA][fU][fU][fU][mU][mC][mU][mU][mU][mU][mGs][mUs][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.

Nucleic Acids and Vectors

The present disclosure also provides a vector encoding at least one 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. In some embodiments, the vector encodes one isolated oligonucleotide disclosed herein. In some embodiments, the vector encodes more than one isolated oligonucleotide disclosed herein. In some embodiments, the vector encodes, two, three, four, or five isolated oligonucleotides disclosed herein. In some embodiments, the vector encodes more than five isolated oligonucleotides disclosed herein.

The disclosure provides nucleic acids comprising the sequences encoding the isolated oligonucleotides (e.g., dsRNAs or siRNAs) targeting HBV 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) and pCpG-siRNA (InvivoGen). Examples of kits for production of PCR-generated shRNA expression cassettes include Silencer Express (Ambion) and siXpress (Mirus)

Viral vectors for the in vivo expression of the isolated oligonucleotides (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 virus. Examples of commercially available viral vectors for shRNA expression include pSilencer adeno (Ambion) and pLenti6/BLOCK-iT™-DEST (Invitrogen). Selection of viral vectors, methods for expressing the siRNA from the vector and methods of delivering the viral vector, for example incorporated within a nanoparticle, are within the ordinary skill of one in the art.

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 at least one isolated oligonucleotide disclosed herein or vector of the present disclosure encoding at least one 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 comprises nanoparticles comprising the isolated oligonucleotides of the present disclosure (e.g., siRNA or dsRNAs) targeting a HBV 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 diblock 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 HBV 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 HBV 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 HBV 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 HBV 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 HBV 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 HBV mRNA and expressing HBV 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 un, 180 nm, 150 nm, 120 nm, 100 un, 90 nm, 80 nm, 70 nm, 60 nm, 50 un, 40 nm, 30 nm or 20 nm. In other embodiments, the nanoparticle has an average characteristic dimension of 10 nm, 20 nu, 30 nm, 40 nm, 50 nm, 60 nm, 70 nu, 80 nm, 90 nm, 100 nm, 120 nm, 150 nm, 180 nm, 200 un, 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 nu, 10-75 nm, 10-50 nm, 50-500 nm, 50-400 nm, 50-300 nm, 50-200 nm, 50-150 nu, 50-100 nm, 50-75 nm 100-500 nm, 100-400 nm, 100-300 nm, 100-250 nm, 100-200 un, 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 nu, 200-500 nm, 200-400 nm or 200-300 nm.

Therapeutic Agents

In some embodiments, the delivery system of the present disclosure is 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 HBV 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, additionally, additional therapeutic agents can be incorporated into the lipid or polymer-based shell of the delivery system, for example via intercalation. Alternatively, additionally, 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 comprising at least one isolated oligonucleotide targeting HBV disclosed herein are formulated in the same composition. For example, the delivery system comprising at least one isolated oligonucleotide of the present disclosure targeting HBV and the additional therapeutic agent can be formulated in the same pharmaceutical composition.

In some embodiments, the additional therapeutic agent and the delivery system comprising at least one isolated oligonucleotide targeting HBV 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 at least one oligonucleotide disclosed herein, a vector of the present disclosure encoding at least one 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 HBV 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 HBV 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 HBV of the present disclosure, and the delivery system and the therapeutic agent are formulated in separate pharmaceutical compositions.

Pharmaceutical compositions of the present disclosure 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 of the present disclosure 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 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 bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. 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) 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 HBV 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 administratio.

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 HBV of the present disclosure and delivery systems comprising same.

The one or more oligonucleotides of (e.g., dsRNAs or siRNAs) targeting HBV 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 HBV 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 HBV 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 HBV 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 HBV 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 HBV in a subject in need thereof, wherein the method comprises administering to the subject an effective amount at least one isolated oligonucleotide disclosed herein, at least one vector disclosed herein, at least one delivery system disclosed herein, or at least one pharmaceutical composition disclosed herein.

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

The present disclosure also provides at least one isolated oligonucleotide disclosed herein, a vector of the of the present disclosure encoding at least one isolated oligonucleotide disclosed herein, a delivery system of the present disclosure, or a pharmaceutical composition of the present disclosure, for use in treatment or prevention of a disease or disorder associated with aberrant or increased expression or activity of HBV or a disease or disorder where HBV plays a role, in a subject in need thereof.

The present disclosure also provides use of at least one isolated oligonucleotide disclosed herein, a vector of the of the present disclosure encoding at least one isolated oligonucleotide disclosed herein, a delivery system of the present disclosure, or a pharmaceutical composition of the present disclosure, in the manufacture of a medicament for treatment or prevention of a disease or disorder associated with aberrant or increased expression or activity of HBV or a disease or disorder where HBV plays a role in a subject in need thereof.

Provided herein are methods of inhibiting or downregulating HBV expression or activity in a cell, comprising contacting the cell with the one or more oligonucleotides (e.g. dsRNA or siRNA) targeting HBV as described herein. The one or more oligonucleotides (e.g., dsRNA or siRNA) targeting HBV as described herein can reduce or inhibit HBV 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 HBV as described herein are capable of inducing RNAi-mediated degradation of an HBV 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 “IC₅₀ value” refers to the concentration of an agent where cell viability is reduced by half. The IC₅₀ is 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 HBV as described herein, and the IC₅₀ value of the oligonucleotides (e.g., dsRNA or siRNA) targeting HBV is calculated after 24, 48 and/or 72 hours to determine its effectiveness in downregulating or inhibiting the level of HBV mRNA or protein to 50%, as compared to the level of HBV mRNA or protein in an untreated cell or in the same cell before initiation of treatment with the isolated oligonucleotide.

Methods of monitoring of HBV mRNA and/or protein expression can be used to characterize gene silencing, and to determine the effectiveness of the compositions described herein. Expression of HBV may be evaluated by any technique known in the art. Examples thereof include immunoprecipitations methods, utilizing HBV antibodies in assays such as ELTSAs, western blotting, or immunohistochemistry to visualize HBV 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 HBV or a unique fragment thereof, or a transcription product (e.g., mRNA) or splicing product of said nucleic acid, northern blotting methods, Southern blotting methods, and various PCR-based methods such as RT-PCR, qPCR or digital droplet PCR. HBV 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 HBV 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 RNAiMAX Lipofectamine kit (Invitrogen) 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) to determine cell viability, and/or HBV mRNA and protein levels can be assessed using the methods described herein.

In some embodiments of the methods of inhibiting or downregulating HBV expression or activity in a cell of the present disclosure, the at least one isolated oligonucleotide, the vector, the delivery system, or the pharmaceutical composition is administered parenterally. In some embodiments, the parenteral administration is intravenous, subcutaneous, intraperitoneal, or intramuscular.

In some embodiments of the methods of inhibiting or downregulating HBV 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 HBV expression or activity in a cell of the present disclosure, the subject experiences symptoms of or suffers from fatigue, poor appetite, stomach pain, inflammation, nausea, vomiting, jaundice, acute HBV infection, chronic HBV infection, hepatitis D virus (LID V) infection, hepatitis infection-induced inflammatory liver disease, liver failure, liver cirrhosis, or liver cancer, such as for example hepatocellular carcinoma, or a related disorder or symptom associated with aberrant or increased expression or activity of HBV or a disease or disorder where HBV plays a role.

In some embodiments of the methods of inhibiting or downregulating HBV expression or activity in a cell of the present disclosure, the method comprises administering the at least one isolated oligonucleotide, the vector, the delivery system, or the pharmaceutical composition, in combination with at least a second therapeutic agent. In some embodiments, the second therapeutic agent is an antibody, a small molecule drug, a peptide, a nucleotide molecule, or a combination thereof. In some embodiments, the second therapeutic agent is an isolated oligonucleotide of the present disclosure.

The present disclosure also provides a method of inhibiting or downregulating the expression or level of HBV 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. In some embodiments, the first and at least second oligonucleotides are administered simultaneously. In some embodiments, the first and at least second oligonucleotides are administered sequentially.

In some embodiments of the methods of inhibiting or downregulating HBV 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 HBV expression or activity in a cell of the present disclosure, the subject experiences symptoms of or suffers from fatigue, poor appetite, stomach pain, inflammation, nausea, vomiting, jaundice, acute HBV infection, chronic HBV infection, hepatitis D virus (HDV) infection, hepatitis infection-induced inflammatory liver disease, liver failure, liver cirrhosis, or liver cancer, such as for example hepatocellular carcinoma, or a related disorder or symptom associated with aberrant or increased expression or activity of HBV or a disease or disorder where HBV plays a role. In some embodiments of the method of treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HBV or a disease or disorder where HBV 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 HBV or a disease or disorder where HBV plays a role of the present disclosure, the disease or disorder is or is associated with fatigue, poor appetite, stomach pain, inflammation, nausea, vomiting, jaundice, acute HBV infection, chronic HBV infection, hepatitis D virus (HDV) infection, hepatitis infection-induced inflammatory liver disease, liver failure, liver cirrhosis, or liver cancer, such as for example hepatocellular carcinoma, or a related disorder or symptom associated with aberrant or increased expression or activity of HBV or a disease or disorder where HBV plays a role. In some embodiments of the use for treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HBV or a disease or disorder where HBV 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 HBV or a disease or disorder where HBV plays a role of the present disclosure, the disease or disorder is or is associated with fatigue, poor appetite, stomach pain, inflammation, nausea, vomiting, jaundice, acute HBV infection, chronic HBV infection, hepatitis D virus (HDV) infection, hepatitis infection-induced inflammatory liver disease, liver failure, liver cirrhosis, or liver cancer, such as for example hepatocellular carcinoma, or a related disorder or symptom associated with aberrant or increased expression or activity of HBV or a disease or disorder where HBV plays a role.

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 HBV of the present disclosure, the subject is a human. In some embodiments, the subject is not a human. In some embodiments, the disease or disorder is or is associated with fatigue, poor appetite, stomach pain, inflammation, nausea, vomiting, jaundice, acute HBV infection, chronic HBV infection, hepatitis D virus (HDV) infection, hepatitis infection-induced inflammatory liver disease, liver failure, liver cirrhosis, or liver cancer, such as for example hepatocellular carcinoma, or a related disorder or symptom associated with aberrant or increased expression or activity of HBV or a disease or disorder where HBV plays a role.

Routes of Administration

Nanoparticles comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HBV 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 HBV, a compositions comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HBV of the present disclosure may be injected directly into cells, injected into the blood stream or body cavities, taken orally or applied through the skin with patches. The dose chosen should be sufficient to constitute effective treatment but not so high as to cause unacceptable side effects. The state of the disease condition and the health of the patient should preferably be closely monitored during and for a reasonable period after treatment.

The compositions comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting HBV mRNA of the present disclosure can be administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperitoneally, 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 HBV. Dosages may vary depending on the age and size of the subject and the type and severity of the disease or disorder associated with HBV 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 HBV or symptoms associated with aberrant or abnormal expression of HBV 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 HBV 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 HBV miRNA 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 HBV 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 HBV 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 kits comprising at least one isolated oligonucleotide disclosed herein, a vector of the present disclosure encoding an isolated oligonucleotide disclosed herein, a delivery system of the present disclosure, or a pharmaceutical composition of the present disclosure. In some embodiments, the vector encodes one isolated oligonucleotide disclosed herein. In some embodiments, the vector encodes more than one isolated oligonucleotide disclosed herein.

The kits are for use in the treatment of diseases related to abnormal or aberrant expression of human HBV. The kits are for use in downregulating or inhibiting expression of HBV partially or completely. In some embodiments, the kit is for use in the treatment of disease or downregulating or inhibiting expression of HBV 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

In some embodiments of the kits of the disclosure, the kit comprises nanoparticles. Nanoparticles comprising the one or more isolated oligonucleotides (e.g., dsRNA or siRNA) targeting 1-BV 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 HBV (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 of the present disclosure include, but are not limited to, instructions for use of the kit in treating diseases related to abnormal or aberrant expression of HBV or diseases related to expression of HBV.

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.

Sequences of the Disclosure

TABLE 1
Exemplary Sequences of the Present Application
SEQ	Antisense			SEQ	Sense
ID	strand (guide)	start	end	ID	strand (passenger)	start	end
NO:	sequence	pos.	pos.	NO:	sequence	pos.	pos.
2	UAUCCUGAUGUGAUGUUCUCCA	158	178	27	GAGAACAUCACAUCAGGAUA	160	178
3	UUAGGAAUCCUGAUGUGAUGUU	163	183	28	CAUCACAUCAGGAUUCCUAA	165	183
4	UUGUAACACGAGAAGGGGUCCU	183	203	29	GACCCCUUCUCGUGUUACAA	185	203
5	UUCAACAAGAAAAACCCCGCCU	203	223	30	GCGGGGUUUUUCUUGUUGAA	205	223
21	UUUGUCAACAAGAAAAACCCCG	206	226	46	GGGUUUUUCUUGUUGACAAA	208	226
6	UUAUUGUGAGGAUUCUUGUCAA	220	240	31	GACAAGAAUCCUCACAAUAA	222	240
7	UAGAGGAAGAUGAUAAAACGCC	389	409	32	CGUUUUAUCAUCUUCCUCUA	391	409
8	UAUAGCAGCAGGAUGAAGAGGA	404	424	33	CUCUUCAUCCUGCUGCUAUA	406	424
9	UAGAAGAUGAGGCAUAGCAGCA	416	436	34	CUGCUAUGCCUCAUCUUCUA	418	436
10	UACAAGAAGAUGAGGCAUAGCA	419	439	35	CUAUGCCUCAUCUUCUUGUA	421	439
22	UUUGGUACAGCAACAGGAGGGA	558	578	47	CCUCCUGUUGCUGUACCAAA	560	578
11	UAGGAAUUUUCCGAAAGCCCAG	621	641	36	GGGCUUUCGGAAAAUUCCUA	623	641
12	UUAGGAAUUUUCCGAAAGCCCA	622	642	37	GGCUUUCGGAAAAUUCCUAA	624	642
13	UACUAGUAAACUGAGCCAGGAG	666	686	38	CCUGGCUCAGUUUACUAGUA	668	686
23	UAUAACUGAAAGCCAAACAGUG	720	740	48	CUGUUUGGCUUUCAGUUAUA	722	740
24	UACCACAUCAUCCAUAUAACUG	734	754	49	GUUAUAUGGAUGAUGUGGUA	736	754
25	UAAAAAGGGACUCAAGAUGCUG	774	794	50	GCAUCUUGAGUCCCUUUUUA	776	794
14	UUAAAAAGGGACUCAAGAUGCU	775	795	39	CAUCUUGAGUCCCUUUUUAA	777	795
26	UUGGUAACAGCGGUAAAAAGGG	787	807	51	CUUUUUACCGCUGUUACCAA	789	807
15	UAGAAAAUUGGUAACAGCGGUA	794	814	40	CCGCUGUUACCAAUUUUCUA	796	814
16	UAAGAAAAUUGGUAACAGCGGU	795	815	41	CGCUGUUACCAAUUUUCUUA	797	815
17	UAAAGAAAAUUGGUAACAGCGG	796	816	42	GCUGUUACCAAUUUUCUUUA	798	816
18	UAAAAGAAAAUUGGUAACAGCG	797	817	43	CUGUUACCAAUUUUCUUUUA	799	817
19	UACAAAAGAAAAUUGGUAACAG	799	819	44	GUUACCAAUUUUCUUUUGUA	801	819
20	UAAAGACAAAAGAAAAUUGGUA	803	823	45	CCAAUUUUCUUUUGUCUUUA	805	823

TABLE 2
Exemplary Sequences of the Present Application and Their HBsAg Potency
Guide							% HBsAg	% HBsAg
Strand			Guide		Passenger		remaining	remaining
posi-			strand		strand		at	at
tion			sequence	SEQ	sequence	SEQ	0.05 nM	0.01 nM
3′	Start	End	(5′-3′)	ID	(5′-3′)	ID	Mean	SEM	Mean	SEM
178	157	177	UUCCUGAUG	52	GGAGAACAUCA	69	68.46	6.73	94.46	2.68
			UGAUGUUCU		CAUCAGGAA
			CCAU
179	158	178	UAUCCUGAU	2	GAGAACAUCAC	27	41.41	1.61	85.62	4.56
			GUGAUGUUC		AUCAGGAUA
			UCCA
184	163	183	UUAGGAAUC	3	CAUCACAUCAG	28	44.04	1.79	70.07	1.58
			CUGAUGUGA		GAUUCCUAA
			UGUU
204	183	203	UUGUAACAC	4	GACCCCUUCUC	29	43.82	2.38	123.10	4.11
			GAGAAGGGG		GUGUUACAA
			UCCU
215	194	214	UAAAACCCCG	86	GUGUUACAGGC	159	86.62	9.08	93.13	14.38
			CCUGUAACAC		GGGGUUUUA
			GA
217	196	216	UUUGUGAGG	53	UUGACAAAAAU	70	34.65	4.95	57.94	1.01
			AUUUUUGUC		CCUCACAAA
			AAGG
219	198	218	UUAUUGUGA	54	GACAAAAAUCC	71	23.86	2.88	40.95	4.23
			GGAUUUUUG		UCACAAUAA
			UCGG
221	200	220	UACAAGAAAA	87	CAGGCGGGGUU	160	96.75	4.77	91.88	7.92
			ACCCCGCCUG		UUUCUUGUA
			UA
224	203	223	UUCAACAAG	5	GCGGGGUUUU	30	44.75	8.62	76.29	2.76
			AAAAACCCCG		UCUUGUUGAA
			CCU
226	205	225	UUGUCAACAA	55	GGGGUUUUUC	72	61.62	7.81	81.46	4.51
			GAAAAACCCC		UUGUUGACAA
			GC
227	206	226	UUUGUCAAC	21	GGGUUUUUCU	46	33.43	5.07	54.44	1.43
			AAGAAAAACC		UGUUGACAAA
			CCG
229	208	228	UUCUUGUCA	88	GUUUUUCUUG	161	76.59	3.86	84.27	8.47
			ACAAGAAAAA		UUGACAAGAA
			CCC
241	220	240	UUAUUGUGA	6	GACAAGAAUCC	31	33.87	4.12	68.75	2.11
			GGAUUCUUG		UCACAAUAA
			UCAA
253	232	252	UUAGACUCU	89	CACAAUACCGC	162	61.48	6.30	72.74	5.24
			GCGGUAUUG		AGAGUCUAA
			UGAG
254	233	253	UCUGAGAGA	56	CGUGGUGGAC	73	31.83	2.91	49.41	4.09
			AGUCCACCAC		UUCUCUCAGA
			GGG
255	234	254	UACUGAGAG	57	GUGGUGGACU	74	43.80	2.20	62.05	0.65
			AAGUCCACCA		UCUCUCAGUA
			CGG
255	234	254	UUCUAGACUC	90	CAAUACCGCAG	163	88.82	4.29	72.92	5.13
			UGCGGUAUU		AGUCUAGAA
			GUG
258	237	257	UAAAACUGAG	58	GUGGACUUCUC	75	81.81	6.10	61.23	2.44
			AGAAGUCCAC		UCAGUUUUA
			GG
269	248	268	UAAGUCCACC	91	CUAGACUCGUG	164	85.52	2.58	84.37	4.12
			ACGAGUCUA		GUGGACUUA
			GAC
276	255	275	UUUGAGAGA	59	CGUGGUGGACU	76	68.03	10.57	81.98	9.07
			AGUCCACCAC		UCUCUCAAA
			GAG
277	256	276	UAUUGAGAG	92	GUGGUGGACU	165	68.17	2.69	79.29	6.91
			AAGUCCACCA		UCUCUCAAUA
			CGA
279	258	278	UAAAUUGAG	93	GGUGGACUUCU	166	73.29	6.76	83.15	3.90
			AGAAGUCCAC		CUCAAUUUA
			CAC
280	259	279	UAAAAUUGA	94	GUGGACUUCUC	167	96.51	4.85	104.70	12.14
			GAGAAGUCCA		UCAAUUUUA
			CCA
282	261	281	UAGAAAAUU	60	GGACUUCUCUC	77	53.00	4.16	98.45	8.17
			GAGAGAAGU		AAUUUUCUA
			CCAC
283	262	282	UUAGAAAAU	61	GACUUCUCUCA	78	58.97	5.58	119.28	8.38
			UGAGAGAAG		AUUUUCUAA
			UCCA
303	282	302	UAGACACACG	95	GGGGAACUACC	168	87.40	7.33	87.14	7.98
			GUAGUUCCCC		GUGUGUCUA
			CU
304	283	303	UAAGACACAC	62	GGGAACUACCG	79	52.14	4.45	94.10	2.35
			GGUAGUUCC		UGUGUCUUA
			CCC
312	291	311	UUUUUGGCC	63	CCGUGUGUCUU	80	51.88	4.54	86.05	2.95
			AAGACACACG		GGCCAAAAA
			GUA
313	292	312	UAUUUUGGC	96	CGUGUGUCUU	169	79.56	2.79	112.33	3.82
			CAAGACACAC		GGCCAAAAUA
			GGU
314	293	313	UAAUUUUGG	97	GUGUGUCUUG	170	90.31	6.45	97.73	4.82
			CCAAGACACA		GCCAAAAUUA
			CGG
318	297	317	UUGCGAAUU	64	GUCUUGGCCAA	81	57.77	4.41	108.23	3.26
			UUGGCCAAG		AAUUCGCAA
			ACAC
334	313	333	UAUUGGAGG	98	GCAGUCCCCAA	171	85.01	5.13	94.65	6.48
			UUGGGGACU		CCUCCAAUA
			GCGA
340	319	339	UUGAGUGAU	99	CCCAACCUCCAA	172	92.21	3.91	116.88	2.45
			UGGAGGUUG		UCACUCAA
			GGGA
364	343	363	UACAAGUUG	100	CUCCUGUCCUC	173	93.49	12.78	110.63	12.30
			GAGGACAGG		CAACUUGUA
			AGGU
367	346	366	UAGGACAAG	65	CUGUCCUCCAA	82	54.31	5.46	104.26	8.94
			UUGGAGGAC		CUUGUCCUA
			AGGA
372	351	371	UUAACCAGGA	101	CUCCAACUUGU	174	90.12	8.20	103.42	3.82
			CAAGUUGGA		CCUGGUUAA
			GGA
383	362	382	UACAUCCAGC	102	CCUGGUUAUCG	175	100.80	9.64	100.32	2.17
			GAUAACCAGG		CUGGAUGUA
			AC
387	366	386	UAGACACAUC	103	GUUAUCGCUGG	176	92.42	10.80	94.19	1.76
			CAGCGAUAAC		AUGUGUCUA
			CA
397	376	396	UAAAACGCCG	104	GAUGUGUCUGC	177	79.42	7.65	109.97	6.38
			CAGACACAUC		GGCGUUUUA
			CA
402	381	401	UAUGAUAAA	105	GUCUGCGGCGU	178	102.01	8.47	105.05	0.57
			ACGCCGCAGA		UUUAUCAUA
			CAC
404	383	403	UAGAUGAUA	106	CUGCGGCGUUU	179	97.94	7.76	118.51	4.20
			AAACGCCGCA		UAUCAUCUA
			GAC
408	387	407	UAGGAAGAU	107	GGCGUUUUAUC	180	81.54	1.23	93.80	5.89
			GAUAAAACGC		AUCUUCCUA
			CGC
410	389	409	UAGAGGAAG	7	CGUUUUAUCAU	32	41.63	2.07	92.34	3.23
			AUGAUAAAAC		CUUCCUCUA
			GCC
411	390	410	UAAGAGGAA	66	GUUUUAUCAUC	83	51.50	6.63	93.61	5.63
			GAUGAUAAA		UUCCUCUUA
			ACGC
425	404	424	UAUAGCAGCA	8	CUCUUCAUCCU	33	42.50	3.29	85.64	3.41
			GGAUGAAGA		GCUGCUAUA
			GGA
434	413	433	UAGAUGAGG	108	CUGCUGCUAUG	181	78.96	0.93	109.80	2.31
			CAUAGCAGCA		CCUCAUCUA
			GGA
437	416	436	UAGAAGAUG	9	CUGCUAUGCCU	34	52.79	9.88	107.18	12.95
			AGGCAUAGCA		CAUCUUCUA
			GCA
440	419	439	UACAAGAAG	10	CUAUGCCUCAU	35	41.07	4.74	69.99	1.12
			AUGAGGCAU		CUUCUUGUA
			AGCA
444	423	443	UACCAACAAG	109	GCCUCAUCUUC	182	79.53	3.92	88.18	5.52
			AAGAUGAGG		UUGUUGGUA
			CAU
445	424	444	UAACCAACAA	110	CCUCAUCUUCU	183	83.87	2.45	89.63	6.16
			GAAGAUGAG		UGUUGGUUA
			GCA
448	427	447	UAAGAACCAA	113	CAUCUUCUUGU	184	94.96	2.74	106.86	5.63
			CAAGAAGAUG		UGGUUCUUA
			AG
457	436	456	UAUAGUCCA	112	GUUGGUUCUU	185	90.71	6.69	100.24	4.21
			GAAGAACCAA		CUGGACUAUA
			CAA
460	439	459	UUUGAUAGU	113	GGUUCUUCUG	186	88.01	3.55	117.80	10.95
			CCAGAAGAAC		GACUAUCAAA
			CAA
464	443	463	UUACCUUGA	114	CUUCUGGACUA	187	99.21	3.43	96.09	4.11
			UAGUCCAGAA		UCAAGGUAA
			GAA
467	446	466	UACAUACCUU	115	CUGGACUAUCA	188	97.83	8.96	95.78	8.06
			GAUAGUCCA		AGGUAUGUA
			GAA
484	463	483	UUAGAGGAC	116	GUUGCCCGUUU	189	94.96	3.73	91.26	2.75
			AAACGGGCAA		GUCCUCUAA
			CAU
487	466	486	UAAUUAGAG	117	GCCCGUUUGUC	190	79.45	5.80	110.56	3.30
			GACAAACGGG		CUCUAAUUA
			CAA
490	469	489	UUGGAAUUA	118	CGUUUGUCCUC	191	76.64	1.78	92.04	3.19
			GAGGACAAAC		UAAUUCCAA
			GGG
500	479	499	UUUGAGGAU	119	CUAAUUCCAGG	192	79.71	4.83	72.39	0.45
			CCUGGAAUU		AUCCUCAAA
			AGAG
533	512	532	UUCAUGCAG	120	CCAUGCCGAAC	193	92.73	5.02	94.03	6.63
			GUUCGGCAU		CUGCAUGAA
			GGUC
538	517	537	UAGUAGUCA	121	CCGAACCUGCA	194	92.77	1.43	100.34	14.51
			UGCAGGUUC		UGACUACUA
			GGCA
544	523	543	UUUGAGCAG	122	CUGCAUGACUA	195	98.04	1.11	89.79	5.49
			UAGUCAUGC		CUGCUCAAA
			AGGU
547	526	546	UUCCUUGAG	123	CAUGACUACUG	196	99.41	7.16	99.49	1.56
			CAGUAGUCA		CUCAAGGAA
			UGCA
555	534	554	UAUAGAGGU	124	CUGCUCAAGGA	197	85.83	3.11	94.87	3.59
			UCCUUGAGCA		ACCUCUAUA
			GUA
557	536	556	UACAUAGAG	125	GCUCAAGGAAC	198	76.27	0.18	114.68	1.71
			GUUCCUUGA		CUCUAUGUA
			GCAG
558	537	557	UUACAUAGA	126	CUCAAGGAACC	199	58.62	0.55	76.33	1.07
			GGUUCCUUG		UCUAUGUAA
			AGCA
563	542	562	UAGGGAUAC	127	GGAACCUCUAU	200	62.83	2.19	97.26	3.37
			AUAGAGGUU		GUAUCCCUA
			CCUU
574	553	573	UACAGCAACA	128	GUAUCCCUCCU	201	74.75	4.76	98.29	3.48
			GGAGGGAUA		GUUGCUGUA
			CAU
578	557	577	UUGGUACAG	129	CCCUCCUGUUG	202	72.88	0.77	118.68	2.44
			CAACAGGAGG		CUGUACCAA
			GAU
579	558	578	UUUGGUACA	22	CCUCCUGUUGC	47	75.27	2.58	78.70	6.57
			GCAACAGGAG		UGUACCAAA
			GGA
580	559	579	UUUUGGUAC	130	CUCCUGUUGCU	203	76.59	2.16	90.66	5.18
			AGCAACAGGA
			GGG		GUACCAAAA
583	562	582	UAGGUUUGG	131	CUGUUGCUGUA	204	94.04	3.71	98.30	4.16
			UACAGCAACA		CCAAACCUA
			GGA
594	573	593	UUUUCCGUCC	132	CCAAACCUUCG	205	92.35	4.49	112.63	4.21
			GAAGGUUUG		GACGGAAAA
			GUA
595	574	594	UAUUUCCGU	133	CAAACCUUCGG	206	101.14	11.62	98.30	5.20
			CCGAAGGUU		ACGGAAAUA
			UGGU
599	578	598	UUGCAAUUU	67	CCUUCGGACGG	84	83.80	11.70	90.14	7.03
			CCGUCCGAAG		AAAUUGCAA
			GUU
605	584	604	UUACAGGUG	134	GACGGAAAUUG	207	95.66	8.55	90.76	7.03
			CAAUUUCCGU		CACCUGUAA
			CCG
607	586	606	UAAUACAGG	135	CGGAAAUUGCA	208	95.58	1.27	122.99	4.36
			UGCAAUUUCC		CCUGUAUUA
			GUC
636	615	635	UUUUCCGAA	136	CAUCCUGGGCU	209	84.74	8.56	84.70	5.27
			AGCCCAGGAU		UUCGGAAAA
			GAU
639	618	638	UAAUUUUCC	137	CCUGGGCUUUC	210	86.23	5.99	103.02	5.02
			GAAAGCCCAG		GGAAAAUUA
			GAU
642	621	641	UAGGAAUUU	11	GGGCUUUCGGA	36	51.00	4.04	83.21	4.86
			UCCGAAAGCC		AAAUUCCUA
			CAG
643	622	642	UUAGGAAUU	12	GGCUUUCGGAA	37	54.52	2.45	129.50	3.15
			UUCCGAAAGC		AAUUCCUAA
			CCA
644	623	643	UAUAGGAAU	138	GCUUUCGGAAA	211	89.20	4.71	102.88	4.71
			UUUCCGAAA		AUUCCUAUA
			GCCC
671	650	670	UAGGAGAAAC	139	GCCUCAGCCCG	212	100.61	4.50	99.43	5.52
			GGGCUGAGG		UUUCUCCUA
			CCC
680	659	679	UAACUGAGCC	140	CGUUUCUCCUG	213	77.12	5.07	94.85	5.83
			AGGAGAAACG		GCUCAGUUA
			GG
681	660	680	UAAACUGAGC	141	GUUUCUCCUGG	214	89.88	5.86	121.84	5.26
			CAGGAGAAAC		CUCAGUUUA
			GG
685	664	684	UUAGUAAAC	142	CUCCUGGCUCA	215	79.36	2.20	86.48	8.36
			UGAGCCAGGA		GUUUACUAA
			GAA
687	666	686	UACUAGUAA	13	CCUGGCUCAGU	38	40.97	5.76	84.81	6.48
			ACUGAGCCAG		UUACUAGUA
			GAG
694	673	693	UAAAUGGCAC	143	CAGUUUACUAG	216	76.23	5.35	91.84	7.29
			UAGUAAACU		UGCCAUUUA
			GAG
696	675	695	UACAAAUGGC	144	GUUUACUAGU	217	75.58	10.39	116.68	2.52
			ACUAGUAAAC		GCCAUUUGUA
			UG
701	680	700	UACUGAACAA	145	CUAGUGCCAUU	218	68.82	1.49	82.14	1.98
			AUGGCACUA		UGUUCAGUA
			GUA
728	707	727	UAAACAGUG	146	GGGCUUUCCCC	219	61.44	4.89	87.99	3.38
			GGGGAAAGCC		CACUGUUUA
			CUA
736	715	735	UUGAAAGCCA	147	CCCCACUGUUU	220	62.17	2.97	89.88	3.41
			AACAGUGGG		GGCUUUCAA
			GGA
738	717	737	UACUGAAAGC	148	CCACUGUUUGG	221	79.85	6.75	94.63	0.81
			CAAACAGUGG		CUUUCAGUA
			GG
739	718	738	UAACUGAAAG	149	CACUGUUUGGC	222	73.89	9.39	97.41	12.38
			CCAAACAGUG		UUUCAGUUA
			GG
741	720	740	UAUAACUGA	23	CUGUUUGGCU	48	57.74	11.19	79.34	1.97
			AAGCCAAACA		UUCAGUUAUA
			GUG
743	722	742	UAUAUAACU	150	GUUUGGCUUU	223	53.83	5.22	80.71	1.82
			GAAAGCCAAA		CAGUUAUAUA
			CAG
747	726	746	UAUCCAUAUA	151	GGCUUUCAGUU	224	96.02	10.16	93.37	1.62
			ACUGAAAGCC		AUAUGGAUA
			AA
749	728	748	UUCAUCCAUA	152	CUUUCAGUUAU	225	77.64	3.91	80.70	3.57
			UAACUGAAAG		AUGGAUGAA
			CC
755	734	754	UACCACAUCA	24	GUUAUAUGGA	49	58.16	8.87	56.22	3.76
			UCCAUAUAAC		UGAUGUGGUA
			UG
771	750	770	UAGACUUGG	153	GGUAUUGGGG	226	80.28	3.46	88.29	2.56
			CCCCCAAUAC		GCCAAGUCUA
			CAC
779	758	778	UUGCUGUAC	154	GGGCCAAGUCU	227	74.44	7.08	85.71	1.74
			AGACUUGGCC		GUACAGCAA
			CCC
780	759	779	UAUGCUGUA	155	GGCCAAGUCUG	228	102.79	12.84	91.66	9.21
			CAGACUUGGC		UACAGCAUA
			CCC
782	761	781	UAGAUGCUG	156	CCAAGUCUGUA	229	90.64	7.48	93.48	5.15
			UACAGACUU		CAGCAUCUA
			GGCC
783	762	782	UAAGAUGCU	68	CAAGUCUGUAC	85	52.68	3.27	97.86	4.02
			GUACAGACU		AGCAUCUUA
			UGGC
793	772	792	UAAAGGGAC	157	CAGCAUCUUGA	230	90.60	9.66	121.66	5.37
			UCAAGAUGC		GUCCCUUUA
			UGUA
795	774	794	UAAAAAGGG	25	GCAUCUUGAG	50	44.10	5.42	65.12	1.10
			ACUCAAGAU		UCCCUUUUUA
			GCUG
796	775	795	UUAAAAAGG	14	CAUCUUGAGUC	39	42.36	5.16	67.89	1.35
			GACUCAAGA		CCUUUUUAA
			UGCU
808	787	807	UUGGUAACA	26	CUUUUUACCGC	51	32.99	0.68	61.83	5.34
			GCGGUAAAA		UGUUACCAA
			AGGG
815	794	814	UAGAAAAUU	15	CCGCUGUUACC	40	14.45	1.88	41.53	0.70
			GGUAACAGC		AAUUUUCUA
			GGUA
816	795	815	UAAGAAAAU	16	CGCUGUUACCA	41	21.65	3.28	50.26	5.26
			UGGUAACAG		AUUUUCUUA
			CGGU
817	796	816	UAAAGAAAA	17	GCUGUUACCAA	42	11.33	1.67	21.08	2.57
			UUGGUAACA		UUUUCUUUA
			GCGG
818	797	817	UAAAAGAAA	18	CUGUUACCAAU	43	17.62	4.23	15.82	1.88
			AUUGGUAAC		UUUCUUUUA
			AGCG
820	799	819	UACAAAAGA	19	GUUACCAAUUU	44	12.13	2.31	25.85	1.76
			AAAUUGGUA		UCUUUUGUA
			ACAG
824	803	823	UAAAGACAA	20	CCAAUUUUCUU	45	20.18	4.11	35.61	2.74
			AAGAAAAUU		UUGUCUUUA
			GGUA
837	816	836	UUAAAUGUA	158	GUCUUUGGGU	231	82.36	7.65	80.11	3.64
			UACCCAAAGA		AUACAUUUAA
			CAA

TABLE 3
Exemplary Sequences of the Present Application
and Their HBV mRNA Potency
Guide							% of gene	% of gene
Strand			Guide		Passenger		remaining	remaining
posi-			strand		strand		at	at
tion			sequence	SEQ	sequence	SEQ	0.5 nM	0.05 nM
3′	Start	End	(5′-3′)	ID	(5′-3′)	ID	Mean	SEM	Mean	SEM
178	157	177	UUCCUGA	52	GGAGAACAUCACAU	69	68.17	2.39	83.13	2.18
			UGUGAUG		CAGGAA
			UUCUCCAU
179	158	178	UAUCCUG	2	GAGAACAUCACAUC	27	59.35	6.35	98.17	4.52
			AUGUGAU		AGGAUA
			GUUCUCCA
184	163	183	UUAGGAA	3	CAUCACAUCAGGAU	28	46.05	4.05	89.70	1.06
			UCCUGAU		UCCUAA
			GUGAUGU
			U
204	183	203	UUGUAAC	4	GACCCCUUCUCGUG	29	75.00	7.55	105.93	4.38
			ACGAGAAG		UUACAA
			GGGUCCU
224	203	223	UUCAACAA	5	GCGGGGUUUUUCU	30	72.47	10.24	94.00	6.74
			GAAAAACC		UGUUGAA
			CCGCCU
226	205	225	UUGUCAA	55	GGGGUUUUUCUUG	72	62.60	4.70	109.57	13.22
			CAAGAAAA		UUGACAA
			ACCCCGC
227	206	226	UUUGUCA	21	GGGUUUUUCUUGU	46	65.27	3.58	87.50	3.41
			ACAAGAAA		UGACAAA
			AACCCCG
217	196	216	UUUGUGA	53	UUGACAAAAAUCC	70	51.37	0.76	75.30	3.55
			GGAUUUU		UCACAAA
			UGUCAAG
			G
219	198	218	UUAUUGU	54	GACAAAAAUCCUCA	71	55.90	2.08	85.70	5.42
			GAGGAUU		CAAUAA
			UUUGUCG
			G
241	220	240	UUAUUGU	6	GACAAGAAUCCUCA	31	62.90	10.56	116.03	6.51
			GAGGAUU
			CUUGUCA
			A		CAAUAA
254	233	253	UCUGAGA	56	CGUGGUGGACUUC	73	75.40	4.10	96.47	8.32
			GAAGUCCA		UCUCAGA
			CCACGGG
276	255	275	UUUGAGA	59	CGUGGUGGACUUC	76	68.00	3.29	99.37	8.68
			GAAGUCCA		UCUCAAA
			CCACGAG
255	234	254	UACUGAG	57	GUGGUGGACUUCU	74	76.57	2.16	97.33	2.64
			AGAAGUCC		CUCAGUA
			ACCACGG
258	237	257	UAAAACU	58	GUGGACUUCUCUCA	75	92.87	5.00	109.13	0.79
			GAGAGAA		GUUUUA
			GUCCACGG
282	261	281	UAGAAAA	60	GGACUUCUCUCAAU	77	93.20	0.58	95.47	3.73
			UUGAGAG		UUUCUA
			AAGUCCAC
283	262	282	UUAGAAA	61	GACUUCUCUCAAUU	78	86.33	3.06	94.53	4.12
			AUUGAGA		UUCUAA
			GAAGUCCA
304	283	303	UAAGACAC	62	GGGAACUACCGUGU	79	80.17	5.14	97.13	2.15
			ACGGUAG		GUCUUA
			UUCCCCC
312	291	311	UUUUUGG	63	CCGUGUGUCUUGG	80	73.63	2.38	84.23	6.10
			CCAAGACA		CCAAAAA
			CACGGUA
318	297	317	UUGCGAA	64	GUCUUGGCCAAAAU	81	86.63	5.45	101.57	3.32
			UUUUGGC		UCGCAA
			CAAGACAC
367	346	366	UAGGACA	65	CUGUCCUCCAACUU	82	69.87	7.97	90.63	7.27
			AGUUGGA		GUCCUA
			GGACAGG
			A
410	389	409	UAGAGGA	7	CGUUUUAUCAUCU	32	62.03	2.86	88.70	7.81
			AGAUGAU		UCCUCUA
			AAAACGCC
411	390	410	UAAGAGG	66	GUUUUAUCAUCUU	83	75.60	1.97	90.97	5.99
			AAGAUGA		CCUCUUA
			UAAAACGC
425	404	424	UAUAGCA	8	CUCUUCAUCCUGCU	33	65.60	1.39	96.97	5.04
			GCAGGAU		GCUAUA
			GAAGAGG
			A
437	416	436	UAGAAGA	9	CUGCUAUGCCUCAU	34	41.30	6.48	63.17	6.08
			UGAGGCA		CUUCUA
			UAGCAGC
			A
440	419	439	UACAAGA	10	CUAUGCCUCAUCUU	35	44.75	4.35	70.27	11.74
			AGAUGAG		CUUGUA
			GCAUAGC
			A
579	558	578	UUUGGUA	22	CCUCCUGUUGCUG	47	55.20	3.35	73.30	4.04
			CAGCAACA		UACCAAA
			GGAGGGA
599	578	598	UUGCAAU	67	CCUUCGGACGGAAA	84	54.60	3.10	68.83	3.22
			UUCCGUCC		UUGCAA
			GAAGGUU
642	621	641	UAGGAAU	11	GGGCUUUCGGAAA	36	36.67	3.72	60.30	2.73
			UUUCCGA		AUUCCUA
			AAGCCCAG
643	622	642	UUAGGAA	12	GGCUUUCGGAAAA	37	46.47	4.74	69.03	3.92
			UUUUCCG		UUCCUAA
			AAAGCCCA
687	666	686	UACUAGU	13	CCUGGCUCAGUUU	38	39.33	2.25	62.93	4.23
			AAACUGA		ACUAGUA
			GCCAGGA
			G
741	720	740	UAUAACU	23	CUGUUUGGCUUUC	48	32.40	1.62	54.27	1.56
			GAAAGCC		AGUUAUA
			AAACAGU
			G
755	734	754	UACCACAU	24	GUUAUAUGGAUGA	49	50.90	5.22	72.57	1.27
			CAUCCAUA		UGUGGUA
			UAACUG
783	762	782	UAAGAUG	68	CAAGUCUGUACAGC	85	69.63	4.80	94.47	6.33
			CUGUACA		AUCUUA
			GACUUGG
			C
795	774	794	UAAAAAG	25	GCAUCUUGAGUCCC	50	92.83	21.39	105.97	10.55
			GGACUCAA		UUUUUA
			GAUGCUG
796	775	795	UUAAAAA	14	CAUCUUGAGUCCCU	39	71.17	1.50	101.70	7.20
			GGGACUC		UUUUAA
			AAGAUGC
			U
808	787	807	UUGGUAA	26	CUUUUUACCGCUG	51	50.43	2.40	90.33	8.00
			CAGCGGU		UUACCAA
			AAAAAGG
			G
815	794	814	UAGAAAA	15	CCGCUGUUACCAAU	40	61.97	1.85	96.73	5.82
			UUGGUAA		UUUCUA
			CAGCGGU
			A
816	795	815	UAAGAAAA	16	CGCUGUUACCAAUU	41	57.73	1.83	84.27	3.54
			UUGGUAA		UUCUUA
			CAGCGGU
817	796	816	UAAAGAA	17	GCUGUUACCAAUU	42	56.23	3.17	76.90	6.33
			AAUUGGU		UUCUUUA
			AACAGCG
			G
818	797	817	UAAAAGA	18	CUGUUACCAAUUU	43	35.03	2.35	55.67	3.17
			AAAUUGG		UCUUUUA
			UAACAGC
			G
820	799	819	UACAAAA	19	GUUACCAAUUUUC	44	44.27	2.31	65.90	2.37
			GAAAAUU		UUUUGUA
			GGUAACA
			G
824	803	823	UAAAGAC	20	CCAAUUUUCUUUU	45	37.67	3.34	64.67	3.71
			AAAAGAA		GUCUUUA
			AAUUGGU
			A

TABLE 4
Exemplary Modified Sequences of the Disclosure
		SEQ ID
—	Sequence	NO:
G (5′-3′)	[mUs][fAs][fU][mC][fC][mU][fG][mA][mU][fG][mU][mG][mA][fU][mG][fU][mU]	SEQ ID NO:
	[mC][mU][mCs][mCs][mA]	232
P (5′-3′)	[mGs][mAs][mG][mA][mA][fC][mA][fU][fC][fA][fC][mA][mU][mC][mA][mG]	SEQ ID NO:
	[mG][mAs][mUs][mA][G1b][G1b][G1b]	242
G (5′-3′)	[mUs][fUs][fA][mG][fG][mA][fA][mU][mC][fC][mU][mG][mA][fU][mG][fU][mG]	SEQ ID NO:
	[mA][mU][mGs][mUs][mU]	233
P (5′-3′)	[mCs][mAs][mU][mC][mA][fC][mA][fU][fC][fA][fG][mG][mA][mU][mU][mC]	SEQ ID NO:
	[mC][mUs][mAs][mA][G1b][G1b][G1b]	243
G (5′-3)	[mUs][fUs][fC][mA][fA][mC][fA][mA][mG][fA][mA][mA][mA][fA][mC][fC][mC]	SEQ ID NO:
	[mC][mG][mCs][mCs][mU]	234
P (5′-3′)	[mGs][mCs][mG][mG][mG][fG][mU][fU][fU][fU][fU][mC][mU][mU][mG][mU]	SEQ ID NO:
	[mU][mGs][mAs][mA][G1b][G1b][G1b]	244
G (5′-3′)	[mUs][fUs][fU][mG][fU][mC][fA][mA][mC][fA][mA][mG][mA][fA][mA][fA][mA]	SEQ ID NO:
	[mC][mC][mCs][mCs][mG]	235
P (5′-3′)	[mGs][mGs][mG][mU][mU][fU][mU][fU] [fC][fU][fU][mG][mU][mU][mG][mA]	SEQ ID NO:
	[mC][mAs][mAs][mA][G1b][G1b][G1b]	245
G (5′-3′)	[mUs][fUs][fA][mU][fU][mG][fU][mG][mA][fG][mG][mA][mU][fU][mC][fU][mU]	SEQ ID NO:
	[mG][mU][mCs][mAs][mA]	236
P (5′-3′)	[mGs][mAs][mC][mA][mA][fG][mA][fA][fU][fC][fC][mU][mC][mA][mC][mA]	SEQ ID NO:
	[mA][mUs][mAs][mA][G1b][G1b][G1b]	246
G (5′-3′)	[mUs][fUs][fA][mG][fG][mA][fA][mU][mU][fU][mU][mC][mC][fG][mA][fA][mA]	SEQ ID NO:
	[mG][mC][mCs][mCs][mA]	237
P (5′-3′)	[mGs][mGs][mC][mU][mU][fU][mC][fG][fG][fA][fA][mA][mA][mU][mU][mC]	SEQ ID NO:
	[mC][mUs][mAs][mA][G1b][G1b][G1b]	247
G (5′-3′)	[mUs][fAs][fU][mA][fA][mC][fU][mG][mA][fA][mA][mG][mC][fC][mA][fA][mA]	SEQ ID NO:
	[mC][mA][mGs][mUs][mG]	238
P(5′-3′)	[mCs][mUs][mG][mU][mU][fU][mG][fG][fC][fU][fU][mU][mC][mA][mG][mU]	SEQ ID NO:
	[mU][mAs][mUs][mA][G1b][G1b][G1b]	248
G (5′-3′)	[mUs][fAs][fG][mA][fA][mA][fA][mU][mU][fG][mG][mU][mA][fA][mC][fA][mG]	SEQ ID NO:
	[mC][mG][mGs][mUs][mA]	239
P (5′-3′)	[mCs][mCs][mG][mC][mU][fG][mU][fU][fA][fC][fC][mA][mA][mU][mU][mU]	SEQ ID NO:
	[mU][mCs][mUs][mA][G1b][G1b][G1b]	249
G (5′-3′)	[mUs][fAs][fA][mA][fA][mG][fA][mA][mA][fA][mU][mU][mG][fG][mU][fA][mA]	SEQ ID NO:
	[mC][mA][mGs][mCs][mG]	240
P (5′-3′)	[mCs][mUs][mG][mU][mU][fA][mC][fC][fA][fA][fU][mU][mU][mU][mC][mU]	SEQ ID NO:
	[mU][mUs][mUs][mA][G1b][G1b][G1b]	250
G (5′-3′)	[mUs][fAs][fC][mA][fA][mA][fA][mG][mA][fA][mA][mA][mU][fU][mG][fG][mU]	SEQ ID NO:
	[mA][mA][mCs][mAs][mG]	241
P (5′-3′)	[mGs][mUs][mU][mA][mC][fC][mA][fA][fU][fU][fU][mU][mC][mU][mU][mU]	SEQ ID NO:
	[mU][mGs][mUs][mA][G1b][G1b][G1b]	251
G: guide strand, P: passenger strand
“m” indicates 2′-O-methyl modification, “f” indicates a 2′-F modification, “s” is a phosphorothioate internucleotide linkage, and “Glb” is a GalNAc Glb moiety (shown below).

EXAMPLES

Example 1: Design and Efficacy of siRNA Compounds Against Hepatitis B Virus (HBV) mRNA

A set of 115 siRNA compounds against hepatitis B virus (HBV) transcript (GenBank Accession No: U95551.1) were designed (see Table 2).

Oligonucleotide 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,

In Vitro Screening

The compounds were diluted into the desired concentration with PBS. The diluted compounds were then transfected into the cultured HepG2.2.15 cells (transfected with HBV plasmid) with Lipofectamine RNAi AX (invitrogen-13778-150) reagents on Day 0. Each compound was tested at concentrations of 0.05 nM and 0.01 nM. After 72 hours incubation post-transfection on Day 3, the culture medium was refreshed. On Day 8, extracellular Hepatitis B surface antigen (HBsAg) in cell culture was tested by ELISA to analyze the compound potency in silencing HBsAg expression, or mRNA was extracted from the transfected cells using RNeasy 96 kit (Qiagen-74182).

The percentage of HBsAg remaining in culture media of HepG2.2.15 cells relative to mock transfection was determined for each compound at a concentration of either 0.05 nM and 0.01 nM. The results identified several compounds that were able to reduce the level of HBsAg at least 50% or more at 0.05 nM and at least 20% or more at 0.01 nM as described in Table 2 and FIG. 1 and FIG. 2. Also, several compounds shown in bold in Table 2 were able to reduce the level of HBsAg in transfected cells by at least 20% at 0.05 nM and 0.01 nM (see Table 2, and FIG. 1 and FIG. 2).

The percentage of hepatitis B virus mRNA remaining in HepG2.2.15 cells relative to mock-transfection when normalized to Gapdh mRNA levels was determined for a subset of compounds from Table 2 at a concentration of 0.5 nM and 0.05 nM. The results identified several compounds that were able to reduce the level of HBV mRNA in transfected cells by 20% to 50% or more than 50% at the defined concentrations as described in Table 3 and FIG. 3 and FIG. 4. Several compounds (shown in bold in Table 3) were able to reduce the level of HBV mRNA in transfected cells by 20% to 50% or at least 50% at 0.05 nM and 0.5 nM (see Table 3, and FIG. 3 and FIG. 4).

In summary, the present disclosure identifies numerous siRNA compounds that reduce the level of hepatitis B HBsAg in target cells when administered at a dosage of 0.05 nM or 0.01 nM, or both concentrations. The present disclosure also identifies numerous siRNA compounds that reduce the level of HBV mRNA in target cells when administered at a dosage of 0.5 nM or 0.05 nM, or at both concentrations.

For a dose-response analysis, a subset of compounds selected from Table 4 were tested at a dose of 1 nM, 0.5 nM, 0.2 nM, and 0.01 nM. All compounds tested were able to reduce the level of HBsAg in HepG2. 15 cells that transiently express HBV genotype B (FIG. 15). In addition, a subset of compounds listed in Table 4 tested at a dose of 1 nM and 0.01 nM were able to reduce the level of HBsAg in HepG2.15 cells which transiently express HBV genotype C (FIG. 16). FIG. 17 shows the potent dose-response of a subset of compounds from Table 4 to effectively reduce the level of HBsAg in HepG2.15 cells that transiently express HBV genotype C.

In Vivo HDI Liver Screening

A subset of compounds with from Table 2 were formulated in 1×PBS dosed on day 1 through subcutaneous dosing to BALB/c female animals (6˜8 weeks old). Female BALB/c mice (6-7 weeks old) mice were used in the HDI liver screening experiments. Day 0 was defined as the day on which the animals were hydrodynamically injected with pAAV2-HBV1.3mer plasmid DNA. pAAV2-HBV1.3mer plasmid DNA was prepared in normal saline and stored at 4° C. until injection. All mice were hydrodynamically injected through tail vein with 10 μg pAAV2-HBV1.3mer plasmid DNA in a volume of normal saline equivalent to 8% of a mouse body weight within 5 seconds. PBS or siRNA compounds were dosed through subcutaneous routes on Day 1. Blood samples were collected submandibular on desired time points. Mice were sacrificed on Day 5 and bled vis cardiac puncture to prepare plasma samples for detection of HBsAg and pregenomic RNA (pgRNA). mRNA samples were collected from liver and test by RT-qPCR.

RT-qPCR Analysis of Liver pgRNA and 2.1 kb/2.4 kb/35 kb mRNA

In brief, the liver tissues were homogenized by the Qiagen Tissue Lyser with TRIZOL. The aqueous phase was collected and extracted twice with chloroform. RNA was precipitated with isopropanol and dissolved in DEPC-treated H₂O. mRNA samples were reverse transcribed into cDNA using FastQuant RT Kit (with gDNase). TaqMAN assays were used to quantify the liver pgRNA, and HBV 2.1 kb/2.4 kb/3.5 kb mRNA.

For dose response study, BALB/c female animals (6-8 weeks old) were dosed subcutaneously at 1 mg/kg with a subset of compounds from Table 2. The control animals were dosed with PBS. The tested compounds were able to reduce plasma HBsAg (FIG. 5) and were able to reduce pgRNA levels in HDI mouse liver (FIG. 6). In addition, the tested compounds were able to reduce HBV 2.1 kb/2.4 kb/3.5 kb+pgRNA levels (FIG. 7) in HDI mouse liver.

In addition, BALB/c female animals (6-8 weeks old) were dosed subcutaneously either at 1 mg/kg (black squares), 3 mg/kg (grey squares), or 6 mg/kg (empty squares) (FIG. 8 and FIG. 9). The control animals were dosed with PBS. The tested compounds were able to reduce plasma HBsAg (FIG. 8) at the three doses tested and were able to reduce pgRNA levels in HDI mouse liver (FIG. 9) at the three doses tested.

Compound Efficacy Test in AAV-HBV Model

C₅₇BL/6 mice (5 weeks old), specific pathogen free, were allowed to acclimate to the new environment for 8 days before the study. The stock rAAV8-1.3HBV genotype D virus of 1×10² viral genome (v.g.)/mL was diluted to 1×10¹¹ v.g./mL with sterile PBS. 2×10¹¹ v.g. AAV/HBV in 200 μL was injected per mouse on Day −24 before dosing of the siRNA compounds. On Days −10 and −3 (before dosing), all 53 mice infected with rAAV8-1.3HBV were submandibular bled (˜100 μL/mouse) for plasma preparation. The blood samples were collected and anti-coagulated with 1K2-EDTA, centrifuged at 4°, 7000 g for 10 minutes for plasma collection. The plasma samples were stored at −80° C. until they were transferred to the in vitro group for determination of HBV DNA, HBsAg and HBeAg level. Day 0 was defined as the day the mice were first dosed with vehicle or a subset of compounds from Table 4.

FIG. 10 shows the efficacy of compounds selected from Table 4 in reducing plasma HBsAg at 3 mg/kg, as compared to PBS control, FIG. 11 shows the efficacy of compounds selected from Table 4 in reducing plasma Hepatitis B e antigen (HBeAg) at 3 mg/kg over time (Day 0 to Day 77), as compared to PBS control. FIG. 12 shows the efficacy of compounds selected from Table 4 in reducing HBV DNA at 3 mg/kg over time (Day 0 to Day 77), compared to PBS control. In addition, FIG. 13 shows the efficacy of compounds selected from Table 4 in reducing HBV total RNA (FIG. 13A), HBV 3.5 kB PNA (FIG. 13B), and HBV DNA copy number (FIG. 13C) in HBV-genotype-D AAV nice at Day 77 at 3 mg/kg.

Liver Total DNA Analysis by qPCR

Total DNA from AAV-HBV mouse liver was isolated using 1N easy Blood&Tissue kit (Invitrogen-15596018 The standard curve DNA of 107 copies/μL was 60-Fold dilution from 5 ng/μL pAAV2-HBV1.3 plasmid, and 10-fold serial dilution with AE buffer from 10⁷ to 10 copies/μL, 2 μL DNA standard curve DNA was added to corresponding wells. HBV primer sets are used to quantify both the HBV virus sequences and AAV/HBV vector sequences, and pAAV primer sets are used to quantify only the remaining AAV/HBV vector sequences in liver tissue.

HBsAg in HDI Mouse Plasma

HBsAg levels from HDI mice plasma were determined using HBsAg ELISA kit (Autobio-CL0310) following the manual. In brief, 50 μL of 100-fold diluted samples, standard or control samples were added to ELISA plates, and followed by addition of 50 μL of enzyme conjugate. After incubated at 37° C. for 1 hr, the plates were washed 5 times, and 50 μL of enzyme substrate was added to each well, followed by incubation at room temperature for another 10 min. The chemiluminescent values were read by Microplate reader SpectraMax ID3 (Molecular Devices).

Detection of HBeAg Levels in AAV-HBV Mice Plasma by ELISA

AAV-HBV mouse plasma samples were diluted for 60-fold (2 μL plasma+118 μL PBS). 50 μL of the 60-fold diluted plasma was used for determination of HBeAg using the HBeAg ELISA kit (Autobio, CL 0312) following the manual. In brief, 60-fold diluted plasma samples were incubated with enzyme conjugate in the well for 1 hr. After being washed 4 times, chemiluminescent substrate was added to each well and incubated for 10 min protected from light. Then the chemiluminescent values were read by a microplate reader.

Measurement of HBsAg in AAV-HBV Mice Plasma by ELISA

Mouse plasma samples were first diluted for 60-fold (2 μL plasma+118 μL PBS), then further diluted for 20-fold (3 μL 60-fold diluted plasma+57 μL PBS). 50 μL of the 1200-fold diluted plasma was used for determination of HBsAg levels using the HBsAg ELISA kit (Autobio, CL 0310) following the manual. In brief, 1200-fold diluted plasma samples were incubated with enzyme conjugate for 1 hr. After 4 times of wash, chemiluminescent substrate was added to each well and incubated for 10 min protected from light. Then the chemiluminescent values were read with a microplate reader.

Detection of ALT and AST in Plasma by Biochemical Assay

ALT and AST levels in plasma samples were detected using the Alanine Aminotransferase Activity Assay Kit (Sigma, MAK052) according to the manual, Automatic biochemical analyzer-AU480 was used for ALT and AST detection. FIG. 14 shows the plasma ALT and AST levels in AAV-HBV mice on Day 7 and Day 28 post dosing of compounds selected from Table 4.

Additional embodiments of the disclosure include the following:

Embodiment 1. 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) 158 to 240;
  • b) 389 to 439;
  • c)⁵58 to 578; and
  • d) 621 to 823,from the 5′ end of a hepatitis B virus (HBV) 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 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) 158 to 240;
  • b) 389 to 439;
  • c) 558 to 578; and
  • d) 621 to 823,from the 5′ end of a hepatitis B virus (HBV) 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) 158 to 240;
  • b) 389 to 439;
  • c)⁵58 to 578; and
  • d) 621 to 823,from the 5′ end of a hepatitis B virus (HBV) 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) 158 to 240;
  • b) 389 to 439;
  • c) 621 to 686; and
  • d) 775 to 823,from the 5′ end of a hepatitis B virus (HBV) 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) 158 to 240;
  • b) 389 to 439;
  • c) 621 to 686; and
  • d) 775 to 823,from the 5′ end of a hepatitis B virus (HBV) 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) 158 to 240;
  • b) 389 to 439;
  • c) 621 to 686; and
  • d) 775 to 823,from the 5′ end of a hepatitis B virus (HBV) 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) 206 to 226;
  • b) 558 to 578; and
  • c) 720 to 807,from the 5′ end of a hepatitis B virus (HBV) 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) 206 to 226;
  • b) 558 to 578; and
  • c) 720 to 807,from the 5′ end of a hepatitis B virus (HBV) 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) 206 to 226;
  • b) 558 to 578; and
  • c) 720 to 807,from the 5′ end of a hepatitis B virus (HBV) mRNA sequence according to SEQ ID NO: 1.Embodiment 10. The isolated oligonucleotide of any one of embodiments 1-9, wherein the isolated oligonucleotide is capable of inducing degradation of the HBV mRNA.

Embodiment 11. The isolated oligonucleotide of any one of embodiments 1-10, wherein either the sense strand or

the antisense strand is a single stranded RNA molecule.

Embodiment 12. The isolated oligonucleotide of any one of embodiments 1-10, wherein both the sense strand and the antisense strand are single stranded RNA molecules.Embodiment 13. The isolated oligonucleotide of any one of embodiments 1-12, wherein the antisense strand comprises a 3′ overhang.Embodiment 14. The isolated oligonucleotide of embodiments 13, wherein the 3′ overhang comprises at least one nucleotide.Embodiment 15. The isolated oligonucleotide of any one of embodiments 1-14, wherein the sense strand comprises an RNA sequence of at least 20 nucleotides in length.Embodiment 16. The isolated oligonucleotide of any one of embodiments 1-15, wherein the antisense strand comprises an RNA sequence of at least 22 nucleotides in length.Embodiment 17. The isolated oligonucleotide of any one of embodiments 1-16, wherein the double stranded region is between 19 and 21 nucleotides in length.Embodiment 18. The isolated oligonucleotide of any one of embodiments 1-17, wherein the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 2-26.Embodiment 19. The isolated oligonucleotide of any one of embodiments 1-18, wherein the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 27-51.Embodiment 20. The isolated oligonucleotide of embodiment 6, wherein the double stranded region comprises:

• • i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UAUCCUGAUGUGAUGUUCUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ GAGAACAUCACAUCAGGAUA 3′);
  • ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5 CAUCACAUCAGGAUUCCUAA 3′);
  • iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UUGUAACACGAGAAGGGGUCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ GACCCCUUCUCGUGUUACAA 3′);
  • iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′);
  • v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 3′);
  • vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UAGAGGAAGAUGAUAAAACGCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CGUUUUAUCAUCUUCCUCUA 3′);
  • vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: (5′ UAUAGCAGCAGGAUGAAGAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUCUUCAUCCUGCUGCUAUA 3′);
  • viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAGAAGAUGAGGCAUAGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ CUGCUAUGCCUCAUCUUCUA 3′);
  • ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′);
  • x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ GGGCUUUCGGAAAAUUCCUA 3′);
  • xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′) and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GGCUUUCGGAAAAUUCCUAA 3′);
  • xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ CCUGGCUCAGUUUACUAGUA 3′);
  • xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUUAA 3′);
  • xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′);
  • xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′);
  • xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ ULAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′);
  • xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′);
  • xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′); or
  • xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′).Embodiment 21. The isolated oligonucleotide of embodiment 9, wherein the double stranded region comprises:
  • i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5 GGGUUUUUCUUGUUGACAAA 3′);
  • ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CCUCCUGUUGCUGUACCAAA 3′);
  • iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′);
  • iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ GUUAUAUGGAUGAUGUGGUA 3′);
  • v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAAAAAGGGACUCAAGAUGCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ GCAUCUUGAGUCCCUUUUUA 3′); or
  • vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUGGUAACAGCGGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′).Embodiment 22. The isolated oligonucleotide of any one of embodiments 1-21, wherein the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.5 nM.Embodiment 23. The isolated oligonucleotide of embodiment 22, wherein the double stranded region comprises:
  • i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UUGUAACACGAGAAGGGGUCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ GACCCCUUCUCGUGUUACAA 3′);
  • ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′);
  • iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUAA 3′);
  • iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUAGCAGCAGGAUGAAGAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUCUUCAUCCUGCUGCUAUA 3′);
  • v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46(5′ GGGUUUUUCUUGUUGACAAA 3′);
  • vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 3′);
  • vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UAGAGGAAGAUGAUAAAACGCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CGUUUUAUCAUCUUCCUCUA 3′);
  • viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′);
  • ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UAUCCUGAUGUGAUGUUCUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ GAGAACAUCACAUCAGGAUA 3′);
  • x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′);
  • xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′);
  • xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CCUCCUGUUGCUGUACCAAA 3′);
  • xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ GUUAUAUGGAUGAUGUGGUA 3′); or
  • xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUGGUAACAGCGGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′).Embodiment 24. The isolated oligonucleotide of any one of embodiments 1-21, wherein the isolated oligonucleotide attenuates expression of the HBV mRNA by at least 50% at a dose of 0.5 nM.Embodiment 25. The isolated oligonucleotide of claim 24, wherein the double stranded region comprises:
  • i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GGCUUUCGGAAAAUUCCUAA 3′);
  • ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′);
  • iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′);
  • iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′);
  • v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAGAAGAUGAGGCAUAGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ CUGCUAUGCCUCAUCUUCUA 3′);
  • vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ CCUGGCUCAGUUUACUAGUA 3′);
  • vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′);
  • viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′UAGGAAUUUUCCGAAAGCCCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ GGGCUUUCGGAAAAUUCCUA 3′);
  • ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′); or
  • x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′).Embodiment 26. The isolated oligonucleotide of any one of embodiments 1-21, wherein the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.05 nM.Embodiment 27. The isolated oligonucleotide of embodiment 26, wherein the double stranded region comprises:
  • i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′);
  • ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CCUCCUGUUGCUGUACCAAA 3′);
  • iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ GUUAUAUGGAUGAUGUGGUA 3′);
  • iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′);
  • v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GGCUUUCGGAAAAUUCCUAA 3′);
  • vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′);
  • vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′);
  • viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAGAAGAUGAGGCAUAGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ CUGCUAUGCCUCAUCUUCUA 3′);
  • ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ CCUGGCUCAGUUUACUAGUA 3′)
  • x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UAGGAAUUUUCCGAAAGCCCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ GGGCUUUCGGAAAAUUCCUA 3′);
  • xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′); or
  • xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′).Embodiment 28. 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) 157 to 177;
  • b) 196 to 410;
  • c) 578 to 598; and
  • d) 762 to 782,from the 5′ end of a hepatitis B virus (HBV) 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 29. The isolated oligonucleotide of embodiment 28, wherein the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.5 nM.Embodiment 30. The isolated oligonucleotide of embodiment 29, wherein the double stranded region comprises:
  • i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ UACUGAGAGAAGUCCACCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 74 (5′ GUGGUGGACUUCUCUCAGUA 3′);
  • ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 66 (5′ UAAGAGGAAGAUGAUAAAACGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 83 (5′ GUUUUAUCAUCUUCCUCUUA 3′);
  • iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ UCUGAGAGAAGUCCACCACGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 73 (5′ CGUGGUGGACUUCUCUCAGA 3′);
  • iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 63 (5′ UUUUUGGCCAAGACACACGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 80 (5′ CCGUGUGUCUUGGCCAAAAA 3′);
  • v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 65 (5′ UAGGACAAGUUGGAGGACAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 82 (5′ CUGUCCUCCAACUUGUCCUA 3′);
  • vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 68 (5′ UAAGAUGCUGUACAGACUUGGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 85 (5′ CAAGUCUGUACAGCAUCUUA 3′);
  • vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 52 (5′ UUCCUGAUGUGAUGUUCUCCAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 69 (5′ GGAGAACAUCACAUCAGGAA 3′);
  • viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 59 (5′ UUUGAGAGAAGUCCACCACGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 76 (5′ CGUGGUGGACUUCUCUCAAA 3′);
  • ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 55 (5′ UUGUCAACAAGAAAAACCCCGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 72 (5′ GGGGUUUUUCUUGUUGACAA 3′);
  • x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ UUAUUGUGAGGAUUUUUGUCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 71 (5′ GACAAAAAUCCUCACAAUAA 3′);
  • xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 67 (5′ UUGCAAUUUCCGUCCGAAGGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 84 (5′ CCUUCGGACGGAAAUUGCAA 3′); or
  • xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGGAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′).Embodiment 31. The isolated oligonucleotide of embodiment 28, wherein the isolated oligonucleotide attenuates expression of the HBV mRNA by 20% to 50% at a dose of 0.05 nM.Embodiment 32. The isolated oligonucleotide of embodiment 31, wherein the double stranded region comprises:
  • i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGGAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′); or
  • ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 67 (5′ UUGCAAUUUCCGUCCGAAGGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 84 (5′ CCUUCGGACGGAAAUUGCAA 3′).Embodiment 33. 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) 158 to 276,
  • b) 389 to 578; and
  • c) 666 to 823,
  • from the 5′ end of a hepatitis B virus (HBV) 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 34. The isolated oligonucleotide of embodiment 33, wherein the isolated oligonucleotide reduces levels of HBsAg by at least 50% at a dose of 0.05 nM.Embodiment 35. The isolated oligonucleotide of claim 34, wherein the double stranded region comprises:
  • i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′);
  • ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′);
  • iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′);
  • iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′);
  • iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′);
  • vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′)
  • vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′UUAUUGUGAGGAUUUUUGUCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 71 (5′ GACAAAAAUCCUCACAAUAA 3′);
  • viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ UCUGAGAGAAGUCCACCACGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 73 (5′ CGUGGUGGACUUCUCUCAGA 3′);
  • ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUGGUAACAGCGGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′);
  • x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ GGGUUUUCUUGUUGACAAA 3′);
  • xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), an d a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAAA 3′);
  • xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGGAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′);
  • xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5 UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ CCUGGCUCAGUUUACUAGUA 3′);
  • xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′);
  • xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UAUCCUGAUGUGAUGUUCUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ GAGAACAUCACAUCAGGAUA 3′);
  • xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UAGAGGAAGAUGAUAAAACGCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CGUUUUAUCAUCUUCCUCUA 3′);
  • xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUAA 3′);
  • xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUAGCAGCAGGAUGAAGAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUCUUCAUCCUGCUGCUAUA 3′);
  • xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ UACUGAGAGAAGUCCACCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 74 (5′ GUGGUGGACUUCUCUCAGUA 3′);
  • xx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UUGUAACACGAGAAGGGGUCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ GACCCCUUCUCGUGUUACAA 3′);
  • xxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′);
  • xxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAAAAAGGGACUCAAGAUGCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ GCAUCUUGAGUCCCUUUUUA 3′); or
  • xxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′).Embodiment 36. The isolated oligonucleotide of embodiment 33, wherein the isolated oligonucleotide reduces levels of HBsAg by at least 20% at a dose of 0.01 nM.Embodiment 37. The isolated oligonucleotide of embodiment 36, wherein the double stranded region comprises:
  • i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′);
  • ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′);
  • iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′);
  • iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′);
  • v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′);
  • vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′);
  • vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ UUAUUGUGAGGAUUUUUGUCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 71 (5′ GACAAAAAUCCUCACAAUAA 3′);
  • viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ UCUGAGAGAAGUCCACCACGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 73 (5′ CGUGGUGGACUUCUCUCAGA 3′)
  • ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUGGUAACAGCGGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′);
  • x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ GGGUUUUUCUUGUUGACAAA 3′);
  • xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 3′);
  • xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGGAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′);
  • xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′);
  • xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′);
  • xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ GUUAUAUGGAUGAUGUGGUA 3′);
  • xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 126 (5′UUACAUAGAGGUUCCUUGAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 199 (5′ CUCAAGGAACCUCUAUGUAA 3′)
  • xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUAA 3′);
  • xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 89 (5′ UUAGACUCUGCGGUAUUGUGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 162 (5′ CACAAUACCGCAGAGUCUAA 3′);
  • xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ UACUGAGAGAAGUCCACCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 74 (5′ GUGGUGGACUUCUCUCAGUA 3′);
  • xx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 92 (5′ UAUUGAGAGAAGUCCACCACGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 165 (5′ GUGGUGGACUUCUCUCAAUA 3′);
  • xxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′);
  • xxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAAAAAGGGACUCAAGAUGCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ GCAUCUUGAGUCCCUUUUA 3′);
  • xxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′);
  • xxiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CCUCCUGUUGCUGUACCAAA 3′);
  • xxv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 119 (5′ UUUGAGGAUCCUGGAAUUAGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 192 (5′ CUAAUUCCAGGAUCCUCAAA 3′);
  • xxvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ UAAAACUGAGAGAAGUCCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 75 (5′ GUGGACUUCUCUCAGUUUUA 3′); or
  • xxvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 90 (5′ UUCUAGACUCUGCGGUAUUGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 163 (5′ CAAUACCGCAGAGUCUAGAA 3′).Embodiment 38. The isolated oligonucleotide of any one of embodiments 1-37, wherein the sense strand or the antisense strand or both comprise one or more modified nucleotide(s).Embodiment 39. The isolated oligonucleotide of any one of embodiments 1-38, wherein the isolated oligonucleotide is capable of reducing levels of HBsAg.Embodiment 40. A vector encoding the isolated oligonucleotide of any one of embodiments 1-39.Embodiment 41. A delivery system comprising the isolated oligonucleotide of any one of embodiments 1-39 or the vector of embodiment 40.Embodiment 42. A pharmaceutical composition comprising at least one isolated oligonucleotide of any one of embodiments 1-39, the vector of embodiment 40, the delivery system of embodiment 41, and a pharmaceutically acceptable carrier, diluent or excipient.Embodiment 43. A kit comprising at least one isolated oligonucleotide of any one of embodiments 1-39, the vector of embodiment 40, the delivery system of embodiment 41, or the pharmaceutical composition of embodiment 42.Embodiment 44. A method of inhibiting or downregulating the expression or level of HBV or HBsAg in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of at least one isolated oligonucleotide of any one of embodiments 1-39, the vector of embodiment 40, the delivery system of embodiment 41, or the pharmaceutical composition of embodiment 42.Embodiment 45. A method of treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HBV or HBsAg or a disease or disorder where HBV or HBsAg plays a role in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of at least one isolated oligonucleotide of any one of embodiments 1-39, the vector of embodiment 40, the delivery system of embodiment 41, or the pharmaceutical composition of embodiment 42.

Claims

1. 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) 158 to 240;b) 389 to 439;c) 558 to 578; andd) 621 to 823,

2.-3. (canceled)

4. The isolated oligonucleotide of claim 1, 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) 158 to 240;b) 389 to 439;c) 621 to 686; andd) 775 to 823,

5.-6. (canceled)

7. The isolated oligonucleotide of claim 1, 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) 206 to 226;b) 558 to 578; andc) 720 to 807,

8.-17. (canceled)

18. The isolated oligonucleotide of claim 1, wherein the antisense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 2-26, 52-68, and 86-158.

19. The isolated oligonucleotide of claim 1, wherein the sense strand comprises a nucleotide sequence according to any one of: SEQ ID NOs: 27-51, 69-85, and 159-231.

20. The isolated oligonucleotide of claim 4, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UAUCCUGAUGUGAUGUUCUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ GAGAACAUCACAUCAGGAUA 3′);ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′);iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UUGUAACACGAGAAGGGGUCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ GACCCCUUCUCGUGUUACAA 3′);iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′);v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 3′);vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UAGAGGAAGAUGAUAAAACGCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CGUUUUAUCAUCUUCCUCUA 3′);vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUAGCAGCAGGAUGAAGAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUCUUCAUCCUGCUGCUAUA 3′);viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAGAAGAUGAGGCAUAGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ CUGCUAUGCCUCAUCUUCUA 3′);ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′);x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UAGGAAUUUUCCGAAAGCCCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ GGGCUUUCGGAAAAUUCCUA 3′);xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GGCUUUCGGAAAAUUCCUAA 3′);xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38(5′ CCUGGCUCAGUUUACUAGUA 3′);xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUAA 3′);xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′);xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′);xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′);xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′);xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′); orxix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′).

21. The isolated oligonucleotide of claim 7, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ GGGUUUUUCUUGUUGACAAA 3′);ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CCUCCUGUUGCUGUACCAAA 3′);iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′);iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ GUUAUAUGGAUGAUGUGGUA 3′);v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAAAAAGGGACUCAAGAUGCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ GCAUCUUGAGUCCCUUUUUA 3′); orvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUGGUAACAGCGGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′).

22. (canceled)

23. The isolated oligonucleotide of claim 221, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UUGUAACACGAGAAGGGGUCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ GACCCCUUCUCGUGUUACAA 3′);ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′);iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUAA 3′);iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUAGCAGCAGGAUGAAGAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUCUUCAUCCUGCUGCUAUA 3′);v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ GGGUUUUUCUUGUUGACAAA 3′);vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 3′);vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UAGAGGAAGAUGAUAAAACGCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CGUUUUAUCAUCUUCCUCUA 3′);viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′);ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UAUCCUGAUGUGAUGUUCUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ GAGAACAUCACAUCAGGAUA 3′);x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′);xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′);xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CCUCCUGUUGCUGUACCAAA 3′);xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ GUUAUAUGGAUGAUGUGGUA 3′);xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUGGUAACAGCGGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′); xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ GGGUUUUUCUUGUUGACAAA 3′), ori) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GGCUUUCGGAAAAUUCCUAA 3′);ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′);iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′);iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′);v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAGAAGAUGAGGCAUAGCAGCA 3′), and a sense strand of nucleic acid according to SEQ ID NO: 34 (5′ CUGCUAUGCCUCAUCUUCUA 3′);vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38(5′ CCUGGCUCAGUUUACUAGUA 3′);vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′);viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UAGGAAUUUUCCGAAAGCCCAG 3′), and a sense strand of nucleic acid according to SEQ ID NO: 36 (5′ GGGCUUUCGGAAAAUUCCUA 3′);ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′); orx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′) ori) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′), and a sense strand of nucleic acid according to SEQ ID NO: 37 (5′ GGCUUUCGGAAAAUUCCUAA 3′);ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′);iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′);iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′);v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAGAAGAUGAGGCAUAGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ CUGCUAUGCCUCAUCUUCUA 3′);vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ CCUGGCUCAGUUUACUAGUA 3′);vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′);viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UAGGAAUUUUCCGAAAGCCCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ GGGCUUUCGGAAAAUUCCUA 3′);ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′); orx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′), ori) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′);ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 47 (5′ CCUCCUGUUGCUGUACCAAA 3′);iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEO ID NO: 49 (5′ GUUAUAUGGAUGAUGUGGUA 3′);iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′);v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 12 (5′ UUAGGAAUUUUCCGAAAGCCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 37 (5′ GGCUUUCGGAAAAUUCCUAA 3′);vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′);vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′);viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 9 (5′ UAGAAGAUGAGGCAUAGCAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 34 (5′ CUGCUAUGCCUCAUCUUCUA 3′);ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38 (5′ CCUGGCUCAGUUUACUAGUA 3′);x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 11 (5′ UAGGAAUUUUCCGAAAGCCCAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 36 (5′ GGGCUUUCGGAAAAUUCCUA 3′);xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′); orxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′).

24.-27. (canceled)

28. 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) 157 to 177;b) 196 to 410;c) 578 to 598; andd) 762 to 782,

29. (canceled)

30. The isolated oligonucleotide of claim 28, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ UACUGAGAGAAGUCCACCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 74 (5′ GUGGUGGACUUCUCUCAGUA 3′);ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 66 (5′ UAAGAGGAAGAUGAUAAAACGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 83 (5′ GUUUUAUCAUCUUCCUCUUA 3′);iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ UCUGAGAGAAGUCCACCACGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 73 (5′ CGUGGUGGACUUCUCUCAGA 3′);iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 63 (5′ UUUUUGGCCAAGACACACGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 80 (5′ CCGUGUGUCUUGGCCAAAAA 3′);v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 65 (5′ UAGGACAAGUUGGAGGACAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 82 (5′ CUGUCCUCCAACUUGUCCUA 3′);vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 68 (5′ UAAGAUGCUGUACAGACUUGGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 85 (5′ CAAGUCUGUACAGCAUCUUA 3′);vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 52 (5′ UUCCUGAUGUGAUGUUCUCCAU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 69 (5′ GGAGAACAUCACAUCAGGAA 3′);viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 59 (5′ UUUGAGAGAAGUCCACCACGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 76 (5′ CGUGGUGGACUUCUCUCAAA 3′);ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 55 (5′ UUGUCAACAAGAAAAACCCCGC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 72 (5′ GGGGUUUUUCUUGUUGACAA 3′);x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ UUAUUGUGAGGAUUUUUGUCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 71 (5′ GACAAAAAUCCUCACAAUAA 3′);xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 67 (5′ UUGCAAUUUCCGUCCGAAGGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 84 (5′ CCUUCGGACGGAAAUUGCAA 3′);xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGGAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′), ori) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGGAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′); orii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 67 (5′ UUGCAAUUUCCGUCCGAAGGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 84 (5′ CCUUCGGACGGAAAUUGCAA 3′.

31.-32. (canceled)

33. 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) 158 to 276;b) 389 to 578; andc) 666 to 823,from the 5′ end of a hepatitis B virus (HBV) 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.

34. (canceled)

35. The isolated oligonucleotide of claim 33, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′);ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′);iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′);iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′);v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′);vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′);vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ UUAUUGUGAGGAUUUUUGUCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 71 (5′ GACAAAAAUCCUCACAAUAA 3′);viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ UCUGAGAGAAGUCCACCACGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 73 (5′ CGUGGUGGACUUCUCUCAGA 3′);ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUGGUAACAGCGGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′);x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 46 (5′ GGGUUUUUCUUGUUGACAAA 3′);xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 3′);xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGGAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′);xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 13 (5′ UACUAGUAAACUGAGCCAGGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 38(5′ CCUGGCUCAGUUUACUAGUA 3′);xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′);xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 2 (5′ UAUCCUGAUGUGAUGUUCUCCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 27 (5′ GAGAACAUCACAUCAGGAUA 3′);xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 7 (5′ UAGAGGAAGAUGAUAAAACGCC 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 32 (5′ CGUUUUAUCAUCUUCCUCUA 3′);xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUAA 3′);xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 8 (5′ UAUAGCAGCAGGAUGAAGAGGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 33 (5′ CUCUUCAUCCUGCUGCUAUA 3′);xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ UACUGAGAGAAGUCCACCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 74 (5′ GUGGUGGACUUCUCUCAGUA 3′);xx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 4 (5′ UUGUAACACGAGAAGGGGUCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 29 (5′ GACCCCUUCUCGUGUUACAA 3′);xxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′);xxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAAAAAGGGACUCAAGAUGCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ GCAUCUUGAGUCCCUUUUUA 3′);xxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′), ori) an antisense strand of nucleic acid sequence according to SEQ ID NO: 17 (5′ UAAAGAAAAUUGGUAACAGCGG 3′), and a sense strand of nucleic acid according to SEQ ID NO: 42 (5′ GCUGUUACCAAUUUUCUUUA 3′);ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 19 (5′ UACAAAAGAAAAUUGGUAACAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 44 (5′ GUUACCAAUUUUCUUUUGUA 3′);iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 15 (5′ UAGAAAAUUGGUAACAGCGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 40 (5′ CCGCUGUUACCAAUUUUCUA 3′);iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 18 (5′ UAAAAGAAAAUUGGUAACAGCG 3′), and a sense strand of nucleic acid according to SEQ ID NO: 43 (5′ CUGUUACCAAUUUUCUUUUA 3′);v) an antisense strand of nucleic acid sequence according to SEQ ID NO: 20 (5′ UAAAGACAAAAGAAAAUUGGUA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 45 (5′ CCAAUUUUCUUUUGUCUUUA 3′);vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 16 (5′ UAAGAAAAUUGGUAACAGCGGU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 41 (5′ CGCUGUUACCAAUUUUCUUA 3′);vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 54 (5′ UUAUUGUGAGGAUUUUUGUCGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 71 (5′ GACAAAAAUCCUCACAAUAA 3′);viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 56 (5′ UCUGAGAGAAGUCCACCACGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 73 (5′ CGUGGUGGACUUCUCUCAGA 3′);ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 26 (5′ UUGGUAACAGCGGUAAAAAGGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 51 (5′ CUUUUUACCGCUGUUACCAA 3′);x) an antisense strand of nucleic acid sequence according to SEQ ID NO: 21 (5′ UUUGUCAACAAGAAAAACCCCG 3′), and a sense strand of nucleic acid sequence according to SEO ID NO: 46 (5′ GGGUUUUUCUUGUUGACAAA 3′);xi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 6 (5′ UUAUUGUGAGGAUUCUUGUCAA 3′), and a sense strand of nucleic acid according to SEQ ID NO: 31 (5′ GACAAGAAUCCUCACAAUAA 3′);xii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 53 (5′ UUUGUGAGGAUUUUUGUCAAGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 70 (5′ UUGACAAAAAUCCUCACAAA 3′);xiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 23 (5′ UAUAACUGAAAGCCAAACAGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 48 (5′ CUGUUUGGCUUUCAGUUAUA 3′);xiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 10 (5′ UACAAGAAGAUGAGGCAUAGCA 3′), and a sense strand of nucleic acid according to SEQ ID NO: 35 (5′ CUAUGCCUCAUCUUCUUGUA 3′);xv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 24 (5′ UACCACAUCAUCCAUAUAACUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 49 (5′ GUUAUAUGGAUGAUGUGGUA 3′);xvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 126 (5′ UUACAUAGAGGUUCCUUGAGCA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 199 (5′ CUCAAGGAACCUCUAUGUAA 3′);xvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 14 (5′ UUAAAAAGGGACUCAAGAUGCU 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 39 (5′ CAUCUUGAGUCCCUUUUUAA 3′);xviii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 89 (5′ UUAGACUCUGCGGUAUUGUGAG 3′), and a sense strand of nucleic acid according to SEQ ID NO: 162 (5′ CACAAUACCGCAGAGUCUAA 3′);xix) an antisense strand of nucleic acid sequence according to SEQ ID NO: 57 (5′ UACUGAGAGAAGUCCACCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 74 (5′ GUGGUGGACUUCUCUCAGUA 3′);xx) an antisense strand of nucleic acid sequence according to SEQ ID NO: 92 (5′ UAUUGAGAGAAGUCCACCACGA 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 165 (5′ GUGGUGGACUUCUCUCAAUA 3′);xxi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 3 (5′ UUAGGAAUCCUGAUGUGAUGUU 3′), and a sense strand of nucleic acid according to SEQ ID NO: 28 (5′ CAUCACAUCAGGAUUCCUAA 3′);xxii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 25 (5′ UAAAAAGGGACUCAAGAUGCUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 50 (5′ GCAUCUUGAGUCCCUUUUUA 3′);xxiii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 5 (5′ UUCAACAAGAAAAACCCCGCCU 3′), and a sense strand of nucleic acid sequence according to SEO ID NO: 30 (5′ GCGGGGUUUUUCUUGUUGAA 3′);xxiv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 22 (5′ UUUGGUACAGCAACAGGAGGGA 3′), and a sense strand of nucleic acid according to SEQ ID NO: 47 (5′ CCUCCUGUUGCUGUACCAAA 3′);xxv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 119 (5′ UUUGAGGAUCCUGGAAUUAGAG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 192 (5′ CUAAUUCCAGGAUCCUCAAA 3′);xxvi) an antisense strand of nucleic acid sequence according to SEQ ID NO: 58 (5′ UAAAACUGAGAGAAGUCCACGG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 75 (5′ GUGGACUUCUCUCAGUUUUA 3′); orxxvii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 90 (5′ UUCUAGACUCUGCGGUAUUGUG 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 163 (5′ CAAUACCGCAGAGUCUAGAA 3′).

36.-41. (canceled)

42. The isolated oligonucleotide of claim 1, 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′.

43. The isolated oligonucleotide of claim 1, 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′.

44. The isolated oligonucleotide of claim 42, wherein the antisense strand comprises any one of: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 232 (5′ [mUs][fAs][fU][mC][fC][mU][fG][mA][mU][fG][mU][mG][mA][fU][mG][fU][mU][mC][mU][mCs][mCs][mA] 3′);ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 233 (5′ [mUs][fUs][fA][mG][fG][mA][fA][mU][mC][fC][mU][mG][mA][fU][mG][fU][mG][mA][mU][mGs][mUs][mU] 3′);iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: 234 (5′ [mUs][fUs][fC][mA][fA][mC][fA][mA][mG][fA][mA][mA][mA][fA][mC][fC][mC][mC][mG][mCs][mCs][mU] 3′);iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: 235 (5′ [mUs][fUs][fU][mG][fU][mC][fA][mA][mC][fA][mA][mG][mA][fA][mA][fA][mA][mC][mC][mCs][mCs][mG] 3′);v) an antisense strand of nucleic acid sequence according to SEQ ID: 236 (5′ [mUs][fUs][fA][mU][fU][mG][fU][mG][mA][fG][mG][mA][mU][fU][mC][fU][mU][mG][mU][mCs][mAs][mA] 3′);vi) an antisense strand of nucleic acid sequence according to SEQ ID: 237 (5′ [mUs][fUs][fA][mG][fG][mA][fA][mU][mU][fU][mU][mC][mC][fG][mA][fA][mA][mG][mC][mCs][mCs][mA] 3′);vii) an antisense strand of nucleic acid sequence according to SEQ ID: 238 (5′ [mUs][fAs][fU][mA][fA][mC][fU][mG][mA][fA][mA][mG][mC][fC][mA][fA][mA][mC][mA][mGs][mUs][mG] 3′);viii) an antisense strand of nucleic acid sequence according to SEQ ID: 239 (5′ [mUs][fAs][fG][mA][fA][mA][fA][mU][mU][fG][mG][mU][mA][fA][mC][fA][mG][mC][mG][mGs][mUs][mA] 3′);ix) an antisense strand of nucleic acid sequence according to SEQ ID: 240 (5′ [mUs][fAs][fA][mA][fA][mG][fA][mA][mA][fA][mU][mU][mG][fG][mU][fA][mA][mC][mA][mGs][mCs][mG] 3′); orx) an antisense strand of nucleic acid sequence according to SEQ ID: 241 (5′ [mUs][fAs][fC][mA][fA][mA][fA][mG][mA][fA][mA][mA][mU][fU][mG][fG][mU][mA][mA][mCs][mAs][mG] 3′), wherein “m” is a 2′-O-methyl modified nucleotide, “f” is a 2′-F modified nucleotide, “s” is a phosphorothioate internucleotide linkage.

45. The isolated oligonucleotide of claim 43, wherein the sense strand comprises any one of: i) a sense strand of nucleic acid sequence according to SEQ ID NO: 242 (5′ [mGs][mAs][mG][mA][mA][fC][mA][fU][fC][fA][fC][mA][mU][mC][mA][mG][mG][mAs][m Us][mA][G1b][G1b][G1b] 3′);ii) a sense strand of nucleic acid sequence according to SEQ ID NO: 243 (5′ [mCs][mAs][mU][mC][mA][fC][mA][fU][fC][fA][fG][mG][mA][mU][mU][mC][mC][mUs][m As][mA][G1b][G1b][G1b] 3′);iii) a sense strand of nucleic acid sequence according to SEQ ID NO: 244 (5′ [mGs][mCs][mG][mG][mG][fG][mU][fU][fU][fU][fU][mC][mU][mU][mG][mU][mU][mGs][m As][mA][G1b][G1b][G1b] 3′);iv) a sense strand of nucleic acid sequence according to SEQ ID NO: 245 (5′ [mGs][mGs][mG][mU][mU][fU][mU][fU][fC][fU][fU][mG][mU][mU][mG][mA][mC][mAs][m As][mA][G1b][G1b][G1b] 3′);v) a sense strand of nucleic acid sequence according to SEQ ID NO: 246 (5′ [mGs][mAs][mC][mA][mA][fG][mA][fA][fU][fC][fC][mU][mC][mA][mC][mA][mA][mUs][m As][mA][G1b][G1b][G1b] 3′);vi) a sense strand of nucleic acid sequence according to SEQ ID NO: 247 (5′ [mGs][mGs][mC][mU][mU][fU][mC][fG][fG][fA][fA][mA][mA][mU][mU][mC][mC][mUs][m As][mA][G1b][G1b][G1b] 3′);vii) a sense strand of nucleic acid sequence according to SEQ ID NO: 248 (5′ [mCs][mUs][mG][mU][mU][fU][mG][fG][fC][fU][fU][mU][mC][mA][mG][mU][mU][mAs][m Us][mA][G1b][G1b][G1b] 3);viii) a sense strand of nucleic acid sequence according to SEQ ID NO: 249 (5′ [mCs][mCs][mG][mC][mU][fG][mU][fU][fA][fC][fC][mA][mA][mU][mU][mU][mU][mCs][m Us][mA][G1b][G1b][G1b] 3′);ix) a sense strand of nucleic acid sequence according to SEQ ID NO: 250 (5′ [mCs][mUs][mG][mU][mU][fA][mC][fC][fA][fA][fU][mU][mU][mU][mC][mU][mU][mUs][m Us][mA][G1b][G1b][G1b] 3′); orx) a sense strand of nucleic acid sequence according to SEQ ID NO: 251 (5′ [mGs][mUs][mU][mA][mC][fC][mA][fA][fU][fU][fU][mU][mC][mU][mU][mU][mU][mGs][m Us][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.

46. The isolated oligonucleotide of claim of claim 44, wherein the double stranded region comprises: i) an antisense strand of nucleic acid sequence according to SEQ ID NO: 232 (5′ [mUs][fAs][fU][mC][fC][mU][fG][mA][mU][fG][mU][mG][mA][fU][mG][fU][mU][mC][mU][mCs][mCs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 242 (5′ [mGs][mAs][mG][mA][mA][fC][mA][fU][fC][fA][fC][mA][mU][mC][mA][mG][mG][mAs][m Us][mA][G1b][G1b][G1b] 3′);ii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 233 (5′ [mUs][fUs][fA][mG][fG][mA][fA][mU][mC][fC][mU][mG][mA][fU][mG][fU][mG][mA][mU][mGs][mUs][mU] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: NO: 243 (5′ [mCs][mAs][mU][mC][mA][fC][mA][fU][fC][fA][fG][mG][mA][mU][mU][mC][mC][mUs][m As][mA][G1b][G1b][G1b] 3′);iii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 234 (5′ [mUs][fUs][fC][mA][fA][mC][fA][mA][mG][fA][mA][mA][mA][fA][mC][fC][mC][mC][mG][mCs][mCs][mU] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 244 (5′ [mGs][mCs][mG][mG][mG][fG][mU][fU][fU][fU][fU][mC][mU][mU][mG][mU][mU][mGs][m As][mA][G1b][G1b][G1b] 3′);iv) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 235 (5′ [mUs][fUs][fU][mG][fU][mC][fA][mA][mC][fA][mA][mG][mA][fA][mA][fA][mA][mC][mC][mCs][mCs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 245 (5′ [mGs][mGs][mG][mU][mU][fU][mU][fU][fC][fU][fU][mG][mU][mU][mG][mA][mC][mAs][m As][mA][G1b][G1b][G1b] 3′);v) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 236 (5′ [mUs][fUs][fA][mU][fU][mG][fU][mG][mA][fG][mG][mA][mU][fU][mC][fU][mU][mG][mU][mCs][mAs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 246 (5′ [mGs][mAs][mC][mA][mA][fG][mA][fA][fU][fC][fC][mU][mC][mA][mC][mA][mA][mUs][m As][mA][G1b][G1b][G1b] 3′);vi) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 237 (5′ [mUs][fUs][fA][mG][fG][mA][fA][mU][mU][fU][mU][mC][mC][fG][mA][fA][mA][mG][mC][mCs][mCs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 247 (5′ [mGs][mGs][mC][mU][mU][fU][mC][fG][fG][fA][fA][mA][mA][mU][mU][mC][mC][mUs][mAs][mA][G1b][G1b][G1b] 3′);vii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 238 (5′ [mUs][fAs][fU][mA][fA][mC][fU][mG][mA][fA][mA][mG][mC][fC][mA][fA][mA][mC][mA][mGs][mUs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 248 (5′ [mCs][mUs][mG][mU][mU][fU][mG][fG][fC][fU][fU][mU][mC][mA][mG][mU][mU][mAs][m Us][mA][G1b][G1b][G1b] 3);viii) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 239 (5′ [mUs][fAs][fG][mA][fA][mA][fA][mU][mU][fG][mG][mU][mA][fA][mC][fA][mG][mC][mG][mGs][mUs][mA] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 249 (5′ [mCs][mCs][mG][mC][mU][fG][mU][fU][fA][fC][fC][mA][mA][mU][mU][mU][mU][mCs][m Us][mA][G1b][G1b][G1b] 3′);ix) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 240 (5′ [mUs][fAs][fA][mA][fA][mG][fA][mA][mA][fA][mU][mU][mG][fG][mU][fA][mA][mC][mA][mGs][mCs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 250 (5′ [mCs][mUs][mG][mU][mU][fA][mC][fC][fA][fA][fU][mU][mU][mU][mC][mU][mU][mUs][m Us][mA][G1b][G1b][G1b] 3′); orx) an antisense strand of nucleic acid sequence according to SEQ ID NO: SEQ ID: 241 (5′ [mUs][fAs][fC][mA][fA][mA][fA][mG][mA][fA][mA][mA][mU][fU][mG][fG][mU][mA][mA][mCs][mAs][mG] 3′), and a sense strand of nucleic acid sequence according to SEQ ID NO: 251 (5′ [mGs][mUs][mU][mA][mC][fC][mA][fA][fU][fU][fU][mU][mC][mU][mU][mU][mU][mGs][m Us][mA][G1b][G1b][G1b] 3′).

47. A vector encoding the isolated oligonucleotide of claim 1.

48. A delivery system comprising the isolated oligonucleotide of claim 1.

49. A pharmaceutical composition comprising at least one isolated oligonucleotide of claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.

50. (canceled)

51. A method of inhibiting or downregulating the expression or level of HBV or HBsAg or treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HBV or HBsAg or a disease or disorder where HBV or HBsAg plays a role in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of at least one isolated oligonucleotide of claim 1.

52. (canceled)

53. The isolated oligonucleotide of claim 28, 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′.

54. The isolated oligonucleotide of claim 28, 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′.

55. A vector encoding the isolated oligonucleotide of claim 28.

56. A delivery system comprising the isolated oligonucleotide of claim 28.

57. A pharmaceutical composition comprising at least one isolated oligonucleotide of claim 28, and a pharmaceutically acceptable carrier, diluent or excipient.

58. A method of inhibiting or downregulating the expression or level of HBV or HBsAg or treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HBV or HBsAg or a disease or disorder where HBV or HBsAg plays a role in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of at least one isolated oligonucleotide of claim 28.

59. The isolated oligonucleotide of claim 33, 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′.

60. The isolated oligonucleotide of claim 33, 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′.

61. A vector encoding the isolated oligonucleotide of claim 33.

62. A delivery system comprising the isolated oligonucleotide of claim 33.

63. A pharmaceutical composition comprising at least one isolated oligonucleotide of claim 33, and a pharmaceutically acceptable carrier, diluent or excipient.

64. A method of inhibiting or downregulating the expression or level of HBV or HBsAg or treating or preventing a disease or disorder associated with aberrant or increased expression or activity of HBV or HBsAg or a disease or disorder where HBV or HBsAg plays a role in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of at least one isolated oligonucleotide of claim 33.