Oligonucleotides and their applications in anti-hepatitis B virus

Abstract

The present invention relates to a modified antisense oligonucleotide or a salt thereof and its use in preparing a medicament for preventing or treating HBV-related diseases or symptoms. The modified antisense oligonucleotide can significantly prolong the duration of action while maintaining the efficiency of reducing HBV expression.

Description

[0001] This application claims priority from Chinese patent application CN202311365471.8, filed on October 20, 2023. The description, drawings, and claims of this priority document are incorporated herein in their entirety and made a part of the original description of this application. The applicant further declares that the applicant reserves the right to amend the description and claims of this application based on this priority document. 1. Technical field

[0002] The present invention belongs to the field of biomedicine, and in particular relates to an antisense oligonucleotide and its application in resisting hepatitis B virus. 2. Background Technology

[0003] Chronic hepatitis B (CHB), a chronic infectious disease caused by hepatitis B virus (HBV), is a global health problem, with a significant number of patients remaining undiagnosed and untreated. HBV-infected hepatocytes produce, in addition to intact HBV virions (Dane particles), numerous small spherical particles and tubular particles composed of these particles. These are primarily non-infectious subviral particles (SVPs) composed of the viral small surface protein (HBsAg), lipids, and carbohydrates. Over 99.9% of the hepatitis B surface antigen (HBsAg) in the blood of chronic HBV-infected patients originates from subviral particles, which can neutralize specific hepatitis B surface antibodies (HBsAbs) secreted by B lymphocytes, leading to immune tolerance. HBsAg seroconversion is a prognostic indicator for achieving functional control of viral infection. Another key factor contributing to the slow clearance and persistent presence of HBV in infected hepatocytes is the presence of cccDNA. cccDNA allows HBV to persist in the cell nucleus, unaffected by the host's innate immune response. Furthermore, the long half-life of infected hepatocytes means that cccDNA is constantly present in the nucleus of infected cells, acting as a reservoir for viral genome replication and reactivation. Transcriptional inhibition or clearance of cccDNA is crucial for the cure or functional cure of HBV infection.

[0004] Currently, the primary treatment for chronic hepatitis B is antiviral therapy, which is categorized into two main types: nucleotide-based therapy and interferon therapy. While these two therapies can improve HBsAg clearance and seroconversion rates, they cannot completely eliminate cccDNA, requiring long-term or even lifelong medication for most patients. New therapies that effectively suppress the hepatitis B virus antigens HBsAg and HBeAg and improve seroconversion rates are urgently needed for chronic HBV infection.

[0005] Antisense oligonucleotides (ASOs) are synthetic DNA fragments that bind to target mRNAs of specific sequences, triggering their fragmentation and inducing gene silencing. Currently, several ASO drugs targeting hepatitis B virus are under development, but they still suffer from various drawbacks, such as low knockdown efficiency, high toxicity, and frequent dosing. The present invention screened and obtained ASO sequences that effectively reduce HBV mRNA levels against hepatitis B virus, and through effective chemical modification, they exhibited a more sustained antiviral effect. 3. Summary of the invention

[0006] By analyzing the gene sequences of different HBV subtypes, the inventors selected three sequences that are relatively conserved across different genotypes and designed the antisense oligonucleotide sequences described in the present invention based on these three sequences. The sequences involved are highly conserved in genotypes B and C. The antisense oligonucleotide sequences of 20-21 nt in length in the present invention are all derived from SEQ ID NOs: 1-3.

[0007] In one aspect, the present invention provides a modified oligonucleotide consisting of 10 to 40 linked nucleosides, wherein the modified oligonucleotide is fully or partially complementary to SEQ ID NOs: 1-3. Preferably, the modified oligonucleotide is at least 96%, 97%, 98%, 99% complementary or fully (100%) complementary to SEQ ID NOs: 1-3. Preferably, the modified oligonucleotide consists of 18-23 linked nucleosides; more preferably, the modified oligonucleotide consists of 20 or 21 linked nucleosides.

[0008] Preferably, the modified oligonucleotide is a single-stranded modified oligonucleotide.

[0009] Preferably, at least one nucleoside in the modified oligonucleotide comprises a modified sugar. Preferably, the modified sugar is a 2'-modified sugar. Preferably, the 2'-modified sugar is a 2'-O-methoxyethyl modification.

[0010] Preferably, at least one nucleoside in the modified oligonucleotide comprises a modified nucleic acid base. Preferably, the modified nucleic acid base is 5-methylcytosine.

[0011] Preferably, the modified oligonucleotide comprises: a gap consisting of linked deoxynucleosides and a 5' wing segment and a 3' wing segment consisting of linked nucleosides, wherein the spacer is located between the 5' wing segment and the 3' wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar. Preferably, the gap consists of 3-16 linked nucleosides. Preferably, the modified oligonucleotide comprises: a gap consisting of 8-12 linked deoxynucleosides and a 5' wing segment and a 3' wing segment consisting of 2-6 linked nucleosides, wherein the gap is located between the 5' wing segment and the 3' wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar. Preferably, the modified oligonucleotide comprises: a gap consisting of 10 linked deoxynucleosides and a 5' wing segment and a 3' wing segment consisting of 5 linked nucleosides, wherein the gap is located between the 5' wing segment and the 3' wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar; wherein each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar or a restricted ethyl sugar.

[0012] Preferably, at least one internucleoside bond in the modified oligonucleotide is a modified internucleoside bond. Preferably, each internucleoside bond in the modified oligonucleotide is a phosphorothioate internucleoside bond. Preferably, the internucleoside bonds in the modified oligonucleotide are all phosphorothioate internucleoside bonds and methylsulfonyl-phosphoramidate (MsPA) internucleoside bonds. Preferably, two of the internucleoside bonds in the modified oligonucleotide are MsPA internucleoside bonds, and the remaining internucleoside bonds are phosphorothioate internucleoside bonds. Preferably, the two MsPA internucleoside bonds are two consecutive internucleoside bonds. Preferably, the two consecutive MsPA internucleoside bonds are located in the gap.

[0013] Particularly preferably, the modified oligonucleotide comprises a single-stranded modified oligonucleotide, the single-stranded modified oligonucleotide consisting of nucleosides having the nucleic acid base sequence SEQ ID NO: 82, and the single-stranded modified oligonucleotide comprises a gap consisting of 10 linked deoxynucleosides and a 5' wing segment and a 3' wing segment each consisting of 5 linked nucleosides, wherein the gap is located between the 5' wing segment and the 3' wing segment, and wherein each nucleoside of each wing segment is a 2'-O-methoxyethyl modified nucleoside, the internucleoside bonds in the entire modified oligonucleotide are phosphorothioate bonds and methylsulfonyl-phosphoramidate bonds, and all cytosines in the entire modified oligonucleotide are 5-methylcytosine.

[0014] More preferably, 2-4 of the internucleoside bonds are methylsulfonyl-phosphoramidate internucleoside bonds, and the remaining internucleoside bonds are phosphorothioate internucleoside bonds.

[0015] Further preferably, the methylsulfonyl-phosphoramidate internucleoside linkage is located in the gap.

[0016] More preferably, the two methylsulfonyl-phosphoramidate internucleoside bonds are 2-4 consecutive internucleoside bonds.

[0017] Preferably, the modified oligonucleotide has the following structure:

[0018] GTGAAGCGAAUGUTUGCACACGG;

[0019] GTGAAGCGAAGTUGUCUACACGG;

[0020] GTGAAGUCUGUAUAGTGCACACGG;

[0021] GCAGAGGTUGUAAGCGAAGTGC;

[0022] CGACGTGCAGUAUGUGUTGAAGCG;

[0023] It comprises a gap consisting of 10 connected deoxynucleosides and a 5' wing segment and a 3' wing segment each consisting of 5 connected nucleosides, wherein the gap is located between the 5' wing segment and the 3' wing segment, and each nucleoside in each wing segment is a 2'-O-methoxyethyl modified nucleoside, "u" indicates that the linkage between adjacent nucleosides is methylsulfonyl-phosphoramidate (MsPA), and the nucleoside bonds at other positions are all thiophosphate (P=S) bonds, and all cytosines in the entire modified oligonucleotide are 5-methylcytosine.

[0024] Preferably, the modified oligonucleotide comprises one or more ligands of N-acetylgalactosamine (GalNAc) derivatives. Preferably, the ligands of N-acetylgalactosamine (GalNAc) derivatives are attached to the oligonucleotide via a linker. Preferably, the linker is a monovalent, divalent or trivalent branched linker.

[0025] In another aspect, the present invention provides a use of the modified oligonucleotide or a salt thereof for preparing a medicament for preventing or treating HBV-related diseases or symptoms, wherein the disease or symptom is jaundice, hepatitis, liver fibrosis, inflammation, cirrhosis, liver failure, liver cancer, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, or transplantation associated with liver disease.

[0026] In another aspect, the present invention provides a pharmaceutical composition comprising the modified oligonucleotide or a salt thereof and a pharmaceutically acceptable carrier. Preferably, the composition comprises another drug for treating HBV-related diseases. Preferably, the composition is an injectable pharmaceutical composition, such as a subcutaneous or intravenous pharmaceutical composition.

[0027] In another aspect, the present invention provides a method for preventing or treating HBV-related diseases or symptoms, comprising administering to a patient a modified oligonucleotide or salt thereof, or a composition thereof, as described herein. Preferably, the compound or composition is co-administered with a second agent. Preferably, the second agent is a nucleoside analog or interferon. 4. Description of the accompanying drawings

[0028] Figure 1 The effect of antisense oligonucleotides with different sequences on reducing HBV gene expression in the HepG2 HBV-Luciferase stable cell line was evaluated by detecting the Luciferase signal with a microplate reader at the end of the test.

[0029] Figure 2 The effect of different concentrations of antisense oligonucleotides on reducing HBV gene expression in HepG2 HBV-Luciferase stably transfected cell lines was evaluated by real-time fluorescence quantitative PCR at the end of the test.

[0030] Figure 3 The effect of MsPA-modified antisense oligonucleotides on reducing HBV gene expression in HepG2 HBV-Luciferase stably transfected cell lines was evaluated by real-time fluorescence quantitative PCR at the end of the test.

[0031] Figure 4 The antiviral effect of MsPA-modified anti-oligonucleotides in the AAV-HBV mouse model was evaluated by detecting the HBsAg content in mouse serum using ELISA. 5. Specific implementation methods

[0032] definition

[0033] "Antisense oligonucleotide" or "ASO" refers to an oligonucleotide having a nucleobase sequence complementary to a target nucleic acid or a region or segment thereof. Antisense oligonucleotides can specifically hybridize to a target nucleic acid or a region or segment thereof, and their hybridization results in RNase H-mediated cleavage of the target nucleic acid.

[0034] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error in the method / device employed to determine the value, or variation between study subjects. Typically, the term "about" is meant to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% variation, depending on the circumstances.

[0035] The term "or" used in the claims is intended to mean "and / or" unless explicitly indicated as referring only to alternatives or the alternatives are mutually exclusive, although this disclosure supports a definition referring only to alternatives and "and / or."

[0036] "2'-deoxyfuranosyl sugar moiety" or "2'-deoxyfuranosyl sugar" means a furanosyl sugar moiety having two hydrogens at the 2'-position. 2'-deoxyfuranosyl sugar moieties can be unmodified or modified and can be substituted or unsubstituted at positions other than the 2'-position. In the context of oligonucleotides, β-D-2'-deoxyribosyl sugar moieties are unsubstituted, unmodified 2'-deoxyfuranosyl sugars and are found in naturally occurring deoxyribonucleic acids (DNA).

[0037] "2'-deoxynucleoside" means a nucleoside comprising a 2'-H(H)furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, the 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).

[0038] "2'-O-methoxyethyl" (also known as 2'-MOE) refers to a 2'-O(CH2)2-OCH3 group in place of the 2'-OH group of the ribosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0039] "2'-MOE nucleoside" (2'-O-methoxyethyl nucleoside) means a nucleoside comprising a 2'-MOE modified sugar moiety.

[0040] "2'-substituted nucleoside" or "2'-modified nucleoside" means a nucleoside comprising a 2'-substituted or 2'-modified sugar moiety. As used herein, "2'-substituted" or "2'-modified" with respect to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH.

[0041] "Gapmer" or "gap body" refers to an antisense oligonucleotide that comprises an internal region having multiple nucleosides that support RNase H cleavage positioned between an external region having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically different from the one or more nucleosides comprising the external region. The internal region can be referred to as a "gap" or "spacer", and the external region can be referred to as a "wing". In certain embodiments, the antisense oligonucleotide is a gapmer.

[0042] "Conjugate group" means a group of atoms that is directly attached to a polynucleotide. In certain embodiments, the conjugate group includes a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the polynucleotide.

[0043] The term "complementary" is used to describe the relationship between nucleotide bases and / or polynucleotides that are capable of hybridizing to each other, for example, when the two nucleotide sequences are aligned in opposite directions, the nucleotide sequence of such a polynucleotide or one or more regions thereof matches the nucleotide sequence of another polynucleotide or one or more regions thereof. As used herein, nucleobase matching or complementary nucleobases include the following pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine ( m C) with guanine (G). Complementary polynucleotides and / or nucleic acids need not have nucleobase complementarity at every nucleoside and may include one or more nucleobase mismatches. Therefore, the present disclosure also encompasses isolated polynucleotides that are complementary to the sequences disclosed or used herein, as well as substantially similar nucleic acid sequences. The extent to which two polynucleotides have matching nucleobases can be expressed in terms of "percent complementarity" or "percent complementarity." Unless otherwise indicated, percent complementarity is the percentage of nucleobases of a shorter sequence that is complementary to a longer sequence.

[0044] "Mismatch" or "non-complementary" means that a nucleobase of the first polynucleotide is not complementary to a corresponding nucleobase of the second polynucleotide or target nucleic acid when the first and second polynucleotides are aligned. For example, a nucleobase (including but not limited to universal nucleobases, inosine, and hypoxanthine) can hybridize to at least one nucleobase but is still a mismatch or non-complementary nucleobase with respect to the nucleobase to which it hybridizes. As another example, a nucleobase of the first polynucleotide that is not capable of hybridizing to a corresponding nucleobase of the second polynucleotide or target nucleic acid when the first and second polynucleotides are aligned is a mismatch or non-complementary nucleobase.

[0045] Nucleobases can be naturally occurring or synthetic. Nucleobases and sugar bases can each independently be modified or unmodified. "Modified nucleoside" means a nucleoside comprising a modified nucleobase and / or a modified sugar moiety. Modified nucleosides can include abasic nucleosides lacking a nucleobase.

[0046] "5-methylcytosine" means cytosine with a methyl group attached to position 5. 5-Methylcytosine is a modified nucleobase.

[0047] In the context of oligonucleotides, "consecutive" refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, "consecutive nucleobases" means nucleobases that are immediately adjacent to each other in the sequence.

[0048] "Linked nucleosides" means adjacent nucleosides linked together by an internucleoside linkage.

[0049] "Internucleoside linkages" are covalent linkages between adjacent nucleosides in a polynucleotide. As used herein, "modified internucleoside linkages" means any internucleoside linkage other than a phosphodiester internucleoside linkage.

[0050] "Phosphorothioate linkage" means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced by a sulfur atom. "Methylsulfonyl-phosphoramidate (MsPA) linkage" means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced by a methylsulfonyl-amino group, the structure of which is as follows:

[0051] Methylsulfonyl-phosphoramidate (MsPA) linkages and phosphorothioate internucleoside linkages are both modified internucleoside linkages.

[0052] "Polynucleotide" means a polymer of linked nucleosides, each of which may be independently modified or unmodified. Unless otherwise indicated, a polynucleotide consists of 8-80 linked nucleosides. "Modified polynucleotide" means a polynucleotide in which at least one sugar, nucleobase, or internucleoside linkage is modified. "Unmodified polynucleotide" means a polynucleotide that does not contain any sugar, nucleobase, or internucleoside modifications.

[0053] Oligonucleotides

[0054] In some embodiments, the present disclosure provides oligonucleotides consisting of 8 to 50 linked nucleosides and having at least 90% sequence complementarity with an equal length portion of SEQ ID NOs: 1-3. In some embodiments, the oligonucleotides consisting of 10 to 30 linked nucleosides and having at least 90% sequence complementarity with an equal length portion of SEQ ID NOs: 1-3. In some embodiments, the oligonucleotides consisting of 17 to 23 linked nucleosides and having at least 90% sequence complementarity with an equal length portion of SEQ ID NOs: 1-3.

[0055] In some embodiments, the oligonucleotide has a nucleobase sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% complementary to an equal length portion of SEQ ID NOs: 1-3.

[0056] In some embodiments, the oligonucleotide comprises at least one modification selected from the group consisting of: at least one modified internucleoside linkage, at least one modified sugar moiety, and at least one modified nucleobase.

[0057] In some embodiments, the oligonucleotide comprises at least one modified internucleoside linkage. Naturally occurring internucleoside linkages in RNA and DNA are 3' to 5' phosphodiester linkages. In some embodiments, oligonucleotides having one or more modified (i.e., non-naturally occurring) internucleoside linkages are generally selected over oligonucleotides having naturally occurring internucleoside linkages due to desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for the target nucleic acid, and increased stability in the presence of nucleases.

[0058] In certain embodiments, any internucleoside connection can be used to link the nucleosides of the modified polynucleotide together. Two main types of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside connections include, but are not limited to, phosphates (also referred to as unmodified or naturally occurring connections) containing a phosphodiester bond ("O"), phosphotriesters, methylphosphonates, phosphoramidates, substituted phosphoramidates (such as mesyl-phosphoramidates), and phosphorothioates ("S") and phosphorodithioates ("HS-S"). Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylene azoimino (-CH2-N(CH3)-O-CH2-), thiodiesters, thiocarbamates (-OC(=O)(NH)-S-); siloxanes (-O-SiH2-O-); and N, N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages can be used to alter (typically increase) the nuclease resistance of polynucleotides compared to naturally occurring phosphate linkages. In some embodiments, internucleoside linkages with chiral atoms are prepared as racemic mixtures or as individual enantiomers. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are known to those skilled in the art.

[0059] Representative chiral internucleoside linkages include, but are not limited to, methylsulfonyl-phosphonamidates and phosphorothioates. The modified polynucleotides disclosed herein containing internucleoside linkages with chiral centers can be prepared as a population of polynucleotides containing stereorandom internucleoside linkages or as a population of polynucleotides containing phosphorothioate linkages in a specific stereochemical configuration. In certain embodiments, the population of polynucleotides contains phosphorothioate internucleoside linkages, wherein all phosphorothioate internucleoside linkages are stereorandom. Such polynucleotides can be produced using synthetic methods that result in a random selection of the stereochemical configuration of each phosphorothioate linkage. Nevertheless, each individual phosphorothioate in each individual polynucleotide molecule has a defined stereoconfiguration. In certain embodiments, the population of polynucleotides is enriched for polynucleotides containing one or more specific phosphorothioate internucleoside linkages in a specific, independently selected stereochemical configuration. Unless otherwise specified, the chiral internucleoside linkages of the RNAi polynucleotides described herein can be stereorandom or in a specific stereochemical configuration. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are known to those skilled in the art.

[0060] In certain embodiments, a polynucleotide (eg, an antisense RNAi polynucleotide and / or a sense RNAi polynucleotide) comprises one or more inverted nucleosides.

[0061] In certain embodiments, the reverse nucleoside is a terminal reverse nucleoside (i.e., the last nucleoside on one end of an oligonucleotide), and thus only one internucleoside linkage described above will be present. In certain such embodiments, additional features (such as conjugate groups) may be attached to the reverse nucleoside. Such terminal reverse nucleosides may be attached to either or both ends of a polynucleotide.

[0062] In certain embodiments, such groups lack a nucleobase and are referred to herein as inverted sugar moieties. In certain embodiments, the inverted sugar moiety is a terminal inverted sugar moiety (i.e., the last nucleoside attached to one end of a polynucleotide), and thus only one internucleoside linkage as described above will be present. In certain such embodiments, additional features (such as conjugate groups) may be attached to the inverted sugar moiety. Such terminal inverted sugar moieties may be attached to either or both ends of a polynucleotide.

[0063] In certain embodiments, nucleic acids may be 2' to 5' linked rather than the standard 3' to 5' link.

[0064] In some embodiments, the polynucleotide comprises modified internucleoside linkages arranged in a defined pattern or modified internucleoside linkage motif along the polynucleotide or its region. In some embodiments, the internucleoside linkages are arranged into a motif with a gap. In such embodiments, the internucleoside linkages in each of the two wing regions may be different from the internucleoside linkages in the gap region. In some embodiments, the internucleoside linkages in the wing are phosphodiester, and the internucleoside linkages in the gap are phosphorothioate. The wing and gap lengths may be the same or different. In some embodiments, the gap further comprises n segregants, which are further divided into n+1 subgaps. Wherein, each segregant comprises 1-3 connected nucleosides, and each subgaps consists of 1-10 connected deoxynucleosides, such as those described in CN114507663 and WO2023 / 131098.

[0065] In some embodiments, the polynucleotide comprises one or more methylsulfonyl-phosphoramidate (MsPA) linkages. In some embodiments, the polynucleotide having a gapmer nucleoside motif comprises all phosphorothioate linkages except 1-5 methylsulfonyl-phosphoramidate (MsPA) linkages. In some embodiments, 1-5 consecutive methylphosphonates are linked in the gap of an oligonucleotide having a gapmer nucleoside motif. In some embodiments, 1-5 consecutive methylphosphonates are linked in the wings of an oligonucleotide having a gapmer nucleoside motif. In some embodiments, 1-5 consecutive methylphosphonates are linked in both the gap and the wings of an oligonucleotide having a gapmer nucleoside motif.

[0066] In some embodiments, the number and position of phosphorothioate internucleoside linkages, methylsulfonyl-phosphoramidate (MsPA), and phosphodiester internucleoside linkages can be arranged in a manner that maintains nuclease resistance.

[0067] In some embodiments, the modified polynucleotide comprises at least one modified sugar moiety. In some embodiments, the at least one modified sugar is a bicyclic sugar, 2'-O-methoxyethyl, 2'-F, or 2'-O-methyl.

[0068] Nucleosides comprising modified sugar moieties (e.g., non-bicyclic modified sugar moieties) are referred to by the position or positions of one or more substitutions on the sugar moiety of the nucleoside. For example, a nucleoside comprising a 2′-substituted or 2-modified sugar moiety is referred to as a 2′-substituted nucleoside or a 2-modified nucleoside. Examples of 2′-substituent groups suitable for non-bicyclic modified sugar moieties include, but are not limited to, 2′-F, 2′-OCH3 (“OMe” or “O-methyl”), and 2′-O(CH2)2OCH3 (“MOE”). In some embodiments, the 2′-substituent group is selected from the group consisting of halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O—C1-C 10 Alkoxy, O-C1-C 10 Substituted alkoxy, O-C1-C 10 Alkyl, O-C1-C 10 substituted alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylene-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C(=O)-N(Rm)(Rn), wherein each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 alkyl, -O(CH2)2ON(CH3)2("DMAOE"), 2'-OCH2OCH2N(CH2)2("DMAEOE"), and 2'-substituent groups described in US6,531,584, US5,859,221, and US6,005,087. Some embodiments of these 2'-substituent groups may be further substituted with one or more substituent groups independently selected from the group consisting of hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. In some embodiments, the 2'-substituted nucleoside or 2'-non-bicyclic modified nucleoside comprises a sugar moiety containing a linear 2'-substituent group selected from the group consisting of F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH2, OCH2CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (OCH2C(=O)-N(Rm)(Rn)), wherein each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 alkyl.

[0069] In certain embodiments, the non-bicyclic modified sugar moiety comprises a substituent group at the 4' position. Examples of suitable 4'-substituent groups include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in WO2015 / 106128. In certain embodiments, the non-bicyclic modified sugar moiety comprises a substituent group at the 3' position. Examples of substituent groups suitable for the 3' position of the modified sugar moiety include, but are not limited to, alkoxy (e.g., methoxy), alkyl (e.g., methyl, ethyl). In certain embodiments, the non-bicyclic modified sugar moiety comprises a substituent group at the 5' position. Examples of substituent groups suitable for the 5' position of the modified sugar moiety include, but are not limited to, alkyl (e.g., methyl (R or S)), vinyl, and 5'-alkoxy (e.g., methoxy). In some embodiments, the non-bicyclic modified sugar comprises more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moiety and modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008 / 101157 and Rajeev et al., US 2013 / 0203836.

[0070] In naturally occurring nucleic acids, sugars are linked to each other in a 3' to 5' sequence. In certain embodiments, a polynucleotide includes one or more nucleosides or sugar moieties linked at alternative positions (e.g., 2' or reverse 5' to 3'). For example, when linked at the 2' position, the 2'-substituent group can alternatively be at the 3' position.

[0071] Certain modified sugar moieties contain a bridging sugar substituent that forms a second ring, resulting in a bicyclic sugar moiety. Nucleosides containing such bicyclic sugar moieties have been referred to as bicyclic nucleosides (BNAs), locked nucleosides, or conformationally constrained nucleosides (CRNs). Certain such compounds are described in U.S. Patent Publication No. 2013 / 0190383 and PCT Publication No. WO 2013 / 036868. In some such embodiments, the bicyclic sugar moiety contains a bridge between the 4' and 2' furanose ring atoms. In certain such embodiments, the furanose ring is a ribose ring. Examples of such 4' to 2' bridged sugar substituents include, but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (referred to as "constrained ethyl" or "cEt" when in the S configuration), 4'-CH2-O-CH2-2', 4'-CH2 -N(R)-2', 4'-CH(CH2OCH3)-O-2' ("constrained MOE" or "cMOE") and the like (see, e.g., Seth et al., US7,399,845, Bhat et al., US7,569,686, Swayze et al., US7,741,457 and Swayze et al., US8,022,193), 4'-C(CH3)(CH3)-O-2' and the like (see, e.g., Seth et al., US7,399,845, Bhat et al., US7,569,686, Swayze et al., US7,741,457 and Swayze et al., US8,022,193), S8,278,283), 4'-CH2-N(OCH3)-2' and its analogs (see, for example, Prakash et al., US8,278,425), 4'-CH2-ON(CH3)-2' (see, for example, Allerson et al., US7,696,345 and Allerson et al., US8,124,745), 4'-CH2-C(H)(CH3)-2' (see, for example, Zhou et al., J. Org. Chem. [Journal of Organic Chemistry], 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and its analogs (see, for example, Seth et al., US8,278,426), 4'-C(RaRb)-N(R)-O-2', 4'-C(RaRb)-ON(R)-2', 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2', wherein each R, Ra and Rb are independently H, a protecting group, or C1-C 12 Alkyl (see, for example, Imanishi et al., US 7,427,672).

[0072] In some embodiments, such 4' to 2' bridges independently comprise 1 to 4 linked groups independently selected from: -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]nO-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra)2-, -S(=O)x-, and -N(Ra)-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each Ra and Rb is independently H, a protecting group, a hydroxyl group, a C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 aryl, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, C5-C7 alicyclic, substituted C5-C7 alicyclic, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and each J1 and J2 are independently H, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C (= O) -H), substituted acyl, heterocyclic, substituted heterocyclic, C1-C 12 Aminoalkyl, substituted C1-C 12 aminoalkyl, or a protecting group.

[0073] Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. N. Am. Am., 1996, 4, 466-468; Proceedings of the National Academy of Sciences of the United States of America, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 20017, 129, 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs [Current Views in Drug Research], 2001, 2, 558-561; Braasch et al., Chem. Biol. [Chemical Biology], 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther. [Current Opinion in Molecular Therapy], 2001, 3, 239-243; Wengel et al., US7,053,207, Imanishi et al., US6,268,490, Imanishi et al., US6,770,748, Imanishi et al., USRE44,779; Wengel et al., US6,794,499, Wengel et al., US6,670,461; Wengel et al., US7,034,133, Wengel et al., US8,080,644; Wengel et al., US8,034,909; Wengel et al., US8,153,365; Wengel et al., US7,572,582; and Ramasamy et al., US6,525,191, Torsten et al., WO 2004 / 106356, Wengel et al., WO 91999 / 014226; Seth et al., WO 2007 / 134181; Seth et al., US 7,547,684; Seth et al., US7,666,854; Seth et al., US8,088,746; Seth et al., US7,750,131; Seth et al., US8,030,467; Seth et al., US8,268,980; Seth et al., US8,546,556; Seth et al., US8,530,640; Migawa et al., US9,012,421; Seth et al., US8,501,805; and U.S. Patent Publication Nos. Allerson et al., US2008 / 0039618 and Migawa et al., US2015 / 0191727.

[0074] In some embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, LNA nucleosides (described herein) can be in the α-L configuration or in the β-D configuration.

[0075] In some embodiments, the sugar surrogate comprises a ring having more than 5 atoms. For example, in some embodiments, the sugar surrogate comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted.

[0076] In some embodiments, the sugar surrogate comprises an acyclic moiety. Examples of nucleosides and polynucleotides (e.g., polynucleotides) comprising such acyclic sugar surrogate include, but are not limited to, peptide nucleic acids ("PNAs"), acyclic butyl nucleic acids (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and the nucleosides and polynucleotides described in Manoharan et al., WO 2011 / 133876.

[0077] In certain embodiments, the sugar surrogate is an "unlocked" sugar structure of a UNA (unlocked nucleic acid) nucleoside. UNA is an unlocked, non-cyclic nucleic acid in which any sugar bonds have been removed, thereby forming an unlocked sugar surrogate. Representative U.S. publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, each of which is hereby incorporated by reference in its entirety.

[0078] In certain embodiments, the sugar substitute is GNA (glycol nucleic acid) as depicted below:

[0079] (S)-GNA

[0080]

[0081] wherein Bx represents any nucleobase.

[0082] Modified nucleobases

[0083] In some embodiments, a polynucleotide comprises at least one modified nucleobase. In some embodiments, the at least one modified nucleobase is 5-methylcytosine. In certain embodiments, a polynucleotide comprises one or more inosine nucleosides (i.e., nucleosides comprising the inosine nucleobase). Nucleobase (or base) modifications or substitutions are structurally distinct from naturally occurring or synthetic unmodified nucleobases but functionally interchangeable with them. Both natural and modified nucleobases are capable of hydrogen bonding. Such nucleobase modifications may confer nuclease stability, binding affinity, or other beneficial biological properties on antisense compounds.

[0084] In some embodiments, the polynucleotides described herein comprise modifications, i.e., modified polynucleotides. In some embodiments, the modified polynucleotides comprise one or more nucleosides comprising unmodified nucleobases. In some embodiments, the modified polynucleotides comprise one or more nucleosides comprising modified nucleobases. In some embodiments, the modified polynucleotides comprise one or more nucleosides comprising no nucleobases (referred to as abasic nucleosides).

[0085] In some embodiments, the modified nucleobase is selected from a 5-substituted pyrimidine, a 6-azapyrimidine, an alkyl or alkynyl substituted pyrimidine, an alkyl substituted purine, and N-2, N-6, and O-6 substituted purines. In some embodiments, the modified nucleobase is selected from the group consisting of: 2-aminopropyladenine, 5-hydroxymethylcytosine, 5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (C₃C-CH₃) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, 8-aza and others 8-substituted purines, 5-halo (especially 5-bromo), 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, mixed bases, size-expanded bases, and fluorinated bases. Additional modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenthiazin-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, for example, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.

[0086] Sugar motif

[0087] In some embodiments, the polynucleotides provided herein comprise one or more types of modified sugars and / or unmodified sugar moieties arranged in a defined pattern or sugar motif along the polynucleotide or a region thereof. In some embodiments, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.

[0088] In some embodiments, the modified polynucleotide comprises or consists of a region having a gapmer motif comprising two outer regions or "wings" and a central or inner region or "gap." The three regions of the gapmer motif (5'-wing, gap, and 3'-wing) form a contiguous sequence of nucleosides, wherein at least some of the sugar moieties of the nucleosides in each of these wings differ from at least some of the sugar moieties of the nucleosides in the gap. Specifically, at least the sugar moiety of the nucleoside closest to the gap in each wing (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) differs from the sugar moiety of the adjacent gap nucleoside, thereby defining a boundary between the wing and the gap (i.e., the wing / gap junction). In some embodiments, the sugar moieties within the gap are identical to one another. In some embodiments, the gap comprises one or more nucleosides having a sugar moiety that differs from the sugar moiety of one or more other nucleosides in the gap. In some embodiments, the sugar motifs of the two wings are identical to one another (symmetrical gapmer). In some embodiments, the sugar motif of the 5'-wing differs from the sugar motif of the 3'-wing (asymmetric gapmer).

[0089] In some embodiments, the wings of the gapmer comprise 1-5 nucleosides. In some embodiments, the wings of the gapmer comprise 2-5 nucleosides. In some embodiments, the wings of the gapmer comprise 3-5 nucleosides. In some embodiments, all nucleosides of the gapmer are modified nucleosides.

[0090] In some embodiments, the gap of a gapmer comprises 7-12 nucleosides. In some embodiments, the gap of a gapmer comprises 7-10 nucleosides. In some embodiments, the gap of a gapmer comprises 8-10 nucleosides. In some embodiments, the gap of a gapmer comprises 10 nucleosides. In certain embodiments, each nucleoside in the gap of a gapmer is an unmodified 2′-deoxynucleoside.

[0091] In some embodiments, the gapmer is a deoxygapmer. In such embodiments, the nucleoside on the gap side of each wing / gap junction is an unmodified 2′-deoxynucleoside, and the nucleoside on the wing side of each wing / gap junction is a modified nucleoside. In some such embodiments, every nucleoside of the gap is an unmodified 2′-deoxynucleoside. In some such embodiments, every nucleoside of each wing is a modified nucleoside.

[0092] In some embodiments, the modified polynucleotide has a fully modified sugar motif, wherein each nucleoside of the modified polynucleotide comprises a modified sugar moiety. In some embodiments, the modified polynucleotide comprises or consists of a region having a fully modified sugar motif, wherein each nucleoside of the region comprises a modified sugar moiety. In some embodiments, the modified polynucleotide comprises or consists of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety (referred to herein as a uniformly modified sugar motif). In some embodiments, the fully modified polynucleotide is a uniformly modified polynucleotide. In some embodiments, each nucleoside of a uniformly modified polynucleotide comprises the same 2′-modification.

[0093] In some embodiments, the compounds provided herein comprise or consist of a polynucleotide (modified or unmodified) and, optionally, one or more conjugate groups and / or terminal groups. A conjugate group consists of one or more conjugate moieties and a conjugate linker that connects the conjugate moieties to the polynucleotide. The conjugate group can be attached to either or both ends of the polynucleotide and / or at any internal position. In some embodiments, the conjugate group is attached to the 2' position of a nucleoside of a modified polynucleotide. In some embodiments, the conjugate group attached to either or both ends of the polynucleotide is a terminal group. In certain such embodiments, the conjugate group or terminal group is attached at the 3' and / or 5' end of the polynucleotide. In certain such embodiments, the conjugate group (or terminal group) is attached at the 3' end of the polynucleotide. In some embodiments, the conjugate group is attached near the 3' end of the polynucleotide. In some embodiments, the conjugate group (or terminal group) is attached at the 5' end of the polynucleotide. In some embodiments, the conjugate group is attached near the 5' end of the polynucleotide.

[0094] In some embodiments, the conjugates / end groups of the polynucleotides comprise capping groups, phosphate moieties, protecting groups, and modified or unmodified nucleosides. In some embodiments, the conjugates / end groups include intercalators, reporters, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamins, polyethylene glycols, thioethers, polyethers, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorophores, and dyes.

[0095] In some embodiments, the conjugate / terminal group of a polynucleotide comprises a targeting moiety. In some embodiments, the targeting moiety is at the 5' end of the polynucleotide. In some embodiments, the targeting moiety is at the 3' end of the polynucleotide. In some embodiments, the targeting moiety targets the polynucleotide to a specific subcellular location and / or a specific cell or tissue type. In some embodiments, the targeting moiety comprises a ligand for a receptor. In some embodiments, the receptor is specific for a certain type of cell and / or tissue. In some embodiments, recognition of the targeting moiety (e.g., ligand) by the receptor mediates endocytosis of the polynucleotide conjugated to the targeting moiety.

[0096] In some embodiments, the targeting moiety targets liver cells (also referred to herein as hepatocytes). In some embodiments, the liver cells are human liver cells. In some embodiments, the liver cells express the asialoglycoprotein receptor (ASGPr) on their cell surface. In some embodiments, the targeting moiety is a ligand for ASGPr. In some embodiments, the targeting moiety comprises an N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the targeting moiety comprises 1 to 5 GalNAc moieties. In some embodiments, the targeting moiety comprises 1, 2, 3, 4, or 5 GalNAc moieties. In some embodiments, the targeting moiety comprises 3 GalNAc moieties. In some embodiments, the targeting moiety comprises 3 GalNAc moieties arranged in a triantennary pattern (triantennary GalNAc). In some embodiments, the polynucleotide comprises triantennary GalNAc at the 5′ position of the polynucleotide.

[0097] The compounds described herein include variations in which one or more atoms are replaced by non-radioactive isotopes or radioactive isotopes of the specified element. For example, a compound herein containing a hydrogen atom encompasses a 1 All possible deuterium substitutions of H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include, but are not limited to: 2 H or 3 H instead 1 H. 13 C or 14 C instead 12 C. 15 N instead 14 N. 17 O or 18 O instead 16 O, and 33 S. 34 S. 35 S or 36 S instead 32In certain embodiments, non-radioactive isotope substitution can impart novel properties to oligomeric compounds that are beneficial for use as therapeutic or research tools. In certain embodiments, radioactive isotope substitution can render the compounds suitable for research or diagnostic purposes, such as imaging.

[0098] "Hepatitis B-related condition" or "HBV-related condition" means any disease, biological symptom, medical symptom, or event that is caused by, associated with, related to, or attributable to hepatitis B infection, exposure, or disease. The term hepatitis B-related condition includes chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, and transplantation associated with liver disease.

[0099] Example 1

[0100] General method for preparing Gap antisense oligonucleotides by solid phase technology

[0101] Unless otherwise stated, all reagents and solutions used to synthesize oligomeric compounds were purchased from commercial sources. Standard phosphoramidite building blocks and solid supports were used to incorporate nucleoside residues, including, for example, T, A, G, and mC residues. Phosphoramidite solutions of all monomers (β-D-2'-deoxyribonucleosides and β-D-2'-(MOE) ribonucleosides) used were 0.06 M solutions in anhydrous acetonitrile.

[0102] A 500 nmol synthesis column made of Universal CPG solid support was loaded on an LK-48E synthesizer, and the specified sequence was synthesized using the phosphoramidite coupling method. For the coupling step, the phosphoramidite monomer was delivered in an amount exceeding 4 times the loading on the solid support and the phosphoramidite condensation was continued for 10 min. All other steps were in accordance with the standard protocol supplied by the manufacturer. A solution of 3% trichloroacetic acid in dichloromethane was used to remove dimethoxytrityl (DMT) from the 5'-hydroxyl of the nucleotide. BTT (0.35 M, containing 0.5% NMI) in anhydrous acetonitrile was used as an activator during the coupling step. The phosphorothioate linkage was introduced by sulfurizing with a 0.2 M solution of diphenylacetyl disulfide (PADS) in 1:1 pyridine / acetonitrile for a contact time of 3 minutes.

[0103] After the synthesis of the designated sequence, the solid support-bound designated sequence was suspended in aqueous ammonia (25 wt%-30 wt%) and heated at 85°C for 2 h. The solid support was then filtered off and the ammonia removed under reduced pressure. The residue was purified by high pressure liquid chromatography to produce the Gap antisense oligonucleotides shown in Table 1.

[0104] The length of the Gap antisense oligonucleotide in Table 1 is 20 nucleosides and is designed as a 5-10-5 gap body. The gap contains 10 2'-deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings each containing 5 nucleosides. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment is 2'-MOE sugar modified, each nucleoside in the gap is 2' deoxy sugar modified, the internucleoside bonds in the entire gap body are phosphorothioate (P = S) bonds, and all cytosine residues in the entire gap body are 5-methylcytosine.

[0105] Table 1 Preparation of Gap antisense oligonucleotides by solid phase technology

[0106]

[0107]

[0108] Example 2

[0109] General method for preparing MsPA-modified antisense oligonucleotides using solid-phase technology

[0110] Unless otherwise stated, all reagents and solutions used to synthesize oligomeric compounds were purchased from commercial sources. Standard phosphoramidite building blocks and solid supports were used to incorporate nucleoside residues, including, for example, T, A, G, and mC residues. Phosphoramidite solutions of all monomers (β-D-2'-deoxyribonucleosides and β-D-2'-(MOE) ribonucleosides) used were 0.06 M solutions in anhydrous acetonitrile.

[0111] The 500nmol synthesis column made by Universal CPG solid support is filled on LK-48E synthesizer, and the phosphoramidite coupling method is used to carry out the specified sequence synthesis. For the coupling step, the phosphoramidite monomer is delivered with the amount exceeding 4 times of the load capacity on the solid support and the phosphoramidite condensation is continued for 10min. All other steps are all according to the standard scheme supplied by the manufacturer. The solution of 3% trichloroacetic acid in dichloromethane is used to remove dimethoxytrityl (DMT) from the 5'-hydroxyl of nucleotide. BTT (0.35M, containing 0.5%NMI) in anhydrous acetonitrile is used as the activator in the coupling step process. The phosphorothioate linkage is introduced by sulfurizing the 0.2M solution of diphenylacetyl disulfide (PADS) in 1:1 pyridine / acetonitrile for 3 minutes. The methylsulfonyl-phosphoramidate (MsPA) linkage is introduced by reacting the 1M solution of methylsulfonyl azide (MsN3) in acetonitrile for 2 minutes.

[0112] After synthesizing the designated sequence, the solid support-bound sequence is suspended in aqueous ammonia (25-30 wt%) and heated at 85°C for 2 hours. The solid support is then filtered off and the ammonia removed under reduced pressure. The residue is purified by high-pressure liquid chromatography to produce an MsPA-modified antisense oligonucleotide.

[0113] Example 3

[0114] Luciferase detection of the antiviral effect of antisense oligonucleotides with different sequences in HepG2-CMV-HBV-Luciferase cell line

[0115] The cells used in this experiment are HepG2 HBV-Luciferase stably transfected cell lines that can stably express HBV. After the cells were passed to the third generation, 3x10 4 Cells / well were plated in a 96-well plate. After 12 hours, different sequence oligonucleotides were transfected into the corresponding wells using Lipofectamine RNAiMax (ThermoFisher) transfection reagent. The final concentration of the oligonucleotide was 100 nM, and the blank control group was transfected with DEPC water. After continued culture at 37°C and 5% CO2 for 48 hours, the medium was changed and 50 μL of fresh culture medium was added to each well. At the same time, 50 μL of Bright-Glo Luciferase detection reagent was added to each well. After incubation at room temperature for 1 hour, the Luciferase signal was detected with an enzyme reader. Compared with the cell group treated with DEPC water, the knockdown efficiency of the oligonucleotide was calculated. The knockdown effect of different sequence oligonucleotides on the HBV gene is shown as follows. Figure 1 The antisense oligonucleotides used are shown in Table 2.

[0116] Table 2 Effects of different sequence antisense oligonucleotides on reducing HBV gene expression

[0117]

[0118]

[0119] Test results such as Figure 1 As shown, among the 122 antisense oligonucleotides with different sequences used in this example, 121 reduced the expression of HBV gene in cells, and the knockdown efficiency ranged from 20% to 90%. The antisense oligonucleotides selected by the present invention successfully inhibited the expression of the corresponding mRNA.

[0120] Example 4

[0121] Real-time fluorescence quantitative PCR detection of the antiviral effect of different concentrations of antisense oligonucleotides in HepG2-CMV-HBV-Luciferase cell line

[0122] In order to further verify the inhibitory effect of the antisense oligonucleotides described in the present invention on the HBV gene, we randomly selected 4 to 9 sequences from antisense oligonucleotides derived from different nucleotide fragments, and performed concentration gradient detection on the selected sequences, that is, detecting the antiviral effect of antisense oligonucleotides of different concentrations in cells.

[0123] The cells used in this example are HepG2-CMV-HBV-Luciferase cells that stably express the HBV gene. These cells were seeded in 24-well plates at 1.5 x 10 5 After 12 hours, cells were transfected with oligonucleotides of different sequences using Lipofectamine RNAiMax (ThermoFisher) transfection reagent into corresponding wells at final concentrations of 33 nM and 100 nM. A blank control group was transfected with DEPC water. After further incubation at 37°C, 5% CO₂ for 24 hours, cells were treated with Trizol (Invitrogen) to extract RNA. The resulting RNA was used as a template for reverse transcription using mmlv reverse transcriptase (Promega) to generate cDNA. HBV gene expression was then detected by real-time quantitative PCR using the cDNA as a template and ACTB as an internal reference gene. The expression of HBV genes was assessed using 2^ -ΔΔCT The data were analyzed using the DEPC water-treated cell group. The knockdown efficiency of oligonucleotides at different concentrations was calculated. Figure 2 and as shown in Table 3.

[0124] Test results such as Figure 2 As shown. This example includes a total of 20 antisense oligonucleotides, each with three different concentrations of 0nM, 33nM and 100nM. According to the results of real-time fluorescence quantitative PCR, the oligonucleotides have a dose-dependent effect on HBV knockdown, among which SG12-73, SG12-79 and SG12-85 have better antiviral effects, showing a higher HBV knockdown efficiency at a final transfection concentration of 33nM.

[0125] Table 3 Antiviral effects of different concentrations of antisense oligonucleotides on HBV

[0126]

[0127]

[0128] Example 5

[0129] Real-time fluorescence PCR detection of the antiviral effect of different numbers of MsPA-linked antisense oligonucleotides at different sites in HepG2-CMV-HBV-Luciferase cell line

[0130] Two, three or four MsPA bonds were introduced into different sites of SG12-73, SG12-79 and SG12-85, respectively, and the antiviral effects of the above-mentioned MsPA-modified antisense oligonucleotides in cells were tested.

[0131] The cells used in this example are HepG2-CMV-HBV-Luciferase cells that stably express the HBV gene. These cells were seeded in 24-well plates at 1.5 x 10 5 After 12 hours, cells were transfected with oligonucleotides of different sequences using Lipofectamine RNAiMax (ThermoFisher) transfection reagent into the corresponding wells at a final concentration of 100 nM. A blank control group was transfected with DEPC water. After a further 24 hours of incubation at 37°C and 5% CO₂, cells were treated with Trizol (Invitrogen) to extract RNA. The resulting RNA was used as a template for reverse transcription using mmlv reverse transcriptase (Promega) to generate cDNA. HBV gene expression was then detected by real-time quantitative PCR using the cDNA as a template and ACTB as an internal reference gene. The expression of HBV genes was assessed using 2^ -ΔΔCT The data were analyzed using the DEPC water-treated cell group and the knockdown efficiency of the oligonucleotides was calculated. Figure 3 The specific MsPA modification sites are shown in Table 4.

[0132] Table 4 Knockdown efficiency of MsPA-modified antisense oligonucleotides

[0133]

[0134]

[0135] The results are as follows Figure 3 As shown, all 58 antisense oligonucleotides with different sequences and sites modified with MsPA in this example exhibited some antiviral efficacy. Compared to antisense oligonucleotides without MsPA modification, the antisense oligonucleotides exhibited varying antiviral efficacy after modification: antisense oligonucleotides with MsPA modification on the SG12-73 and SG12-85 sequences exhibited improved efficacy in some cases, but the antiviral efficacy of most was significantly reduced. Surprisingly, antisense oligonucleotides with MsPA modification on the SG12-79 sequence exhibited generally stable efficacy, demonstrating stronger or comparable antiviral efficacy.

[0136] Example 6

[0137] Antiviral effect of MsPA-modified antisense oligonucleotides in an AAV-HBV mouse model

[0138] In this embodiment, a mouse model of persistent HBV infection was obtained by injecting rAAV8-1.3HBV into the tail vein of C57 mice. The injection dose of AAV virus was 1E+11vg / mouse. Blood was collected 3 weeks after the virus injection to detect the HBsAg content in the serum. The mice were randomly divided into 3 groups after determining the stable replication of HBV virus in the individual based on the results of the mouse serum HBsAg test. The first group was a normal saline control group, with a total of 4 mice; the second group was a positive control group, with a total of 3 mice, and the positive control drug was SG12-79; the third group was given an antisense oligonucleotide modified with MsPA at positions 3 and 4 (SG12-79-2), with a total of 4 mice. Each group of mice was given a single dose of 100 mg / kg by subcutaneous injection, and after the administration was completed, blood was collected every 5 days to detect the HBsAg content in the serum. That is, the day of administration was defined as D0, and mice in each group were administered on D0. At the same time, blood was collected on D5, D10, D15, D20, D25, D30 and D35 to detect the HBsAg content in the serum of each model mouse, and compared with the HBsAg content in the serum on D-2 to determine the anti-HBV effect of different oligonucleotides in the mouse model. Figure 4 and as shown in Table 5.

[0139] After a single dose of AAV-HBV model mice, the HBsAg content in the serum was significantly reduced. Among them, the antiviral effect of the MsPA-modified antisense oligonucleotide sequence in mice was similar to that of the positive control drug, but the duration of the effect was significantly longer. Figure 4 As shown, the antiviral effect of the positive drug control group gradually decreased after the completion of a single dose of the drug, and the HBsAg content in the serum of the model mice gradually recovered. On the 30th day after administration, the HBsAg content in the serum had returned to the level before administration. On the 35th day after administration, the HBsAg content in the serum of this group of model mice increased by 0.11log10 compared with before administration; while the antisense oligonucleotide containing 3- and 4-position MsPA modifications showed a more lasting antiviral effect in the model mice. After 30 days of drug treatment, the HBsAg content in the serum of the model mice was still reduced by 0.52log10 compared with before administration. After 35 days of drug treatment, the HBsAg content in the serum of the model mice was still reduced by 0.32log10 compared with before administration.

[0140] Table 5 In vivo HBV antiviral effect of MsPA modified antisense oligonucleotides

[0141]

Claims

1. A single-stranded modified oligonucleotide having the following structure: GTGAAGCGAA U G U T U GCACACGG; GTGAAGCGAAGT U G U C U ACACGG; GTGAAG U C U G U A U AGTGCACACGG; GCAGAGGT U G U AAGCGAAGTGC; CGACGTGCAG U A U G U G U TGAAGCG; It comprises a gap consisting of 10 linked deoxynucleosides and a 5' wing segment and a 3' wing segment each consisting of 5 linked nucleosides, wherein the gap is located between the 5' wing segment and the 3' wing segment, and wherein each nucleoside in each wing segment is a 2'-O-methoxyethyl modified nucleoside, "u" indicates that the linkage between adjacent nucleosides is methylsulfonyl-phosphoramidate (MsPA), and the nucleoside bonds at other positions are all phosphorothioate (P=S) bonds, and all cytosines in the entire modified oligonucleotide are 5-methylcytosine.

2. The oligonucleotide according to claim 1, wherein the modified oligonucleotide comprises one or more ligands of N-acetylgalactosamine derivatives.

3. A pharmaceutical composition comprising the oligonucleotide or a salt thereof according to claim 1 and a pharmaceutically acceptable carrier.

4. Use of the oligonucleotide or a salt thereof according to claim 1 in the preparation of a medicament for preventing or treating HBV-related diseases or symptoms.

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