JPWO2019079781A5 - - Google Patents

Description

Other aspects of the present disclosure provide oligonucleotides for reducing expression of hepatitis B virus surface antigen (HBsAg) mRNA, the oligonucleotides comprising a sense strand forming a duplex region with an antisense strand. The sense strand comprises the sequence set forth in GACAAAAUCCUCCACAAUAAGCAGCCGAAAGGCUGC (SEQ ID NO: 8), the antisense strand comprises the sequence set forth in UUAUUGUGAGGAUUUUUGUCGG (SEQ ID NO: 5.1), and the antisense and sense strands each comprise one or more 2'-fluoro and 2'-O-methyl modified nucleotides and at least one phosphorothioate linkage. wherein the 4'-carbon of the 5' nucleotide sugar of the antisense strand contains a phosphate analogue, and the sense strand is conjugated to one or more N-acetylgalactosamine (GalNAc) moieties.

Further provided herein are oligonucleotides for reducing expression of hepatitis B virus surface antigen (HBsAg) mRNA.当該オリゴヌクレオチドは、アンチセンス鎖と二重鎖領域を形成するセンス鎖を含み、当該オリゴヌクレオチド中で：当該センス鎖はＧＡＣＡＡＡＡＡＵＣＣＵＣＡＣＡＡＵＡＡＧＣＡＧＣＣＧＡＡＡＧＧＣＵＧＣ（配列番号８）に規定される配列を含み、３、８～１０、１２、１３及び１７位にある２'‐フルオロ修飾ヌクレオチド、１、２、４～７、１１、１４～１６、１８～２６、及び３１～３６位にある２'‐Ｏ‐メチル修飾ヌクレオチド、並びに少なくとも１つのホスホロチオエートヌクレオチド間結合を含み、当該センス鎖は、１つ以上のＮ‐アセチルガラクトサミン（ＧａｌＮＡｃ）部分にコンジュゲートされる；当該アンチセンス鎖はＵＵＡＵＵＧＵＧＡＧＧＡＵＵＵＵＵＧＵＣＧＧ（配列番号５．１）に規定される配列を含み、２、３、５、７、８、１０、１２、１４、１６及び１９位にある２'‐フルオロ修飾ヌクレオチド、１、４、６、９、１１、１３、１５、１７、１８及び２０～２２位にあるＯ‐メチル修飾ヌクレオチド、並びに少なくとも３つのホスホロチオエートヌクレオチド間での結合を含み、当該アンチセンス鎖における５'‐ヌクレオチドの糖の４'‐炭素はホスフェート類似体を含み、当該センス鎖は、１位と２位のヌクレオチド間にホスホロチオエート結合を含む。

In some embodiments the oligonucleotide comprises at least one modified nucleotide. In some embodiments, modified nucleotides include 2'-modifications. In some embodiments, the 2' modification is a modification selected from: 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid. In some embodiments, all nucleotides of the oligonucleotide are modified nucleotides. In some embodiments the oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, the at least one modified internucleotide linkage is a phosphorothioate linkage. The 4'-carbon of the 5'-nucleotide sugar in the antisense strand contains a phosphate analogue.

Deoxyribonucleotide: As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has a hydrogen in place of a hydroxyl at the 2' position of its pentose sugar as compared to a ribonucleotide. Modified deoxyribonucleotides are deoxyribonucleotides that have one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions in the sugar, phosphate group or base, or modifications or substitutions of the sugar, phosphate group or base.

Hepatocyte: As used herein, the term “hepatocyte(s)” refers to cells of the parenchyma of the liver. These cells make up approximately 70-85% of the mass of the liver and produce serum albumin, fibrinogen, and the prothrombin group of clotting factors (except factors 3 and 4). Markers of hepatocyte lineage cells include, but are not limited to, transthyretin (Ttr), glutamine synthase (Glul), hepatocyte nuclear factor 1a (Hnf1a), and hepatocyte nuclear factor 4a (Hnf4a). Markers of mature hepatocytes include, but are not limited to, cytochrome P450 (Cyp3a11), fumarylacetoacetate hydrolase (Fah), glucose 6- phosphate (G6p), albumin (Alb), and OC2-2F8. See, eg, Huch et al. (2013) Nature 494(7436):247-250. Its content regarding hepatocyte markers is incorporated herein by reference.

Loop: As used herein, the term "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) flanked by two antiparallel regions of nucleic acid that are sufficiently complementary to each other such that under appropriate hybridization conditions (e.g., in a phosphate buffer, in cells), the two antiparallel regions flanking the unpaired region hybridize to form a duplex (referred to as a "stem").

Modified nucleotide: As used herein, the term "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to the corresponding reference nucleotide selected from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides and thymidine deoxyribonucleotides. In some embodiments, modified nucleotides are non-naturally occurring nucleotides. In some embodiments, a modified nucleotide has one or more chemical modifications in its sugar, nucleobase and/or phosphate groups. In some embodiments, a modified nucleotide has one or more chemical moieties conjugated to the corresponding reference nucleotide. Typically, modified nucleotides impart one or more desirable properties to the nucleic acid in which they are present. For example, modified nucleotides can improve thermostability, degradation resistance, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, and the like.

Phosphate analogue: As used herein, the term “ phosphate analogue” refers to chemical moieties that mimic the electrostatic and/or steric properties of a phosphate group. In some embodiments, the phosphate analog is located at the 5' terminal nucleotide of the oligonucleotide in place of the 5'- phosphate , which often undergoes enzymatic removal. In some embodiments, the 5' phosphate analogue comprises a phosphatase-resistant linkage. Examples of phosphate analogs include 5' phosphonic acids such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). In some embodiments, oligonucleotides have a phosphate analogue (referred to as a "4'- phosphate analogue") at the 4' carbon position of the sugar of the 5' terminal nucleotide. An example of a 4'- phosphate analogue is an oxymethylphosphonate, where the oxygen atom of the oxymethyl group is attached to the sugar moiety (eg at its 4'-carbon) or analogue thereof. See, for example, US Provisional Application No. 62/383,207, filed September 2, 2016, and US Provisional Application No. 62/393,401, filed September 12, 2016. The contents of each of these regarding phosphate analogues are incorporated herein by reference. Other modifications have been developed for the 5′ end of oligonucleotides (see, for example, WO 2011/133871, US Pat. No. 8,927,513, and Prakash et al. (2015) Nucleic Acids Res., 43(6):2993-3011, the contents of each of which regarding phosphate analogs are incorporated herein by reference). cited).

Region of Complementarity: As used herein, the term “region of complementarity” refers to a nucleotide sequence of a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel sequence of nucleotides to allow hybridization between the two sequences of nucleotides under suitable hybridization conditions, such as in a phosphate buffer, in a cell, etc.

Ribonucleotide: As used herein, the term "ribonucleotide" refers to a nucleotide having ribose as its pentose sugar containing a hydroxyl group at its 2' position. Modified ribonucleotides are ribonucleotides that have one or more modifications or substitutions of atoms other than the 2'-position, such as ribose, phosphate group or base modifications or substitutions.

In some embodiments, the oligonucleotide for reducing HBsAg mRNA expression comprises a sense strand that forms a duplex region with the antisense strand, the sense strand comprising a sequence set forth in any one of SEQ ID NOs:6-8.1, and the antisense strand comprising a sequence set forth in any one of SEQ ID NOs:4-5.1. In some embodiments, the sense strand comprises 2'-fluoro and 2'-O-methyl modified nucleotides and at least one phosphorothioate internucleotide linkage. In some embodiments, the sense strand is conjugated to N-acetylgalactosamine (GalNAc) moieties. In some embodiments, the antisense strand comprises 2'-fluoro and 2'-O-methyl modified nucleotides and at least one phosphorothioate internucleotide linkage. In some embodiments, the 4'-carbon of the 5'-nucleotide sugar of the antisense strand comprises a phosphate analogue. In some embodiments, each of the antisense and sense strands comprises 2′-fluoro and 2′-O-methyl modified nucleotides and at least one phosphorothioate internucleotide linkage, the 4′-carbon of the 5′-nucleotide sugar of the antisense strand comprises a phosphate analogue, and the sense strand is conjugated to an N-acetylgalactosamine (GalNAc) moiety.

In some embodiments, the antisense strand comprising the sequence set forth in any one of SEQ ID NOS: 4-5.1 comprises 2'-fluoro modified nucleotides at positions 2, 3, 5, 7, 8, 10, 12, 14, 16, and 19. In some embodiments, the antisense strand comprises 2'-O-methyl modified nucleotides at positions 1, 4, 6, 9, 11, 13, 15, 17, 18, and 20-22. In some embodiments, the antisense strand comprises three phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 3 and 4, between nucleotides 20 and 21, and between nucleotides 21 and 22. In some embodiments, the 4'-carbon of the 5'-nucleotide sugar of the antisense strand comprises a phosphate analogue.
ii. double-stranded oligonucleotide

Oligonucleotides may be modified in a variety of ways to improve or control specificity, stability, delivery, bioavailability, resistance to nuclease degradation, immunogenicity, base-pairing properties, RNA distribution and cellular uptake, and other characteristics relevant to therapeutic or research applications. See, eg, Bramsen et al., Nucleic Acids Res. 2009, 37, 2867-2881; Bramsen and Kjems (Frontiers in Genetics, 3 (2012): 1-22). Thus, in some embodiments, oligonucleotides of the present disclosure may contain one or more suitable modifications. In some embodiments, a modified nucleotide has modifications to its base (or nucleobase), sugar (eg, ribose, deoxyribose), or phosphate group.

In some embodiments, the terminal 3′ terminal group (e.g., 3′-hydroxyl) is a phosphate group or other group, which can be used, for example, to attach a linker, adapter or label, or to directly attach an oligonucleotide to another nucleic acid.
b. 5' terminal phosphate

In some embodiments, the 5' terminal phosphate group of the oligonucleotide facilitates interaction with Argonaute-2. However, oligonucleotides containing a 5' phosphate group are susceptible to degradation by phosphatases or other enzymes, which can limit their bioavailability in vivo. In some embodiments, oligonucleotides include 5' phosphate analogs that are resistant to such degradation. In some embodiments, the phosphate analog can be oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 5′ end of the oligonucleotide chain is attached to a chemical moiety (“ phosphate mimetic”) that mimics the electrostatic and steric properties of the natural 5′- phosphate group (eg, Prakash et al. (2015) Nucleic Acids Res. Nucleic Acids Res. 2015 Mar 31;43(6):2993-3011). , the content of which regarding phosphate analogues is incorporated herein by reference). A number of phosphate mimetics have been developed that can be attached to the 5' terminus (see, eg, US Pat. No. 8,927,513, the contents of which regarding phosphate analogues are incorporated herein by reference). Other modifications have also been developed for the 5' end of oligonucleotides (see, eg, WO2011/133871, the content of which regarding phosphate analogues is incorporated herein by reference). In certain embodiments, a hydroxyl group is attached to the 5' end of the oligonucleotide.

In some embodiments, oligonucleotides have a phosphate analogue (referred to as a "4'- phosphate analogue") at the 4'-carbon position of the sugar. See, for example, U.S. Provisional Application No. 62/383,207 entitled "4'-Phosphate Analogs and Oligonucleotides Composing the Same," filed September 2, 2016, and "4'-Phosphate Analogs and Oli," filed September 12, 2016. See U.S. Provisional Application No. 62/393,401, entitled "Gonucleotides Composing the Same". Each of its contents relating to phosphate analogues is incorporated herein by reference. In some embodiments, oligonucleotides provided herein include a 4'- phosphate analogue at the 5' terminal nucleotide. In some embodiments, the phosphate analogue is an oxymethylphosphonate, where the oxygen atom of the oxymethyl group is attached to the sugar moiety (eg, at its 4' carbon) or analogue thereof. In other embodiments, the 4'- phosphate analogue is a thiomethylphosphonate or aminomethylphosphonate, wherein the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is attached to the 4'-carbon of the sugar moiety or analogue thereof. In certain embodiments, the 4'- phosphate analogue is oxymethylphosphonate. In some embodiments, the oxymethylphosphonate has the formula -O-CH2 _- PO ₍ OH)2 or -O-CH2 _-PO (OR _{)2 ,} where R _is independently H, _CH3 , an alkyl group, _CH2CH2CN , _CH2OCOC ( _CH3 ) ₃ , _CH2OCH2CH2Si ( _CH3 ) ₃ , or from _a protecting group. selected. In certain _embodiments , an alkyl group is _CH2CH3 . More _typically , R is independently selected from H, _CH3 , or _CH2CH3 .

を、例えば、ガイド（アンチセンス）鎖の最初の位置で使用してもよく、この修飾ヌクレオチドは［Ｍｅホスホネート‐４Ｏ‐ｍＵ］又は５’‐メトキシ，ホスホネート‐４’オキシ‐２’‐Ｏ‐メチルウリジンと称する。
ｃ．修飾ヌクレオシド間結合 In certain embodiments, the phosphate analog attached to the oligonucleotide is methoxyphosphonate (MOP). In certain embodiments, the phosphate analog attached to the oligonucleotide is a 5' mono-methyl protected MOP. In some embodiments, the following uridine nucleotides with phosphate analogs:

may be used, for example, at the first position of the guide (antisense) strand, and this modified nucleotide is referred to as [Mephosphonate-4O-mU] or 5'-methoxy,phosphonate-4'oxy-2'-O-methyluridine.
c. Modified internucleoside linkage

In some embodiments, phosphate modifications or substitutions can result in oligonucleotides containing at least one (eg, at least one, at least two, at least three, or at least five) modified internucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1-10 (e.g., 1-10, 2-8, 4-6, 3-10, 5-10, 1-5, 1-3, or 1-2) modified internucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

In some embodiments, the reversibly modified nucleotide comprises a glutathione sensitive moiety. Typically, nucleic acid molecules are chemically modified with cyclic disulfide moieties to mask the negative charge created by the internucleotide diphosphate linkage, improving cellular uptake and nuclease resistance. U.S. Published Application No. 2011/0294869 originally granted to Traversa Therapeutics, Inc. (“Traversa”); Solstice Biologies, Inc. (“Solstice”) PCT Publication No. WO2015/188197; 2014, 32:1256-1263 (“Meade”), Merck Sharp & Dohme PCT Publication No. WO 2014/088920. each of which is incorporated by reference for disclosure of such modifications. This reversible modification of the internucleotide diphosphate bond is designed to be cleaved intracellularly by the reducing environment of the cytosol (eg, glutathione). Early examples include the neutralization of phosphotriester modifications reported to be intracellularly cleavable (Dellinger et al., J. Am. Chem. Soc. 2003, 125:940-950).

Various formulations have been developed to facilitate the use of oligonucleotides. For example, oligonucleotides can be delivered to a subject or cellular environment using formulations that minimize degradation, facilitate delivery and/or uptake, or provide other beneficial properties to the oligonucleotides in the formulation. In some embodiments, provided herein are compositions comprising oligonucleotides (eg, single-stranded or double-stranded oligonucleotides) for reducing expression of HBV antigens (eg, HBsAg). Such compositions can be suitably formulated so that when administered either in the immediate environment of target cells or systemically in a subject, a sufficient amount of the oligonucleotides will enter the cells to reduce HBV antigen expression. As disclosed herein, any of a variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for reducing HBV antigens. In some embodiments, oligonucleotides are formulated in buffers such as phosphate buffered saline, liposomes, micellar structures, and capsids.

Claims (38)

An oligonucleotide comprising a sense strand forming a duplex region with the antisense strand,
The sense strand consists of the sequence defined by GACAAAAAUCCUCCACAAUAAGCAGCCGAAAGGCUGC (SEQ ID NO: 8), and 2'-fluoro modified nucleotides at positions 3, 8-10, 12, 13 and 17; containing an ate bond,
each said nucleotide of said -GAAA- sequence on said sense strand is conjugated with a monovalent GalNac moiety;
said antisense strand consists of the sequence defined by UUAUUGUGAGGAUUUUUGUCGG (SEQ ID NO: 5.1) and is 2'-fluoro modified nucleotides at positions 2, 3, 5, 7, 8, 10, 12, 14, 16 and 19; and phosphorothioate linkages between nucleotide positions 3, 3 and 4, 20 and 21, and 21 and 22, and the 4'-carbon of the 5'-nucleotide sugar of said antisense strand comprises a methoxyphosphonate (MOP). The 5'-nucleotide of the antisense strand has the following structure:

The oligonucleotide of claim 1, having The -GAAA- motif has the following structure:

contains, in the formula:
3. The oligonucleotide of claim 1 or 2, wherein L is a bond, a click chemistry handle, or a linker of 1 to 20 consecutive covalent atoms in length and is a linker selected from the group consisting of substituted and unsubstituted alkylene, substituted and unsubstituted alkenylene, substituted and unsubstituted alkynylene, substituted and unsubstituted heteroalkylene, substituted and unsubstituted heteroalkenylene, substituted and unsubstituted heteroalkynylene, and combinations thereof, and X is O, S, or N. 4. The oligonucleotide of Claim 3, wherein L is an acetal linker. 5. The oligonucleotide of claim 3 or 4, wherein X is O. The -GAAA- sequence has the following structure:

The oligonucleotide according to any one of claims 1 to 5, comprising An oligonucleotide comprising a sense strand forming a duplex region with the antisense strand,
said sense strand consists of the sequence defined by GACAAAAAUCCUCCACAAUAAGCAGCCGAAAGGCUGC (SEQ ID NO: 8), and 2'-fluoro modified nucleotides at positions 3, 8-10, 12, 13 and 17; containing a phosphorothioate internucleotide linkage,
each said nucleotide of said -GAAA- sequence of said sense strand is conjugated with a monovalent GalNac moiety;
The -GAAA- sequence has the following structure:

wherein the antisense strand consists of the sequence defined by UUAUUGUGAGGAUUUUUGUCGG (SEQ ID NO: 5.1), and 2'-fluoro modified nucleotides at positions 2, 3, 5, 7, 8, 10, 12, 14, 16 and 19; comprising phosphorothioate internucleotide linkages between nucleotides at positions 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22; and
The 5'-nucleotide of said antisense strand has the following structure:

An oligonucleotide characterized by having A composition comprising an oligonucleotide according to any one of claims 1-7. 9. The composition of claim 8, comprising a counterion. 10. A composition according to claim 8 or 9, comprising a pharmaceutically acceptable carrier. A composition according to any one of claims 8-10, comprising an excipient. A composition according to any one of claims 8 to 11, comprising phosphate buffered saline. An in vitro method for reducing expression of hepatitis B virus (HBV) surface antigen (HBsAg) in a cell, comprising delivering an oligonucleotide according to any one of claims 1 to 7 to said cell. 14. The method of claim 13, wherein said cells are hepatocytes. 15. The method of claim 13 or 14, wherein said oligonucleotide is delivered in the form of a transgene genetically engineered to express said oligonucleotide within said cell. Use of an oligonucleotide according to any one of claims 1-7 or a composition according to any one of claims 8-12 in the manufacture of a medicament for treating HBV infection in a patient . 17. Use according to claim 16, wherein said oligonucleotide or composition is administered subcutaneously to said patient . 17. Use according to claim 16, wherein said oligonucleotide or composition is administered intravenously to said patient . 19. Use according to claim 18, wherein the medicament comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is a solvent or dispersion medium comprising water. A double-stranded oligonucleotide,
sense strand:
5' mG-S-mA-fC-mA-mA-mA-mA-fA-fU-fC-mC-fU-fC-mA-mC-mA-fA-mU-mA-mA-mG-mC-mA-mG-mC-mC-[ademG-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC -mU-mG-mC 3′ and
Antisense strand:
5' [MePhosphonate-4O-mU]-S-fU-S-fA-S-mU-fU-mG-fU-fG-mA-fG-mG-fA-mU-fU-mU-fU-mU-mG-fU-mC-S-mG-S-mG 3';
including
here,
"-" between nucleosides means a phosphorodiester internucleoside linkage,
internucleoside "-S-" means a phosphorothioate internucleoside linkage,
mA means 2′-O-methyladenosine ribonucleoside;
mG means 2'-O-methylguanosine ribonucleoside;
mC means 2′-O-methylcytidine ribonucleoside;
mU means 2′-O-methyluridine ribonucleoside;
fA means 2′-fluoroadenosine deoxyribonucleoside;
fG means 2'-fluoroguanosine deoxyribonucleoside;
fC means 2′-fluorocytidine deoxyribonucleoside;
fU means 2′-fluorouridine deoxyribonucleoside;
[ademG-GalNAc]

means
[ademA-GalNAc]

means and
[MePhosphonate-4O-mU] is

A double-stranded oligonucleotide, characterized in that it means A sodium salt of a double-stranded oligonucleotide, the double-stranded oligonucleotide comprising:
sense strand:
5' mG-S-mA-fC-mA-mA-mA-mA-fA-fU-fC-mC-fU-fC-mA-mC-mA-fA-mU-mA-mA-mG-mC-mA-mG-mC-mC-[ademG-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC -mU-mG-mC 3′ and
Antisense strand:
5' [MePhosphonate-4O-mU]-S-fU-S-fA-S-mU-fU-mG-fU-fG-mA-fG-mG-fA-mU-fU-mU-fU-mU-mG-fU-mC-S-mG-S-mG 3';
including
here,
"-" between nucleosides means a phosphorodiester internucleoside linkage,
internucleoside "-S-" means a phosphorothioate internucleoside linkage,
mA means 2′-O-methyladenosine ribonucleoside;
mG means 2'-O-methylguanosine ribonucleoside;
mC means 2′-O-methylcytidine ribonucleoside;
mU means 2′-O-methyluridine ribonucleoside;
fA means 2′-fluoroadenosine deoxyribonucleoside;
fG means 2'-fluoroguanosine deoxyribonucleoside;
fC means 2′-fluorocytidine deoxyribonucleoside;
fU means 2′-fluorouridine deoxyribonucleoside;
[ademG-GalNAc]

means
[ademA-GalNAc]

means and
[MePhosphonate-4O-mU] is

A sodium salt of a double-stranded oligonucleotide, characterized in that it means the following formula,



A double-stranded oligonucleotide having the structure of A composition comprising the double-stranded oligonucleotide of any one of claims 20-22. 24. The composition of claim 23, further comprising a counterion. 25. The composition of claim 24, wherein said counterion is a Na+ counterion. 24. The composition of Claim 23, further comprising a pharmaceutically acceptable carrier. 27. The composition of claim 26, wherein said pharmaceutically acceptable carrier is water. 24. The composition of claim 23, further comprising Na+ counterions and water. Use of the double-stranded oligonucleotide of any one of claims 20-22 or the composition of any one of claims 23-28 for the manufacture of a medicament for treating chronic hepatitis B infection in a patient. 30. Use according to claim 29, wherein said medicament is administered subcutaneously to said patient. Use according to any one of claims 16-19, 29 and 30, wherein said patient is an HBVe-antigen (HBeAg) positive patient. 32. Use according to claim 31, wherein said HBeAg positive patient has serum HBV surface antigen (HBsAg)>1,000 IU/mL. Use according to any one of claims 16-19, 29 and 30, wherein said patient is an HBeAg negative patient. 34. Use according to claim 33, wherein said HBeAg negative patient has serum HBsAg>500 IU/mL. Use according to any one of claims 16-19, 29 and 30, wherein said patient has serum HBV DNA>20,000 IU/mL. 23. An in vitro method of reducing expression of hepatitis B virus (HBV) surface antigen (HBsAg) in a cell, comprising delivering to said cell an oligonucleotide according to any one of claims 20-22. 37. The method of claim 36, wherein said cells are hepatocytes. 38. The method of claim 36 or 37, wherein said oligonucleotide is delivered in the form of a transgene genetically engineered to express said oligonucleotide in said cell.