HBV-targeting small nucleic acid

Abstract

The present disclosure relates to an RNAi active agent for inhibiting hepatitis B virus (HBV) and use thereof. The RNAi active agent can be used for treating and / or preventing HBV-related diseases or disorders, for example, hepatitis B virus infection or related diseases, such as acute or chronic hepatitis, such as hepatitis B or hepatitis D, cirrhosis, or liver cancer, such as hepatocellular carcinoma.

Description

Small nucleic acids targeting HBV

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202510092717.1, filed on January 21, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of small nucleic acid drugs, and more specifically to small nucleic acid drugs targeting hepatitis B virus (HBV) and their applications, wherein the small nucleic acid drugs can be used to treat and / or prevent HBV-related diseases or conditions, such as hepatitis B virus infection or related diseases such as acute or chronic hepatitis, hepatitis B or hepatitis D, cirrhosis or liver cancer such as hepatocellular carcinoma. Background Technology

[0004] RNAi

[0005] Oligonucleotides are polymers of nucleotides (RNA, DNA, and their analogues). Nucleic acid inhibitor molecules are oligonucleotides that regulate intracellular RNA levels and have shown promise in the treatment of genetic diseases, metabolic diseases, cancer, and viral infections. Nucleic acid inhibitor molecules can regulate RNA expression through a different set of mechanisms, including RNA interference (RNAi).

[0006] RNAi is a conserved pathway found in most eukaryotes. A typical form of RNAi inhibitor is a double-stranded RNA (dsRNA) molecule consisting of an antisense strand and a sense strand that form a double-stranded region. This double-stranded RNA (dsRNA) molecule can inhibit the expression of a target gene that is complementary to the antisense strand of the dsRNA. In a typical RNAi pathway, a longer dsRNA molecule can be cleaved by Dicer into a shorter RNA double strand (also known as “small interfering RNA,” siRNA). It has been shown that siRNA binds to Dicer, trans-activating response RNA-binding protein (TRBP), and Argonaute 2 (Ago2) to form a complex known as the RNA-induced silencing complex (RISC). Ago2 is a nuclease that uses the antisense strand (also known as the guide strand) of the siRNA to direct sequence-specific cleavage of the target mRNA, thereby cleaving the target mRNA.

[0007] Various RNAi molecules have been developed over the years. For example, early work on RNAi inhibitor molecules focused on double-stranded nucleic acid molecules mimicking natural siRNA, where each strand has 19-25 nucleotides and includes at least one 3' overhang with 1 to 5 nucleotides (see, for example, U.S. Patent No. 8,372,968). Subsequently, longer dsRNA molecules were developed, which are cleaved in vivo by Dicer into active RNAi inhibitor molecules (see, for example, U.S. Patent No. 8,883,996). Subsequent work developed double-stranded nucleic acid inhibitor molecules with extension modifications at the 5' and / or 3' ends, such modifications including, for example, targeting ligands, pharmacokinetic modulators, and thermodynamically stable tetracyclic structures (see, for example, WO2015 / 173208 and WO2016100401). These extensions can be single-stranded or double-stranded.

[0008] In recent years, the application of modified nucleosides in siRNA has increased significantly. Examples include modifications to the sugar rings of ribonucleic acid, such as 2'-fluoro (2'-F) modification, 2'-O-methyl (2'-OMe) modification, 2'-O-methoxyethyl (2'-O-MOE) modification, non-locked nucleotide (UNA) modification, locked nucleotide (LNA) modification, 4'-CH(CH3)-O-2'(cEt) modification, and diol nucleic acid (GNA) modification; and the replacement of 5'-phosphate esters with phosphatase resistance analogs, such as 5'-(E)-vinylphosphonate (5'-(E)-VP). It has been demonstrated that, in some cases, depending on the RNAi molecule used, chemical modifications can be introduced at specific sites on the inhibitor molecule to modulate its properties under application conditions, such as stability, target cell uptake, or specificity or inhibitory activity against the target gene.

[0009] While computer predictions can be helpful in RNAi molecular design to some extent, they still have significant limitations. For example, they cannot accurately predict properties such as RNA secondary structure, and they struggle to simulate the dynamics and complexity of the actual cellular environment. These factors all affect the practical effectiveness of computationally predicted RNAi. Therefore, selecting the antisense and sense strand sequences of RNAi and their chemical modifications to achieve high-level gene silencing efficacy against target mRNA genes, as well as desired properties such as high stability and specificity, and the elimination or reduction of off-target effects, remains a significant challenge in this field.

[0010] HBV

[0011] Hepatitis B virus (HBV) is a hepatotropic virus belonging to the Hepatoviridae family. A complete viral particle is a spherical object with a diameter of 42 nm. The HBV genome is generally about 3.2 kb in length and contains four open reading frames (ORFs): S, C, P, and X, which have significant sequence overlap. ORF-C encodes HBe antigen (HBeAg) and HBc antigen (HBcAg), ORF-S encodes HBs antigen (HBsAg), ORF-P encodes viral DNA polymerase, and ORF-X encodes the X protein. HBcAg plays a crucial role in HBV infection, reflecting the presence of Dane particles in serum and HBV replication in the liver. It can also interact and complement other HBV serological markers. However, due to the strong affinity of HBcAg antibodies, they rapidly bind to HBcAg in serum, forming immune complexes, making it difficult to detect free HBcAg in serum. Mutations in the pre-C region gene can affect HBeAg production, resulting in negative antigen tests. This does not reflect a reduction or disappearance of HBV replication; rather, it may increase the risk of severe hepatitis. The X protein plays a significant role in regulating HBV DNA replication. The X gene region contains core promoter and enhancer elements related to transcription; mutations in this region can affect HBV transcriptional and translational levels. HBsAg is the antigen that HBV infects through the sodium ion-taurocholic acid cotransporter (NTCP) on the hepatocyte membrane. This antigen appears in the blood circulation of patients in the early stages of HBV infection and can last for months, years or even a lifetime. It is the most commonly used indicator for diagnosing HBV infection.

[0012] The spontaneous error rate and lack of proofreading activity of HBV viral polymerase are the reasons for the numerous subtypes of the virus. Based on genotypic differences of >7.5%–8%, HBV is mainly divided into 10 genotypes, A, J, and D. The predominant subtypes and subtypes of the virus circulating in different regions vary greatly, as do natural infection histories, clinical manifestations, and responses to antiviral drugs. HBV infection is a global epidemic, with HBV types A, B, C, and D being the main infectious subtypes worldwide, accounting for approximately 80% of all infections.

[0013] HBV is highly adaptable to the external environment, surviving for at least 6 months at 30℃-32℃ and up to 15 years at ~20℃. Several key immunological characteristics of HBV allow it to persist for a long time and be difficult to eliminate. Its replication within hepatocytes goes undetected by the innate immune system, generating and secreting large amounts of viral antigens, gradually altering and depleting the function of HBV-specific T and B cells. Therefore, infection caused by this virus has become a global public health problem, with over 350 million people worldwide chronically infected. Hepatitis B patients can be classified as follows: a) HBeAg-positive chronic hepatitis B: positive serum HBsAg, HBV DNA, and HBeAg, negative anti-HBe, persistently or repeatedly elevated serum ALT, or hepatitis lesions on liver histology. b) HBeAg-negative chronic hepatitis B: positive serum HBsAg and HBV DNA, persistently negative HBeAg, positive or negative anti-HBe, persistently or repeatedly abnormal serum ALT, or hepatitis lesions on liver histology. Studies have found that 51.3% of patients with chronic hepatitis B (CHB) are HBeAg negative. As HBsAg is the main route of HBV infection, serum HBsAg positivity is one of the markers of HBV infection. Therefore, detecting serum HBsAg expression levels is particularly important for evaluating the effectiveness of HBV treatment.

[0014] Currently, there is no cure for chronic hepatitis B (CHB). Nucleotide analogues (NAs) and pegylated interferon (PEG-IFN) are the mainstream treatment options. NA treatment effectively inhibits HBV DNA replication, but has almost no effect on reducing HBsAg levels and requires lifelong medication; the disease rebounds once medication is discontinued. PEG-IFN treatment can provide long-term immune control, but the rate of achieving immune control is low. Some reports indicate that only 3% of individuals receiving PEG-IFN treatment achieved HBsAg clearance after 48 weeks. Furthermore, it has been found that the probability of HBsAg clearance is even lower in CHB patients with high HBsAg expression treated with PEG-IFN.

[0015] Invention Description

[0016] This disclosure describes the design of RNAi, such as siRNA, based on conserved regions of HBV. It has been found that these RNAi exhibit antiviral activity against HBV in multiple animal models and can significantly reduce the expression levels of HBV DNA, HBV transcripts, and / or HBV proteins.

[0017] Therefore, this disclosure provides novel RNAi agents and conjugates thereof that inhibit HBV gene expression via RNA interference. The RNAi agents or conjugates thereof according to this disclosure exhibit good HBV gene inhibitory activity and / or sustained efficacy, and in some further preferred embodiments, further enhanced sustained efficacy is achieved through optimization of sequence nucleotide modification, delivery, and / or modification. The RNAi agents or conjugates thereof according to this disclosure are suitable for the prevention and / or treatment of HBV-related diseases or conditions, such as hepatitis B virus infection or related diseases such as acute or chronic hepatitis such as hepatitis B (e.g., acute or chronic hepatitis B) or hepatitis D (e.g., acute or chronic hepatitis D), cirrhosis, or liver cancer such as hepatocellular carcinoma.

[0018] Therefore, in a first aspect, this disclosure provides an RNAi activator, wherein the RNAi activator comprises an antisense strand and a sense strand complementary to at least a portion of a sequence in the antisense strand to form a double-stranded region, wherein the antisense strand comprises a sequence motif complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or all of a sequence of target genes selected from the following sequences, preferably, the complementarity refers to at least 70%, at least 80%, at least 85%, at least 90%, or 100% complementarity:

[0019] (i) The nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6;

[0020] (ii) At least 15-35 consecutive nucleotides, such as 18, 19, 20, or 21 consecutive nucleotides, starting from position 1096, 1097, 1098, 1099, or 1100 of the HBV genome; or

[0021] (iii) At least 15-35 consecutive nucleotides, such as 18, 19, 20 or 21 consecutive nucleotides, starting from position 972, 973, 974, 975 or 976 of the HBV genome.

[0022] In some embodiments, the RNAi activator according to this disclosure comprises an antisense strand whose sequence motif is perfectly complementary to at least 15, 16, 17, 18, or 19 consecutive nucleotides selected from the target gene sequences listed below:

[0023] (i) The nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6;

[0024] (ii) At least 15-35 consecutive nucleotides, such as 18, 19, 20, or 21 consecutive nucleotides, starting from position 1096, 1097, 1098, 1099, or 1100 of the HBV genome; or

[0025] (iii) At least 15-35 consecutive nucleotides, such as 18, 19, 20 or 21 consecutive nucleotides, starting from position 972, 973, 974, 975 or 976 of the HBV genome.

[0026] In some implementations, the sense strand includes a region complementary to at least 15 or at least 19 consecutive nucleotides in the antisense strand.

[0027] In some implementations, the length of the double-stranded region formed by the complementarity of the sense strand and the antisense strand is at least 15, 16, 17, 18 or 19 nucleotides, preferably at least 19 nucleotides.

[0028] In some embodiments, the antisense strand is 17-30 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides; and / or the sense strand is 17-30 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0029] In some embodiments, the RNAi activator according to this disclosure comprises a double strand with a protruding end consisting of an antisense strand and a sense strand, preferably wherein the protruding end is a 3' protruding end consisting of 1, 2, 3 or 4 nucleotides from the terminal end of the 3' of the antisense strand, preferably a 3' protruding end consisting of 2 nucleotides, preferably the 2 nucleotides being GG.

[0030] In some embodiments, the RNAi activator according to this disclosure comprises an antisense strand and a sense strand, wherein: the antisense strand comprises a sequence motif differing by 0, 1, 2, or 3 nucleotides from the antisense strand sequence of any compound in Table A or Table 1 or the antisense strand sequence shown in SEQ ID NO: 3 or 4; and / or the sense strand comprises a sequence motif differing by 0, 1, 2, or 3 nucleotides from the sense strand sequence of any compound in Table A or Table 1 or the sense strand sequence shown in SEQ ID NO: 1 or 2.

[0031] In some embodiments, the RNAi activator according to this disclosure comprises an antisense strand and a sense strand, wherein: the antisense strand comprises a sequence motif differing by 0, 1, 2, or 3 nucleotides from the antisense strand sequence of at least 15, 16, 17, 18, 19, 20, or 21 of the compounds in Table AC or Tables 1-3; and the sense strand comprises a sequence motif differing by 0, 1, 2, or 3 nucleotides from the sense strand sequence of at least 15, 16, 17, 18, or 19 of the corresponding compounds in Table AC or Tables 1-3.

[0032] In some embodiments, the RNAi activator according to this disclosure comprises an antisense strand and a sense strand, wherein the lengths of the antisense strand and the sense strand are between 17 and 30 nucleotides, or between 18 and 30 nucleotides, or between 19 and 30 nucleotides, respectively. In some embodiments, the antisense strand is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long, and / or the sense strand is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the antisense strand and the sense strand form a double-stranded region of 17-30, 18-30, 19-30, 17-25, 18-25, 19-25, 17-23, 18-23, or 19-23 nucleotide pairs in length. More preferably, the double-stranded region is 15-25 nucleotide pairs long, for example, 17-21 nucleotide pairs, 18-21 nucleotide pairs, 19-21 nucleotide pairs, or 15, 16, 17, 18, or 19 nucleotide pairs. More preferably, the antisense strand is 21 nucleotides long, the sense strand is 19 nucleotides long, and the double-stranded region is 19 nucleotide pairs long. In some embodiments, the antisense strand and the sense strand are at least 70%, 80%, 90%, or 100% complementary in the double-stranded region.

[0033] In some embodiments, preferably, the antisense strand comprises an antisense strand sequence selected from any compound selected from Table A or Table 1; and / or the sense strand comprises a sense strand sequence selected from any compound selected from Table A or Table 1.

[0034] In some embodiments, the antisense strand comprises a sequence motif that differs from at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides in the antisense strand sequence shown in SEQ ID NO:3 or SEQ ID NO:4 by 0, 1, 2, or 3 nucleotides; and / or the sense strand comprises a sequence motif that differs from at least 15, 16, 17, 18, or 19 consecutive nucleotides in the sense strand sequence shown in SEQ ID NO:1 or SEQ ID NO:2 by 0, 1, 2, or 3 nucleotides.

[0035] In some embodiments, the antisense strand comprises a sequence motif that differs from at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides in the antisense strand sequence shown in SEQ ID NO:3 by 0, 1, 2, or 3 nucleotides; and / or the sense strand comprises a sequence motif that differs from at least 15, 16, 17, 18, or 19 consecutive nucleotides in the sense strand sequence shown in SEQ ID NO:1 by 0, 1, 2, or 3 nucleotides.

[0036] In some embodiments, the antisense strand comprises a sequence motif that differs from at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides in the antisense strand sequence shown in SEQ ID NO:4 by 0, 1, 2, or 3 nucleotides; and / or the sense strand comprises a sequence motif that differs from at least 15, 16, 17, 18, or 19 consecutive nucleotides in the sense strand sequence shown in SEQ ID NO:2 by 0, 1, 2, or 3 nucleotides.

[0037] In some embodiments, more preferably, the antisense strand comprises or is composed of the nucleotide sequence shown in SEQ ID NO:3, and the sense strand comprises or is composed of the nucleotide sequence shown in SEQ ID NO:1; or the antisense strand comprises or is composed of the nucleotide sequence shown in SEQ ID NO:4, and the sense strand comprises or is composed of the nucleotide sequence shown in SEQ ID NO:2.

[0038] In some embodiments, more preferably, the unmodified nucleotide sequence of the antisense strand is as shown in SEQ ID NO:3, and the unmodified nucleotide sequence of the sense strand is as shown in SEQ ID NO:1; or the antisense strand is as shown in SEQ ID NO:4, and the sense strand is as shown in SEQ ID NO:2.

[0039] In a second aspect, this disclosure provides an RNAi activator, which is a modified form of the RNAi activator according to the first aspect of this disclosure.

[0040] In some embodiments, the modification includes at least one phosphate backbone modification and / or at least one nucleotide modification.

[0041] In some embodiments, the RNAi activator comprises at least one phosphate thioester or methyl phosphate linker.

[0042] In some preferred embodiments, the RNAi activator contains one or two phosphate thioester links at the 5' and / or 3' ends (preferably the 5' and 3' ends) of the antisense strand.

[0043] In some embodiments, the first to third nucleotides at the 5' end of the antisense and sense strands, as well as the first to third nucleotides at the 3' end, are linked by a phosphate thioester bond.

[0044] In some embodiments, any one or more G-base nucleosides in the RNAi activator are replaced by nucleoside analogs. In some embodiments, one G-base nucleoside in the RNAi activator is replaced by a nucleoside analog. The nucleoside analog can be any structural analog of a nucleoside containing a nucleobase and a sugar, known in the art, wherein 1-3 phosphate groups are linked to the nucleoside. In some embodiments, the nucleoside analog is a methylene-substituted C-nucleotide mimic (E) or L-2'-deoxy-2'-fluororibonucleotide (CLV).

[0045] In some embodiments, the G-base nucleoside of the RNAi activator selected from the following positions is replaced by a nucleoside analog:

[0046] (i) One or two G base nucleosides, preferably two G base nucleosides, from the 3' end of the antisense strand (e.g., the G base at the 20th or 21st position from the 5' end);

[0047] (ii) The G base nucleoside in the sense strand corresponding to the 9th position from the 5' end of the antisense strand;

[0048] (iii) The G base nucleoside in the sense strand corresponding to the 13th position from the 5' end of the antisense strand;

[0049] (iv) Any one or more G-base nucleotides at other positions in the positive chain; or

[0050] (v) Combinations of the above positions.

[0051] In some embodiments, the nucleoside analog is linked to its adjacent nucleotide via a phosphate thioester group. In some embodiments, the nucleoside analog is directly linked to its adjacent nucleotide.

[0052] In some preferred embodiments, the two G-nucleotides at the 3' end of the antisense strand of the RNAi activator are replaced by nucleoside analogs, optionally the two nucleoside analogs are linked by a thiophosphate group, and optionally the nucleoside analog is linked to the third nucleotide at the 3' end of the antisense strand via a thiophosphate group.

[0053] In some preferred embodiments, one or two of the sense strands corresponding to the 9th position from the 5' end or the 13th position from the 5' end of the antisense strand of the RNAi activator, preferably one G base nucleoside, are replaced by a nucleoside analog.

[0054] In some preferred embodiments, the nucleoside analog is a methylene-substituted carbon glycoside nucleotide analog (E).

[0055] In some embodiments, any one of the G bases of the RNAi activator is replaced by a methylene-substituted C-nucleotide mimic (E); in some embodiments, one G base of the RNAi activator is replaced by a methylene-substituted C-nucleotide mimic (E). In some embodiments, the two G bases at the 3' end of the antisense strand of the RNAi activator are replaced by a methylene-substituted C-nucleotide mimic (E). In some embodiments, one or two, preferably one, G bases of the sense strand corresponding to the 9th or 13th position from the 5' end of the antisense strand of the RNAi activator are replaced by a methylene-substituted C-nucleotide mimic (E).

[0056] In some embodiments, the structure of the methylene-substituted carbon glycoside nucleotide mimic (E) is shown in A18.

[0057] Where X includes O, S, Se, CH2, CH-CH3, and R includes H, OH, OMe, F, OMOE, etc.

[0058] Preferably, X represents CH2 and R represents H, and its structure is shown in A19:

[0059] In some embodiments, the RNAi activator comprises at least one 2'-modified nucleotide, particularly selected from 2'-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl (2'-O-MOE).

[0060] In some embodiments, the RNAi activator further comprises at least one modified nucleotide selected from the following: deoxyribonucleotides, nonlocked nucleotides (UNA), locked nucleotides (LNA), and threonucleotides (TNA).

[0061] In some embodiments, the RNAi activator comprises a 5'-terminal phosphate modification, preferably wherein the modification is a 5'-(E)-vinylphosphonate modification at the 5' end of the antisense strand, and / or a threonucleotide modification at the 5' end of the sense strand.

[0062] In some embodiments, the RNAi activator comprises a 4'-modified threonucleotide located at the 5' terminal of the positive strand, particularly a 4'-modified threonucleotide having the following formula. Wherein, Base represents a natural or modified nucleoside base, wherein the natural nucleoside base is A, T, C, G or U, and R represents an alkyl group having 1-30 carbon atoms, preferably, R represents a straight-chain alkyl group with 12 carbon atoms.

[0063] In some preferred embodiments, the RNAi activator comprises a modified sense strand (SS) and a modified antisense strand (AS) of any of the compounds shown in Table 2 or Table B. More preferably, the RNAi activator comprises a modified sense strand (SS) and a modified antisense strand (AS) of any of the compounds selected from Table B.

[0064] In a third aspect, this disclosure provides an RNAi conjugate comprising a non-nucleoside conjugate portion conjugated to a double-stranded oligonucleotide of an RNAi activator according to a first or second aspect of this disclosure.

[0065] In some embodiments, the conjugation occurs at the 5' and / or 3' end of the sense strand of the RNAi and optionally at the 3' end of the antisense strand. Optionally, the conjugation is performed via thiophosphate group linkage or phosphate group linkage.

[0066] In some preferred embodiments, the RNAi conjugate comprises an asialic acid glycoprotein receptor (ASGPR) ligand, particularly a GalNAc ligand, such as a monovalent, divalent, trivalent, or tetravalent ligand. In this document, the double-stranded RNAi conjugates of this disclosure comprising such ligands are also referred to simply as GalNAc-siRNA conjugates, G, or GalNAc-siRNA.

[0067] In some particularly preferred embodiments, the GalNAc ligand has a P36 structure according to the present disclosure; preferably, the GalNAc-siRNA conjugate has two GalNAc ligands located at the 5' and 3' ends of the positive strand, respectively; or the GalNAc-siRNA conjugate has two GalNAc ligands located at the 5' end of the positive strand.

[0068] In some other particularly preferred embodiments, the GalNAc ligand has an L96 structure according to the present disclosure, and preferably, the GalNAc-siRNA conjugate has the GalNAc ligand located at the 3' end of the positive strand.

[0069] In other particularly preferred embodiments, the GalNAc ligand has a P34 structure according to this disclosure. Preferably, the GalNAc-siRNA conjugate has the GalNAc ligand located at the 5' end of the positive strand, for example, the GalNAc-siRNA conjugate has two GalNAc ligands located at the 5' end of the positive strand.

[0070] In some other particularly preferred embodiments, the GalNAc ligand has a P67 structure according to this disclosure. Preferably, the GalNAc-siRNA conjugate has the GalNAc ligand located at the 5' end of the positive strand. Preferably, the GalNAc-siRNA conjugate has a GalNAc ligand with a P36 structure and a GalNAc ligand with a P67 structure located at the 5' end of the positive strand.

[0071] In some preferred embodiments, the RNAi conjugate comprises a modified sense strand (SS) and a modified antisense strand (AS) with a nonnucleotide moiety conjugated to any of the compounds shown in Table 3 or Table C. More preferably, the RNAi conjugate comprises a modified sense strand (SS) and a modified antisense strand (AS) with a nonnucleotide moiety conjugated to any of the compounds selected from Table C.

[0072] In some more preferred embodiments, this disclosure provides compounds selected from any of Table 3 or Table C.

[0073] In a fourth aspect, this disclosure provides compositions comprising an RNAi active agent or RNAi conjugate according to this disclosure, such as pharmaceutical compositions, and their uses.

[0074] In one embodiment, the use is for use as a medicine, or for use in the preparation of a medicine. In some embodiments, the medicine is used to reduce HBV levels and / or expression in a subject, or for the prevention or treatment of HBV-mediated diseases or conditions.

[0075] In a fifth aspect, this disclosure also provides a method for reducing HBV levels and / or expression in a subject, or for preventing or treating HBV-mediated diseases or conditions, comprising administering to a subject in need an effective amount of an RNAi activator or RNAi conjugate according to this disclosure, particularly siRNA conjugates as shown in Table 3 of this disclosure.

[0076] In some implementations, the disease or condition includes HBV-mediated diseases or conditions, such as hepatitis B virus infection or related diseases, such as acute or chronic hepatitis, such as hepatitis B (e.g., acute or chronic hepatitis B) or hepatitis D (e.g., acute or chronic hepatitis D), cirrhosis, or liver cancer such as hepatocellular carcinoma.

[0077] In some implementations, the subject is a mammal, particularly a human individual.

[0078] In some embodiments, the method includes administering the RNAi activator or RNAi conjugate according to this disclosure subcutaneously or intravenously.

[0079] In some embodiments, the RNAi activator or RNAi conjugate described herein is in the form of a salt.

[0080] Table A below provides examples of some representative RNAi sequence motifs according to this disclosure. In Table A, "SS" represents the sense strand and "AS" represents the antisense strand. In some cases, RNAi activators according to this disclosure that contain RNAi sequence motifs (unmodified nucleotide sequences) provided in Table A below are more preferred in the first to fifth aspects of this disclosure described above.

[0081] Table A: Sequence motifs of representative siRNA duplexes

[0082] Table B below provides examples of some representative RNAi according to this disclosure, where “SS” represents the sense strand and “AS” represents the antisense strand. In some cases, RNAi activators according to this disclosure that include modified sense and modified antisense strands as provided in Table B below are more preferred in the first to fifth aspects described above.

[0083] Table B: Modified siRNA duplexes

[0084] Table C below provides examples of some representative RNAi conjugates according to this disclosure, where "SS" represents the sense strand and "AS" represents the antisense strand. In some cases, the RNAi conjugates provided in Table C below are more preferred in the first to fifth aspects of this disclosure described above.

[0085] Table C: siRNA duplexes modified with conjugates Attached image description:

[0086] Figure 1: Expression of HBV DNA in plasma in an AAV-HBV mouse model.

[0087] Figure 2: Plasma HBsAg expression in the AAV-HBV mouse model.

[0088] Figure 3: Plasma HBsAg expression in the AAV-HBV mouse model.

[0089] Figure 4: Plasma HBV DNA expression in the AAV-HBV mouse model.

[0090] Figure 5: Plasma HBeAg expression in the AAV-HBV mouse model.

[0091] Figure 6: Serum HBsAg expression in HBV transgenic mice.

[0092] definition

[0093] In this article, unless the context clearly indicates otherwise, the singular forms “a,” “one,” and “the” cover both singular and plural referents.

[0094] In this document, the terms "about" or "approximately," when referring to a measurable value (e.g., a parameter, quantity, duration, etc.), are intended to encompass both the specified value and variations relative to that specified value, such as variations of + / -10% or less, +1-5% or less, + / -1% or less, and + / -0.1% or less relative to the specified value, provided that such variations apply to the disclosed technical solution. It should be understood that the specific value referred to by the terms "about" or "approximately" is itself specifically and preferably disclosed.

[0095] In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the term comprises the mentioned elements, integers, or steps. For example, when referring to an oligonucleotide that “comprising” a specific sequence, it is also intended to cover oligonucleotides that comprise that specific sequence.

[0096] For clarity, when referring to the sequence or sequence motif of RNAi or oligonucleotides, only the nucleotide sequence within that sequence or motif is indicated. Since the pairing of nucleoside bases A and U corresponds to the pairing of nucleoside bases A and T, in this article, for modified RNAi molecules or oligonucleotides containing a deoxynucleotide T replacing a nucleotide U, the substitution position is indicated by a U base when referring to their sequence.

[0097] In this document, for clarity, unless otherwise specified, references to RNAi or oligonucleotides should be understood to refer not only to the nucleoside base sequence (i.e., sequence) of the nucleotide chain that makes up the molecule, but also to any chemical modifications present thereon (if such modifications are present). Such modifications can include modifications to the phosphate backbone, nucleoside, and sugars of the nucleotide chain, as well as conjugations of the nucleotide chain to non-nucleoside compounds. Therefore, in this disclosure, as those skilled in the art will understand, an “RNAi conjugate” can be considered a specific RNAi activator having a conjugated portion modification. In this document, HBV mRNA refers to the mRNA of all proteins encoded by the genome of the hepatitis B virus (HBV). HBV proteins encompass HBV surface protein (HBsAg), HBV core proteins (HBcAg and HBeAg), HBV polymerase (Pol), and HBV X protein (HBx). Following transcription, the HBV genome expresses a series of viral mRNAs via the host's translation system, including 3.5kb precore RNA (PreC RNA) and pregenomic RNA (pgRNA), 2.4kb RNA, 2.1kb RNA, and 0.7kb RNA. These mRNAs encode the aforementioned viral proteins. Due to the high overlap of HBV DNA transcripts, the RNAi compounds disclosed herein can directly target the HBV 3.5kb precore RNA (PreC RNA) and pregenomic RNA (pgRNA), 2.4kb RNA, and 2.1kb RNA transcripts; and indirectly regulate the abundance of 0.7kb RNA by directly regulating pgRNA expression. Therefore, in this disclosure, when referring to HBV mRNA as a target gene, it generally refers to mRNA that directly targets all proteins except HBV HBx. In some embodiments, this disclosure primarily uses the level of HBsAg protein as one of the biomarkers to evaluate the antiviral efficacy of the compounds. In some implementations, the HBV mRNA suitable as a target gene is the mRNA corresponding to accession number NC_003977.2.

[0098] In this document, RNAi activators for targeting HBV specifically refer to double-stranded oligonucleotide RNA molecules capable of binding to all HBV transcript sequences (including the HBsAg transcript) except for HBx mRNA and acting through an RNAi mechanism to reduce or knock down HBV mRNA expression. These double-stranded oligonucleotides are referred to as antisense and sense strands, respectively, based on their complementarity with the target HBV gene sequence. In this document, such RNAi activators with oligonucleotide double strands are also referred to as double-stranded RNAi or dsRNA. In some embodiments, when referring to RNAi activators, double-stranded RNAi or dsRNA, such as siRNA, is also included.

[0099] In this article, when referring to RNAi activators or RNAi conjugates, their salt forms are also included.

[0100] In this paper, the term "antisense" in relation to nucleic acid molecules refers to a nucleotide sequence containing a region complementary to the target gene sequence. Therefore, for an RNAi molecule containing an antisense strand targeting all HBV transcripts except HBx mRNA (including the HBsAg transcript), it will contain a nucleotide sequence complementary to several (e.g., 15-30) consecutive nucleotide sequences in the "sense" nucleic acids encoding the HBV HBsAg protein, HBV core proteins (HBcAg and HBeAg), and HBV polymerase (Pol) (e.g., several (e.g., 15-30) consecutive nucleotide sequences in the coding strand of the double-stranded DNA of the HBV HBsAg gene, HBV core protein (HBcAg and HBeAg) gene, and HBV polymerase (Pol) gene, or several (e.g., 15-30) consecutive nucleotide sequences in HBV-associated mRNA).

[0101] In this document, "target sequence" or "target gene sequence" refers to a continuous nucleotide portion in the mRNA molecule formed during the transcription of a target gene (e.g., the HBV gene). In this disclosure, the target gene sequence refers to a continuous nucleotide portion in which all transcripts (including HBsAg transcripts) transcribed from HBV mRNA, except for HBx mRNA, are produced. The target gene sequence associated with RNAi of this disclosure should be at least long enough to be used as a substrate for RNAi-guided nucleic acid cleavage, thereby causing a cleavage at or near the location of that sequence in the mRNA molecule formed by the transcription of the target gene. For example, the length of the target sequence can be, for example, 15-36 nucleotides ("nt"), or any sub-length therein. As a non-limiting example, the length of the target sequence can be 15-30 nt, 15-26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17 nt, 18-30 nt, 18-26 nt, 18-23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 18 nt, 19-30 nt, 19-26 nt, The target sequence length is 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 19 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 20 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides. In some embodiments of this disclosure, the target sequence length is preferably at least 18 nucleotides, more preferably at least 19 nucleotides. In some embodiments of this disclosure, the target sequence length is about 19 to about 30 nucleotides. In some embodiments of this disclosure, the target sequence length is about 19 to about 25 nucleotides. In some embodiments of this disclosure, the target sequence length is about 19 to about 23 nucleotides. In some embodiments of this disclosure, the target sequence length is about 21 to about 23 nucleotides. In the antisense strand of RNAi, the sequence region complementary to consecutive nucleotides in the target gene sequence is also referred to herein as a "sequence motif." Based on this core sequence motif, and applying the Watson and Crick base pairing rules, those skilled in the art who read this disclosure can readily design RNAi activator molecules that specifically target HBV. The antisense strand of such RNAi activators can be, for example, about 15, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 nucleotides in length.Depending on the form of the designed RNAi activator, the antisense strand sequence outside the "sequence motif" can, in some cases, be designed to be completely or substantially complementary to the extension of the target sequence on HBV mRNA, or not according to the Watson and Crick base pairing rules. All RNAi activators designed in this way are an aspect of this disclosure.

[0102] In this document, unless otherwise specified, the terms "complementarity" or "complementarity" refer to the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions and form a double-stranded structure. Those skilled in the art can determine the optimal complementarity of the two sequences and the conditions used to determine this complementarity based on the final application of the hybridized oligonucleotide or polynucleotide. Therefore, in this document, when describing the base pairing between the sense and antisense strands of RNAi, or the base pairing between the antisense strand and the target sequence of RNAi, the terms "complementarity" or "complementarity" should be understood to cover not only 100% complementarity (i.e., perfect complementarity) but also cases of less than 100% complementarity, i.e., the presence of base mismatches in the complementary double-stranded nucleic acid region that do not substantially affect the RNAi's intended function. As those skilled in the art will appreciate, in double-stranded nucleic acid molecules, when a base on one strand forms a Watson-Crick base pair with a corresponding base on the other strand in a complementary manner, the bases at that position on both strands are considered to be "complementarily paired" or "matched." For example, the purine base adenine (A) is complementary to the pyrimidine base thymine (T) or uracil (U); the purine base guanine (C) is complementary to the pyrimidine base cytosine (G). Correspondingly, a "mismatch" refers to a situation in double-stranded nucleic acids where corresponding bases on one strand are not complementary to each other. However, it should be understood that nucleotides modified in the base portion of RNA nucleosides should also be considered complementary if Watson-Crick base pairing is permitted. Therefore, in this paper, nucleoside base “complementarity” encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, for example, Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

[0103] In this document, for the purposes of this disclosure, the expression “complementary” or “complementarity” associated with double-stranded RNAi activators (such as siRNA as described herein) is preferably not less than 70%, meaning that at least 70% of the base positions in the double-stranded region formed by complementary hybridization are complementary, i.e., the number of mismatched positions in the continuous nucleotide sequence forming the double-stranded region is less than 30%. For example, for a 19-base-pair double-stranded region, not less than 70% complementarity means that the double-stranded region forms no more than 5, 4, 3, 2, 1, or 0 mismatched base pairs during hybridization. Preferably, the presence of insertions and deletions is not allowed when calculating the complementarity % of the continuous nucleotide sequence in the double-stranded region. Accordingly, in this document, the expression associated with RNAi activators, “complementary (antisense) sequence” to the target sequence, or “complementary (sense) sequence” to a portion of the antisense sequence, can be “completely complementary” or “substantially complementary.” “Completely complementary” means that the two sequences have 100% complementarity. When the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may contain one or more, but typically no more than 30%, 20%, or 10%, mismatched base pairs in the hybridized duplex, and still retain the ability to hybridize under conditions most relevant to its final application (e.g., repressing gene expression via a RISC pathway). It should be understood here that when the two oligonucleotides of an RNAi are designed to form one or more single-stranded overhangs during hybridization, such overhangs will not be considered mismatches when determining complementarity. For example, for the purposes described herein, an RNAi comprising a 19-nucleotide sense oligonucleotide chain and a 21-nucleotide antisense oligonucleotide chain may still be considered “perfectly complementary” if the longer antisense oligonucleotide contains a 19-nucleotide sequence that is perfectly complementary to the shorter sense oligonucleotide.

[0104] In this document, the term "protruding end" is used to describe an unpaired nucleotide located at the 3' or 5' end of the double-stranded region of a double-stranded oligonucleotide. In some embodiments according to this disclosure, the protruding end is 1 to 4 nt long, for example 1 or 2 nt, and is preferably located at the 3' end of the antisense strand of the RNAi.

[0105] In this paper, a "nucleotide difference" between two nucleotide sequences refers to a change in the type of bases at the same position of the nucleotides compared to the former. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then a nucleotide difference at that position is considered to exist between the two nucleotide sequences. In some embodiments, replacing the nucleotide at the original position with a baseless nucleotide or its equivalent can also be considered a nucleotide difference at that position.

[0106] In this article, the nucleosides and nucleotides that make up the nucleotide chain in nucleic acid molecules (such as RNAi or siRNA molecules) may be referred to as “units” or “monomers”.

[0107] In this document, "nucleotide" refers to the structural unit of oligonucleotides and polynucleotides, and for the purposes of this disclosure, includes naturally occurring nucleotides and modified nucleotides. In nature, RNA nucleotides comprise a sugar moiety (ribose), a nucleobase moiety, and a phosphate ester group. In this document, a modified nucleotide refers to a nucleotide that, corresponding to a natural RNA nucleotide, has modifications in its sugar moiety and / or nucleobase moiety.

[0108] In this document, the term "modified nucleoside" or "nucleoside modification" refers to a modified nucleoside formed by introducing one or more sugar moieties and / or one or more (nucleo)base moieties, compared to the corresponding RNA nucleoside. Therefore, the term "modified nucleoside" may also be used interchangeably with the term "modified nucleotide." In some preferred embodiments according to this disclosure, the modified nucleoside comprises a modified sugar moieties.

[0109] In this paper, the term "nucleobase" refers to the base portion of nucleosides and nucleotides. In natural nucleic acids, nucleobases are the purine portion (e.g., adenine and guanine) and the pyrimidine portion (e.g., uracil, thymine, and cytosine) of nucleosides. In this paper, the term "nucleobase" also encompasses modified nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this case, "nucleobase" refers to naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-natural variants. Descriptions of such variants can be found, for example, in Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1. In some cases, the nucleobase moiety can be modified by changing the purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolylcytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolyluracil, 2-thiouracil, 2'-thiothymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine. The nucleobase moiety can be indicated by a letter code (e.g., A, T, G, C, or U) for each corresponding nucleobase, wherein each letter may optionally include a functionally equivalent modified nucleobase. For example, in the oligonucleotides of the RNAi molecules exemplified in Table A of this disclosure, the nucleobase portion is selected from A, T, G, C, and U.

[0110] For naturally occurring oligonucleotides, the internucleotide bond includes a phosphate ester group that forms a phosphate ester bond between adjacent nucleosides. In this document, the term "modified internucleotide bond" is defined as a bond that covalently links two nucleosides together, other than a phosphate ester (PO) bond. The oligonucleotide chain of RNAi according to this disclosure may contain one or more internucleotide bonds derived from natural phosphate esters, a modification also referred to herein as "phosphate ester backbone modification." Modified internucleotide bonds contemplated according to this disclosure include, but are not limited to: thiophosphate bonds, dithiophosphate bonds, methylphosphate bonds, selenophosphate bonds, phosphoramidite bonds, etc. In some embodiments, the modified internucleotide bond can increase the stability of the oligonucleotide, such as nuclease resistance, compared to a phosphate ester bond. Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using a nuclease resistance assay (e.g., snake venom phosphatase (SVPD)) in a manner well known in the art. In some embodiments, preferably, the modified internucleotide bond in the oligonucleotide used for RNAi according to this disclosure is a thiophosphate bond. In some embodiments, the oligonucleotides of the sense and / or antisense strands may have one or two phosphate-thioester nucleoside bonds at their 5' and / or 3' ends. In some embodiments, the nucleoside linking the oligonucleotide of the disclosed RNAi to a non-nucleotide functional group such as a conjugate may be a phosphate ester bond, or in some cases, a phosphate-thioester bond.

[0111] In this article, structural fragments These terms can be used interchangeably to indicate phosphate linkage (where X is oxygen) or thiophosphate linkage (where X is sulfur).

[0112] In this document, the term "phosphate ester bond" or "phosphate ester linkage" refers to a portion having the following chemical structure:

[0113] These terms can be used interchangeably in this article.

[0114] In this article, the term "thiophosphate bond" or "thiophosphate link" refers to a portion having the following chemical structure:

[0115] Similarly, formula A22 can be used with Used interchangeably.

[0116] In this document, unless otherwise stated, the term "conjugation" refers to the covalent connection between two or more chemical moieties, each having a specific function; correspondingly, "conjugated compound" refers to a compound formed by covalently connecting these chemical moieties. Accordingly, in this document, "RNAi conjugated compound" (e.g., "siRNA conjugated compound") refers to a compound formed by covalently linking one or more chemical moieties having a specific function to an oligonucleotide chain of RNAi (such as siRNA). In this document, the specific chemical moieties covalently linked to the oligonucleotide chain of RNAi (such as siRNA), or the specific compounds that can conjugate such specific chemical moieties to RNAi via a reaction, are also referred to as "conjugated moieties." In some embodiments according to this disclosure, the conjugated moieties are non-nucleoside or non-nucleotide chemical moieties, but this does not preclude the possibility that the conjugated moieties are linked to oligonucleotides via nucleosides or nucleotides or their analogues or derivatives.

[0117] In this document, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes both the possibility that the event or condition occurs and the possibility that it does not occur. For example, "alkyl" in "optionally substituted" includes "alkyl" and "substituted alkyl" as defined below. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically infeasible, and / or inherently unstable.

[0118] In this document, "alkyl" refers to a straight-chain or branched chain having a specified number of carbon atoms, which can be from 1 to 30 carbon atoms, for example, 1 to 20 carbon atoms, or 12 to 16 carbon atoms. When referring to an alkyl residue having a specific number of carbon atoms, it is intended to cover all branched and straight-chain forms having that number of carbon atoms, and optionally substituted. When an alkyl group is substituted with one or more hydroxyl groups, it may be called a hydroxyalkyl group. In particular, the term "hydroxyethyl" refers to an ethyl group substituted with one hydroxyl group.

[0119] Accordingly, the term "alkylene" refers to a divalent group derived from an alkane, such as a straight-chain or branched alkane having a specified number of carbon atoms, by removing two hydrogen atoms. Examples include C... 1-20 Alkylene, C 1-12 Alkylene, C 1-8 Alkylene, C 1-6 Alkylenes, etc. When an alkylene group is replaced by one or more hydroxyl groups, it may be called a hydroxyalkylene group. In particular, the term "hydroxyethylene" refers to an ethylene substituted with one hydroxyl group, wherein the two bonding sites of the group may be located on one carbon atom or on two separate carbon atoms.

[0120] In this document, "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group having a specified number of carbon atoms and containing at least one double bond. Specifically, alkenyl groups have 2 to 18, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms, and are optionally substituted. For example, as used herein, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, propenyl, allyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.

[0121] In this document, "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon group having a specified number of carbon atoms and containing at least one triple bond. Specifically, an alkynyl group has 2 to 18, for example 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms, and is optionally substituted. For example, as used herein, the term "C2-C6 alkynyl" refers to a straight-chain or branched alkynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-methyl-1-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, etc.

[0122] The terms "alkoxy" and "alkyl-O-" are used interchangeably to refer to an alkyl group as defined above, linked by an oxygen atom. Preferably, the alkoxy group has 1-6 carbon atoms (C... 1-6 alkoxy group), 1-4 carbon atoms (C 1-4 alkoxy group or 1-3 carbon atoms (C 1-3 Alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, etc.), pentoxy (including n-pentoxy, isopentoxy, neopentoxy, etc.), hexoxy, heptoxy, octoxy, etc.

[0123] The term "cycloalkyl" refers to a fully or partially saturated non-aromatic monocyclic or bicyclic hydrocarbon group consisting of carbon and hydrogen atoms, preferably fully saturated. Preferably, the cycloalkyl group has 3-8 ring carbon atoms (C6H ... 3-8 cycloalkyl groups or 5-6 cyclic carbon atoms (C 5-6 Cycloalkyl groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. When a cycloalkyl group is replaced by one or more hydroxyl groups, it may be called a "hydroxycycloalkyl group," including C14 and C24. 3-8 Hydroxycycloalkyl or C 5-6 Hydroxycycloalkyl.

[0124] Accordingly, the term "cycloalkylene" refers to a fully or partially saturated non-aromatic monocyclic or bicyclic divalent hydrocarbon group consisting of carbon and hydrogen atoms, preferably fully saturated. Preferably, the cycloalkylene group has 3-8 ring carbon atoms (C... 3-8 (cycloalkylene) or 5-6 ring carbon atoms (C 5-6 Cycloalkylene groups. Examples of cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclopentenylene, cyclohexylene, and cyclohexenylene.

[0125] The term "heterocyclic alkyl" refers to a fully saturated non-aromatic monocyclic or bicyclic group containing one or more, for example, 1, 2, 3, or 4 heteroatoms independently selected from N, O, or S, with the remaining ring members being carbon. Preferably, the heterocyclic alkyl is a 3-8 membered heterocyclic alkyl, more preferably a 5-6 membered heterocyclic alkyl. The heterocyclic group can be attached to the rest of the molecule by a carbon atom or a heteroatom, as long as it is chemically feasible. Examples include pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazoyl, isothiazolyl, tetrahydropyridinyl, hexahydropyridinyl, hexahydropyrimidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiaranyl, morpholinyl, and thiomorpholinyl. Nitrogen-containing heterocyclic alkyl refers to a heterocyclic alkyl containing one or more nitrogen heteroatoms with the remaining ring members being carbon, such as pyrrolidinyl, pyrazolyl, imidazoyl, tetrahydropyridinyl, hexahydropyrimidinyl, piperazinyl, etc.

[0126] Accordingly, the term "heterocyclic alkyl" refers to a fully saturated non-aromatic monocyclic or bicyclic divalent group containing one or more, for example, 1, 2, 3, or 4 heteroatoms independently selected from N, O, or S, and with the remaining ring members being carbon. Preferably, the heterocyclic alkyl is a 3-8 membered heterocyclic alkyl, more preferably a 5-6 membered heterocyclic alkyl. The heterocyclic group can be attached to the rest of the molecule by a carbon atom or a heteroatom, as long as it is chemically feasible. Examples include pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, tetrahydropyridinyl, hexahydropyridinyl, hexahydropyrimidinyl, piperazine, tetrahydropyranyl, tetrahydrothiaranyl, morpholinyl, and thiomorpholinyl. Nitrogen-containing heterocyclic alkyl groups refer to heterocyclic alkyl groups containing one or more nitrogen heteroatoms and whose remaining ring members are carbon atoms, such as pyridine alkyl, pyrazolidine alkyl, imidazolyl alkyl, tetrahydropyridinyl, hexahydropyridazinyl, hexahydropyrimidinyl, piperazine, etc.

[0127] In this document, the term "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6-20, for example, 6-12 carbon atoms in the ring moiety, which may be substituted or unsubstituted. Preferably, the aryl group is (C6-C6) 10Aryl. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl, each of which may optionally be substituted with 1 to 4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, mercapto, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamide, heterocyclic, etc. Preferably, the aryl group is an optionally substituted phenyl group.

[0128] In this document, the term "heteroaryl" refers to a 5- to 20-membered (e.g., 5-14-, 5-8-, 5-6-membered) aromatic monocyclic or polycyclic ring system containing 1-3 heteroatoms selected from N, O, or S, which may be substituted or unsubstituted. Preferably, a heteroaryl is a 5- to 6-membered heteroaryl containing 1 to 3 heteroatoms independently selected from N, O, or S. Representative heteroaryl groups include thienyl (e.g., 2- or 3-thienyl), furanyl (e.g., 2- or 3-furanyl), pyrroleyl (e.g., 2- or 3-pyrroleyl), imidazoleyl (e.g., 2-, 4-, or 5-imidazolyl), pyrazolyl (3-, 4-, or 5-pyrazolyl), thiazolyl (e.g., 2-, 4-, or 5-thiazolyl), isothiazolyl (e.g., 3-, 4-, or 5-isothiazolyl), and oxazolyl (e.g., 2-, 4-, or 5-thiazolyl). -oxazolyl), isoxazolyl (such as 3-, 4- or 5-isooxazolyl), triazolyl (such as 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl), tetrazolyl, pyridyl (such as 2-, 3- or 4-pyridyl), pyrazinyl (such as 3- or 4-pyridyl), pyrazinyl (such as 3-, 4- or 5-pyrazinyl, 2-pyrazinyl), pyrimidinyl (such as 2-, 4- or 5-pyrimidinyl).

[0129] The term "heteroaryl" refers to a divalent group derived by removing two hydrogen atoms from a heteroaryl group as defined herein. Examples include 5-14-membered, 5-8-membered, or 5-6-membered heteroaryl groups. Preferably, the heteroaryl group is a 5- to 6-membered heteroaryl group comprising 1 to 3 heteroatoms independently selected from N, O, and S, such as thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl subunit, oxazolyl, isoxazolyl subunit, triazolyl, tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, etc.

[0130] The term "subunit" or "sub...unit" refers to a divalent group derived by removing two hydrogen atoms from a molecule.

[0131] The terms "-CO-" or "-C(O)-" represent carbonyl groups.

[0132] In this article, valence bond When present in a group or part, it indicates that the group or part is connected to the rest of the molecule via the wavy valence bond. When present in the molecule, it indicates that the chiral carbon atom connected by the wavy valence bond can have any chiral configuration, such as R and / or S. Additionally, when the wavy line passes through the valence bond originating from a group or part, for example in… In this context, it indicates that the group or part of it is connected to the rest of the molecule via the valence bond.

[0133] In this document, the term "ligand" refers to a chemical moiety conjugated to dsRNA that can alter the distribution, targeting, or lifetime of the dsRNA. In a preferred embodiment, such a ligand provides enhanced affinity for selected targets (e.g., molecules, cells or cell types, compartments (e.g., cell or organ compartments, tissues, organs, or regions of the body) compared to, for example, dsRNA without said ligand. In this document, the term "ligand" includes a chemical moiety consisting of a conjugating portion and (if any) a linking group.

[0134] In this document, "GalNAc ligand" refers to an asialic acid glycoprotein receptor (ASGPR) ligand containing a structural moiety of N-acetylgalactosamine (GalNAc) or a derivative thereof. This term encompasses monovalent, divalent, trivalent, tetravalent, and multivalent GalNAc ligands providing one, two, three, four, or more structural moieties of GalNAc or GalNAc derivatives.

[0135] In this document, the designation of equation (I) encompasses any of its sub-equations or instances, such as those selected from (Ia), (Ia-1), (Ib), (Ib-1), (Ib-2), (Ic-1), (Ic-2), (Ic-3), (Ic-4), L96, P36 (including sP36 or P36s), P36P36, P36P67, and P34P34. Other designations are understood similarly.

[0136] In this document, the term "isolated" as a modifier for a compound means that the compound is artificially prepared or that the compound is completely or at least partially isolated from the natural environment in which the compound is naturally present. It should be understood that, unless otherwise stated, the compounds used in this disclosure (whether chemical or biochemical) are isolated. In some cases, isolated compounds are "purified" or "partially purified" from their production or preparation environment, and thus do not contain at least some of the components present in the environment in which said compound is produced or prepared. In some embodiments, the isolated compound has a purity of at least 90%. "Purified" or "partially purified" compounds can be combined with one or more other compounds to form compositions that achieve a particular purpose. Therefore, "purified" or "partially purified" does not exclude combinations of such compositions, such as the combination of an RNAi conjugate with a second active agent for the treatment of HBV-related diseases or conditions.

[0137] In this document, the terms “subject,” “individual,” and “patient” are used interchangeably and refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, primates (e.g., human and non-human primates), laboratory animals (e.g., rodents, such as mice and rats), farm animals (e.g., cattle, pigs, sheep, and horses), grazing animals, and pets (such as dogs and cats).

[0138] In this document, the term "treatment" of a symptom and / or disease in mammals means (i) prevention of the symptom or disease, i.e., avoidance of any symptoms of the disease or symptom; (ii) suppression of the symptom or disease, i.e., prevention of the occurrence or progression of symptoms; and / or (iii) relief of the symptom or disease, i.e., inducing symptom resolution. This term encompasses both therapeutic and preventative treatments. Therefore, in some aspects, an RNAi active agent or RNAi conjugate according to this disclosure may be administered therapeutically to inhibit, reduce, relieve, stop, or reverse the progression of HBV-related diseases or symptoms or their symptoms in a subject, or to stabilize the development or progression of said disease or symptom or its symptoms.

[0139] In this document, the term "prevention" refers to reducing or decreasing the likelihood of a subject developing a disease or disease symptoms. Therefore, in some aspects, the RNAi active agent or RNAi composition of this disclosure may be administered prophylactically to prevent the occurrence or recurrence of HBV-related diseases or conditions or their symptoms in a subject. In some aspects, the subject does not yet have, but is at risk of having, said disease or condition, or is susceptible to developing said disease or condition.

[0140] In this article, "effective amount" means a predetermined amount of active agent that can elicit a desired biological or medical response in an organization, system, animal, or human, and / or an amount that prevents, inhibits, delays, or reverses the progression of a disease state or any other adverse symptom, or otherwise improves a disease state or symptom to achieve the desired therapeutic effect.

[0141] In this document, "therapeutic effective dose" and "preventive effective dose" refer to the amount that effectively achieves the desired therapeutic or preventive outcome at the required dose and for the required duration. Therapeutic and preventive effective doses can vary depending on various factors such as the disease to be treated or prevented, the individual's age, sex, and weight. Therapeutic and preventive effective doses are amounts in which any toxic or harmful effects are less than the beneficial therapeutic / preventive effects. Compared to subjects who have not received the drug, "therapeutic effective doses" and "preventive effective doses" preferably reduce measurable parameters (e.g., serum HBV levels or LDL-C levels) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%. The ability of the RNAi active agent or RNAi conjugate of this disclosure to reduce said measurable parameters can be evaluated in in vitro or animal model systems that predict therapeutic efficacy in humans. Typically, prophylactic administration is performed in subjects before the onset of disease symptoms, or before or at an earlier stage of the disease. Detailed Implementation

[0142] This disclosure provides RNAi activators (especially siRNAs with oligonucleotide double strands) that silence HBV genes using an RNAi mechanism, their conjugates, compositions, and uses. In cell-based and animal studies, this disclosure has demonstrated that the RNAi activators, such as siRNA molecules, specifically and efficiently mediate RNAi, resulting in a significant inhibition of HBV gene expression. Therefore, methods and compositions comprising these RNAi activators, such as siRNAs or RNAi conjugates, can be used to treat diseases or conditions that would benefit from downregulation of HBV expression (such as hyperlipidemia and cardiovascular disease). Using these RNAi activators, such as siRNAs or RNAi conjugates, allows for targeted degradation of HBV mRNAs involved in the regulation of LDL receptors and circulating cholesterol levels.

[0143] The following provides a detailed description of various aspects of this disclosure. Those skilled in the art will understand that any combination of any technical features of these aspects is within the scope of this disclosure unless the context explicitly indicates otherwise. Furthermore, those skilled in the art will understand that, unless the context explicitly indicates otherwise, RNAi active agents, conjugates, compositions, methods, pharmaceuticals, and uses according to any aspect of this disclosure may include any such combination of features.

[0144] RNAi activator

[0145] In a first aspect, this disclosure provides RNAi activators that target HBV mRNA (particularly any HBV transcript other than HBx mRNA). According to this disclosure, “RNAi” or “RNAi activator” is an activator molecule containing RNA (or a derivative thereof), wherein the activator is capable of mediating the targeted cleavage of messenger RNA (mRNA) (i.e., HBV (particularly HBsAg) mRNA) via the RNA-induced silencing complex (RISC) pathway.

[0146] Intrinsic RNAi (RNA interference) mechanisms in organisms typically involve a series of processes, including: Dicer processing long dsRNA into short 19-21 base pairs (bp) siRNA; siRNA binding to Ago protein to form an RNA-induced silencing complex (RISC); the AGO protein cleaving the sense strand of the siRNA and releasing it; subsequently, the mature RISC bound to the antisense strand cleaves the mRNA that is anticomplementary to the antisense strand through a sequence complementation mechanism. Based on this RNA interference mechanism, various artificial RNAi molecules with different structures have been developed. These structures can enter the RNAi pathway at different stages to achieve sequence-specific cleavage of target gene transcripts. See, for example, Molecules 2019, 24, 2211; doi:10.3390 / molecules24122211 (this literature is hereby incorporated herein by reference in its entirety for the purposes of this disclosure). Artificial RNAi molecules with such structures include, for example, siRNA molecules having a double-stranded region and one or two overhangs, long-chain siRNA molecules that can serve as substrates for the Dicer enzyme, short hairpin RNA (shRNA) that can be processed by Dicer to produce siRNA structures, and long single-stranded siRNA molecules containing only the antisense strand. It is understood that these molecular forms all fall within the scope of the RNAi activators disclosed herein.

[0147] In some aspects, preferably, the RNAi activator according to this disclosure refers to an siRNA molecule comprising an oligonucleotide duplex, i.e., a sense strand (sense oligonucleotide) and an antisense strand (antisense oligonucleotide), wherein the sense strand is complementary to at least a portion of the sequence (e.g., at least 15, 16, 17, 18, or 19 consecutive nucleotides) of the antisense strand to form an oligonucleotide duplex region. For example, siRNA molecules having a 19-21 nt duplex region and a 2-nt 3' overhang, or siRNA molecules comprising a 20-22 nt antisense strand and a 15-16 nt short sense strand, thus having an atypical long overhang, are all within the scope of this disclosure. Furthermore, molecules in which the sense and antisense strands of the RNAi molecule of this disclosure are covalently linked together by a nucleotide single strand or other linkage (e.g., the shRNA molecule described below) are also within the scope of this disclosure.

[0148] In this paper, the RNAi-related term "antisense strand" refers to an oligonucleotide chain in an RNAi activator containing a region complementary to a consecutive nucleotide of the target sequence. The RNAi-related term "sense strand" refers to an oligonucleotide chain in an RNAi activator containing a region complementary to at least a portion of the antisense strand to form a double-stranded region. In this paper, the antisense strand may also be referred to as the "guide" strand, and the sense strand as the "passenger" strand.

[0149] For the purpose of inhibiting target mRNA expression, as those skilled in the art will know, the oligonucleotide used as the sense strand does not participate in direct complementary binding to the target gene, and does not need to have perfectly complementary base pairing with the antisense strand oligonucleotide in the duplex region. Therefore, in some aspects, the sense strand (passenger strand) according to this disclosure may include at least one or more of the following properties: substantially complementary to the consecutive nucleotides of the antisense strand in the duplex region with the antisense strand, for example, at least 70%, at least 80%, at least 90%, or 100% complementary; having one or more additional nucleotides forming a protrusion or loop relative to the consecutive nucleotides of the antisense strand in the duplex region; and having one or more nucleotide gaps or vacancies relative to the consecutive nucleotides of the antisense strand in the duplex region. Similarly, for the purpose of inhibiting target mRNA expression, as those skilled in the art will understand, the antisense strand serving as the guide RNAi for specific binding to the target mRNA may also contain sequences that are not 100% complementary to the continuous nucleotide regions of the target mRNA; for example, the complementarity may be at least 80%, at least 90%, or 95% complementary; however, in some cases, 100% complementarity is preferred. For the purposes of this disclosure, in some aspects, when considering the sequence motif of the antisense strand to be complementary to the continuous nucleotide regions of the target gene sequence, the presence of insertions and deletions is preferably not permitted. With regard to the sense and antisense strands of this disclosure, in some aspects, when the complementary region is not perfectly complementary to the said continuous nucleotide region, the mismatch may be located inside or at the end of that region, for example, a mismatch of 3, 2, or 1 nucleotides at the 5' and / or 3' ends.

[0150] In some embodiments, this disclosure provides an RNAi activator whose antisense strand comprises a sequence motif complementary to at least 18, at least 19, at least 20, at least 21, or all of a consecutive nucleotide in a target gene sequence. In some embodiments, the complementarity refers to at least 80% complementarity, i.e., nucleotide mismatches at no more than 20% of the positions in the consecutive nucleotide regions of the target gene sequence. In other embodiments, the complementarity refers to at least 85%, 90%, or 95% complementarity. In still other embodiments, the complementarity refers to 100% complementarity. In some embodiments, the target gene sequence is selected from any of the following sequences:

[0151] (i) The nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6;

[0152] (ii) At least 15-35 consecutive nucleotides, such as 18, 19, 20, or 21 consecutive nucleotides, starting from position 1096, 1097, 1098, 1099, or 1100 of the HBV genome; or

[0153] (iii) At least 15-35 consecutive nucleotides, such as 18, 19, 20 or 21 consecutive nucleotides, starting from position 972, 973, 974, 975 or 976 of the HBV genome.

[0154] In some embodiments, this disclosure also covers cases where the sequence motif of the antisense strand is complementary to at least 18, at least 19, at least 20, or all of the consecutive nucleotides in a region containing the target gene sequence within two nucleotides upstream and downstream. In some embodiments, the length of the antisense strand is, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 nucleotides.

[0155] In some embodiments, the antisense strand comprises 19 consecutive nucleotides that are completely complementary to SEQ ID NO:5 or SEQ ID NO:6.

[0156] In some embodiments, this disclosure also provides a sense strand complementary to at least a portion of the sequence in the antisense strand according to this disclosure, and a double-stranded RNAi activator comprising such an antisense strand and a sense strand. In some embodiments, the at least portion of the sequence in the antisense strand complementary to the sense strand has, for example, at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides. In some embodiments, the complementarity refers to at least 70% complementarity, i.e., nucleotide mismatches at no more than 20% of the positions in the consecutive nucleotide regions of the target gene sequence. In other embodiments, the complementarity refers to at least 80%, 85%, 90%, 95%, or 100% complementarity. In some embodiments, the length of the sense strand is, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 nucleotides.

[0157] In this document, when referring to double-stranded RNAi such as siRNA, the double-stranded region formed by the hybridization of the sense and antisense strands can be of any length that allows for the specific degradation of HBV target mRNA (e.g., any one or more or all HBV transcripts other than HBx mRNA) via the RISC pathway. In some embodiments, this length is 15 to 36 base pairs (“bp”), or can be any length within this range, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 bp, and any subranges therein, including but not limited to 15-30 bp, 15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp, 15-17 bp, 18-30 bp, 18- 26bp, 18-23bp, 18-22bp, 18-21bp, 18-20bp, 19-30bp, 19-26bp, 19-23bp, 19-22bp, 19-21bp, 19-20bp, 19bp, 20-30bp, 20-26bp, 20-25bp, 20-24bp, 20-23bp, 20-22bp, 20-21bp, 20bp, 21-30bp, 21-26bp, 21-25bp, 21-24bp, 21-23bp, 21-22bp, 21bp, 22bp, or 23bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 17 to about 30 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 17 to about 27 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 17 to about 25 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 17 to about 23 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 17 to about 21 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 18 to about 30 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 18 to about 27 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 18 to about 25 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 18 to about 23 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 18 to about 21 bp.In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 19 to about 30 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 19 to about 27 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 19 to about 25 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 19 to about 23 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 19 to about 21 bp. In some embodiments, the double-stranded RNAi and siRNA according to this disclosure have a double-stranded region of about 17, 18, 19, 20, or 21 bp.

[0158] In some embodiments, the oligonucleotide double strands (i.e., the sense strand and the antisense strand) forming the double-stranded region in the RNAi molecule according to this disclosure need not be, but may be, covalently linked. Hereinafter, the structure is generally referred to as “shRNA” when the two strands are covalently linked by a hairpin loop. When the two strands are covalently linked by a means other than a hairpin loop, the linking structure is referred to as a “connector.” For siRNA molecules having an shRNA structure, the nucleotide single strand constituting the “hairpin loop” is located between the 3’ end of one strand forming the double-stranded structure and the 5’ end of the corresponding other strand, and may contain at least one unpaired nucleotide. In some embodiments, the hairpin loop may contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides.

[0159] In some embodiments, the double-stranded RNAi, such as siRNA, according to this disclosure may have one or two overhangs. When the 3' end of one strand of the double-stranded oligonucleotide constituting the double-stranded RNAi or siRNA extends beyond the 5' end of the other strand, or when the 5' end of one strand extends beyond the 3' end of the other strand, unpaired nucleotides form overhangs. The length of the overhang can be at least one nucleotide; optionally, the overhang can contain at least two, three, four, five, or more nucleotides. The overhang can contain nucleotides or modified nucleotides, or consist of nucleotides or modified nucleosides. The overhang can be located on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhang can be located at the 5' end and / or the 3' end of the antisense strand or the sense strand. In some preferred embodiments, the RNAi according to this disclosure comprises a double-stranded body with overhangs consisting of an antisense strand and a sense strand, and preferably, the overhang is a single 3' overhang consisting of one, two, three, or four nucleotides from the terminal 3' end of the antisense strand. More preferably, the overhang is a single 3' overhang consisting of the last two nucleotides of the antisense strand. In some embodiments, the overhang on the antisense strand may be fully or substantially complementary to the extension of the target gene sequence on HBV (especially HBsAg) mRNA.

[0160] The RNAi activator according to this disclosure can also have zero overhangs. In the case of zero overhangs, the RNAi activator has blunt ends, also known as a "blunt RNAi activator," and such molecules lack 3' or 5' single-stranded nucleotide overhangs.

[0161] In some embodiments, this disclosure provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand comprises a sequence motif complementary to at least 18, 19, 20, 21, or all consecutive nucleotides of a target gene sequence, and wherein the target gene sequence is selected from:

[0162] (i) The nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6;

[0163] (ii) At least 15-35 consecutive nucleotides, such as 18, 19, 20, or 21 consecutive nucleotides, starting from position 1096, 1097, 1098, 1099, or 1100 of the HBV genome; or

[0164] (iii) At least 15-35 consecutive nucleotides, such as 18, 19, 20 or 21 consecutive nucleotides, starting from position 972, 973, 974, 975 or 976 of the HBV genome;

[0165] Preferably, the antisense strand is completely complementary to the target gene sequence.

[0166] In some embodiments, this disclosure provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and sense strand each comprise a sequence motif differing by 0, 1, 2, or 3 nucleotides from the antisense and sense strand sequences of at least 17, 18, 19, 20, or all consecutive nucleotides of any of the compounds in Table A or Table 1. In some embodiments, the differing nucleotides are nucleotide substitutions, for example, occurring at the 5' and 3' terminal 1 to 3 residues of the sequence motif.

[0167] In some embodiments, this disclosure provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand comprises a sequence motif complementary to at least 18, 19, 20, 21, or all consecutive nucleotides in a target gene sequence, and wherein:

[0168] a. The target gene sequence is the sequence shown in SEQ ID NO:5, or at least 15-35 consecutive nucleotides, such as 18, 19, 20 or 21 consecutive nucleotides, starting from position 1096, 1097, 1098, 1099 or 1100 of HBV mRNA (e.g., the mRNA corresponding to accession number NC_003977.2); and preferably the antisense strand contains a sequence motif that differs from at least 18, 19, 20 or 21 consecutive nucleotides in the sequence shown in SEQ ID NO:3 by 0, 1, 2 or 3 nucleotides, and more preferably the sense strand contains a sequence motif that differs from at least 18, 19, 20 or 21 consecutive nucleotides in the sequence shown in SEQ ID NO:1 by 0, 1, 2 or 3 nucleotides;

[0169] b. The target gene sequence is the sequence shown in SEQ ID NO:6, or at least 15-35 consecutive nucleotides, such as 18, 19, 20 or 21 consecutive nucleotides, starting from position 972, 973, 974, 975 or 976 of HBV mRNA (e.g., the mRNA corresponding to accession number NC_003977.2); and preferably the antisense strand contains a sequence motif that differs from at least 18, 19, 20 or 21 consecutive nucleotides in the sequence shown in SEQ ID NO:4 by 0, 1, 2 or 3 nucleotides, and more preferably the sense strand contains a sequence motif that differs from at least 18, 19, 20 or 21 consecutive nucleotides in the sequence shown in SEQ ID NO:2 by 0, 1, 2 or 3 nucleotides.

[0170] In some embodiments, the differential nucleotide is a nucleotide substitution, and preferably occurs at the 5' and / or 3' terminal 1 to 3 residues of the sequence motif.

[0171] In some preferred embodiments, this disclosure provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise an antisense strand sequence, a sense strand sequence, or preferably an antisense strand sequence and a sense strand sequence selected from any of the compounds in Table 1 or Table A.

[0172] In some preferred embodiments, the RNAi activator comprises an antisense strand and a sense strand, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:3 and the sense strand comprises the nucleotide sequence shown in SEQ ID NO:1; or the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:4 and the sense strand comprises the nucleotide sequence shown in SEQ ID NO:2.

[0173] In some preferred embodiments, the RNAi activator comprises an antisense strand and a sense strand, wherein the nucleotide sequence of the antisense strand is as shown in SEQ ID NO:3 and the nucleotide sequence of the sense strand is as shown in SEQ ID NO:1; or the nucleotide sequence of the antisense strand is as shown in SEQ ID NO:4 and the nucleotide sequence of the sense strand is as shown in SEQ ID NO:2.

[0174] In addition to the RNAi activators described above, this disclosure also considers any other RNAi activators targeting at least 10-50, for example 15-35, consecutive nucleotides, such as 18, 19, 20, or 21 consecutive nucleotides, near (left or right, covering position 1096 or 972) of HBV mRNA (e.g., the mRNA corresponding to accession number NC_003977.2). Such RNAi activator molecules can be designed according to the Watson and Crick base pairing rules. The RNAi activator may contain an antisense oligonucleotide that is a portion of the target gene sequence region. For example, the antisense oligonucleotide may be, for example, about 5, 10, 15, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 nucleotides in length. In some embodiments, preferably, the RNAi activator is a double-stranded RNAi or siRNA having the characteristics described above.

[0175] In some embodiments, the RNAi disclosed herein, having the aforementioned antisense and sense strands, possesses one or more of the following properties: binding to the coding region of HBV (particularly HBsAg); not binding or hardly binding to mRNA or transcripts of other genes (with little or no "off-target effects"); not inducing or hardly inducing immunogenicity; capable of binding to conserved HBV mRNA sequence segments across multiple animal species (including humans, mice, rats, cynomolgus monkeys, etc.), thereby facilitating the testing of RNAi activity in experimental animals; and / or lacking specific sequences known or suspected of reducing RNAi activity. In one embodiment, the HBV-specific RNAi activator is a siRNA possessing any one or more of these properties.

[0176] Modification of RNAi activators

[0177] Those skilled in the art will recognize that the RNAi molecules according to this disclosure can be unmodified (i.e., containing naturally occurring RNA nucleosides) but can also be (and preferably are) modified, as long as they retain the desired functional activity (i.e., capable of forming the desired double-stranded structure and allowing or mediating the specific degradation of the target RNA via the RISC pathway). Such RNA modifications can occur at the base portion, sugar portion, and / or phosphate linker portion of the nucleotide.

[0178] As a non-limiting example, modified RNAi activators can be constructed using methods known in the art, employing chemical synthesis and enzymatic ligation reactions. For instance, modified RNAi activators can be chemically synthesized using naturally occurring nucleotides or nucleotides with various modifications (designed to reduce off-target effects and / or increase the biological stability of the molecule, or to increase the physical stability of the double strand formed between antisense and sense nucleic acids).

[0179] As another non-limiting example, the oligonucleotide chain of the RNAi molecule may include at least one modified nucleoside, including but not limited to: 2'-O-methyl modified nucleosides, nucleosides containing a 5'-thiophosphate group, terminal nucleosides linked to a cholesterol derivative or a dodecanoic acid didecanoic acid group, locked nucleosides, baseless nucleosides, 2'-deoxyribonucleosides, 2'-fluorinated nucleosides, 2'-amino-modified nucleosides, 2'-alkyl-modified nucleosides, morpholino nucleosides, non-locked nucleosides (UNA, see, for example, WO 2008 / 147824), aminophosphates, or nucleosides containing non-natural bases, or any combination thereof. Alternatively, the oligonucleotide of the RNAi molecule may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty or more modified nucleosides, or all nucleosides of the oligonucleotide may be modified nucleosides. For each of the multiple modified nucleosides in an RNAi molecule, the modifications do not need to be identical.

[0180] In some respects, this disclosure covers modified forms of any RNAi activator specifically described herein (e.g., Table 2). In some embodiments, variants of the modifications contain the same sequence (i.e., the nucleoside base sequence) but contain modifications in the phosphate backbone, ribose, or bases. Examples of applicable modifications include, for example: 2'-O-methyl nucleotide modifications, 2'-fluoronucleotide modifications, 2'-deoxyribonucleotide modifications, locked nucleotide (LNA) modifications, unlocked nucleotide (UNA) modifications, threononucleotide (TNA) modifications, conformation-restricted nucleotide modifications, 2'-O-methoxyethyl nucleotide modifications, debaseted nucleotide modifications, 2'-amino nucleotide modifications, 2'-O-allyl nucleotide modifications, 2'-C-alkyl nucleotide modifications, 2'-O-alkyl nucleotides, morpholinonucleotides, aminophosphonucleotides, etc. Amide nucleotide modifications, nucleotide modifications of non-natural bases, tetrahydropyranonucleotide modifications, 1,5-dehydrated hexadiol nucleotide modifications, cyclohexenyl nucleotide modifications, nucleotide modifications containing thiophosphate groups, nucleotide modifications containing methylphosphate groups, nucleotide modifications containing 2'-phosphates, nucleotide modifications containing 5'-phosphates, thermostable nucleotide modifications, ethylene glycol nucleotide (GNA) modifications, nucleotide modifications containing methylene-substituted carbon glycoside nucleotide mimics, and 2-O-(N-methylacetamide) nucleotide modifications; and combinations thereof.

[0181] In some embodiments, the RNAi activator according to this disclosure may have one or more modifications within the nucleic acid molecule or at one or both ends thereof. Examples of nucleoside base modifications, ribose moiety modifications, and phosphate backbone modifications that can be used for RNAi according to this disclosure are described below. Furthermore, a summary list of some oligonucleotide modifications known in the art can be found in PCT Publication WO 200370918. However, it is understood that the modifications that can be used for RNAi according to this disclosure are not limited thereto.

[0182] Nucleoside base modification

[0183] “G,” “C,” “A,” “T,” and “U” typically represent nucleotides containing guanine, cytosine, adenine, thymine nucleotides, and uracil as bases, respectively. However, those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be replaced by other parts without substantially altering the base-pairing properties of the oligonucleotide containing the nucleotide with such a substitution. Examples of such nucleoside base modifications that can be used to generate RNAi activators include the substitution of nucleotides containing uracil, guanine, or adenine with nucleotides containing, for example, inosine; and the replacement of adenine and cytosine in oligonucleotides with guanine and uracil, respectively, to form a GU Wobble base pair with the target mRNA. In addition, other examples of modified nucleoside bases that can be used to generate RNAi activators include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-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-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl queosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. All of these modified nucleoside bases are within the scope of consideration in this disclosure.

[0184] Sugar modification

[0185] Compared to the ribose moiety present in natural RNA, the oligonucleotides of the RNAi disclosed herein may contain one or more nucleosides with modified sugar moieties (i.e., glycosidic modifications). Numerous glycosidic modifications with ribose moieties have been developed, primarily with the aim of improving certain properties of the oligonucleotides, such as affinity and / or nuclease resistance, and off-target effects. Such modifications include, for example, modifications to the ribocyclic structure by replacing one of the following: a hexose ring (HNA), a threonose ring (TNA), a locked nucleic acid (LNA, a bicyclic ring with a bimolecular bridge between the C2 and C4 carbons), or a non-locked nucleic acid (UNA, a ribocyclic ring lacking a bond linking the C2 and C3 carbons). Examples of usable glycosidic modifications also include, for example, bicyclic hexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798). Modified nucleosides also include those in which the sugar moiety is replaced by a non-sugar moiety, such as in peptide nucleic acids (PNAs) or morpholinonucleotides. Sugar modification also includes modifications by replacing the naturally occurring 2'-OH group on the ribose ring of the RNA nucleoside with other groups. Furthermore, substituents can be introduced, for example, at the 2', 3', 4', or 5' positions of the sugar ring.

[0186] In some preferred embodiments, the RNAi activator of this disclosure comprises at least one 2'-modified nucleotide (i.e., a 2' sugar-modified nucleoside). Such modified nucleosides include nucleosides having a substituent at the 2' position other than –OH (2'-substituted nucleosides). A variety of 2'-substituted nucleosides have been developed for use in RNAi molecules, and several 2'-substituted nucleosides have been found to possess beneficial properties when incorporated into oligonucleotides. For example, 2'-modified sugars can provide oligonucleotides with enhanced binding affinity and / or increased nuclease resistance. Examples of 2'-substituted modified nucleosides are 2'-O-alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA nucleosides (MOE), 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, and 2'-F-ANA nucleosides. Other examples can be found, for instance, in Freier and Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213 and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. In some embodiments, the RNAi activator according to this disclosure comprises at least one 2'-modified nucleotide. In some embodiments, the 2'-modification is selected from 2'-deoxy, 2'-fluorinated, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-allyl, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), and 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, the RNAi activator according to this disclosure comprises at least one 2'-modified nucleoside selected from the group consisting of 2'-O-alkyl-RNA nucleoside, 2'-O-methyl-RNA nucleoside, 2'-alkoxy-RNA nucleoside, 2'-O-methoxyethyl-RNA nucleoside (MOE), 2'-amino-DNA nucleoside, 2'-fluoro-RNA nucleoside, and 2'-F-ANA nucleoside.

[0187] In some embodiments, the RNAi activator according to this disclosure comprises one or more deoxyribonucleosides. In this case, the RNAi activator may comprise, for example, one or more (e.g., 2-3) deoxyribonucleosides located at the overhang or in the double-strand region.

[0188] Nucleotide analogues

[0189] In some embodiments, the RNAi activator disclosed herein comprises at least one nucleotide analog.

[0190] Examples of nucleotide analogs that can be used for RNAi include, but are not limited to, LNA nucleotides, UNA nucleotides, and TNA nucleotides.

[0191] “LNA nucleotides” are 2’-modified nucleotides containing C2’ and C4’ bimoles (also called “2’-4’ bridges”) that link the ribose ring of the nucleoside to the ribose ring, which restrict or lock the conformation of the ribose ring. In the literature, these nucleotides are also referred to as bridging nucleic acids or bicyclic nucleic acids (BNAs). Non-restricted exemplary LNA nucleotides are disclosed in the following literature: Morita et al., Bioorganic & Med. Chem. Lett. 12, 73-76; Seth et al., J. Org. Chem. 2010, Vol 75(5) pp. 1569-81; and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238. “UNA nucleosides” are non-locked acyclic nucleotides. Preferably, in this disclosure, UNA nucleotides refer to nucleotides in which the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039). Further descriptions of UNA can be found in US 8,314,227; and US 2013 / 0096289; US 2013 / 0011922; and US 2011 / 0313020. All cited references are incorporated herein by reference in their entirety for the purposes of this disclosure.

[0192] "Threonucleotide" (TNA) is an artificial nucleotide in which the natural pentose sugar present in RNA nucleotides is replaced by a non-natural 4-carbon threose sugar, and TNA is capable of efficient base pairing with complementary bases of RNA nucleosides. In some preferred embodiments according to this disclosure, the RNAi activator according to this disclosure comprises a threonucleotide. Preferably, the threonucleotide is located at the 5' terminal of the positive strand of the RNAi activator, and more preferably, the threonucleotide is a 4'-modified threonucleotide. For a description of 4'-modified threonucleotides that can be used in this disclosure, see the applicant's co-pending Chinese application (CN202311244208.3) or PCT (PCT / CN2024 / 121099). This application is hereby incorporated herein by reference in its entirety for the purposes of this disclosure.

[0193] Phosphate backbone modification

[0194] Various phosphate backbone modifications for use in RNAi molecules are known in the art. Such modifications include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylphosphonates and chiral phosphonates), phosphonites, and aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates). Furthermore, modifications to inter-nucleotide links or backbones that do not contain phosphorus atoms are also within the scope of this disclosure. A description of phosphate backbone modifications can be found in WO 2023 / 076451, which is incorporated herein by reference for the purposes of this disclosure. In some embodiments, a thiophosphate backbone modification is introduced into the nucleoside of the oligonucleotide of the RNAi molecule of this disclosure. This modification can enhance the nuclease stability of said oligonucleotide.

[0195] In some implementations, the thiophosphate bond is as shown in formula A22:

[0196] In some embodiments, the RNAi activator according to this disclosure may optionally also include a chemical modification at the 5' and / or 3' ends, i.e., a non-nucleotide or nucleoside chemical portion linked to the end of the oligonucleotide chain (sense and / or antisense strand) of RNAi. Examples of chemical portions linked to the 3' end of the oligonucleotide chain can be found, for example, in WO 2005 / 021749 and WO 2007 / 128477. Examples of chemical portions linked to the 5' end of the oligonucleotide chain may include, but are not limited to, a 5'-terminal phosphate ester modification, preferably said modification being selected from: 5'-(E)-vinylphosphonate (5'-(E)-VP), 5'-methylphosphonate (5'-MP), (S)-5'-C-methyl analogues, and 5'-thiophosphate (5'-PS). In some preferred embodiments, the RNAi activator according to this disclosure includes a 5'-(E)-vinylphosphonate modification at the 5' end of the antisense strand.

[0197] nucleoside analogues

[0198] In some embodiments, any one or more G-base nucleosides in the RNAi activator are replaced by nucleoside analogs. In some embodiments, one G-base nucleoside in the RNAi activator is replaced by a nucleoside analog.

[0199] Nucleoside analogs can be any structural analog of a nucleoside known in the art that comprises a nucleobase and a sugar, wherein 1-3 phosphate groups are linked to the nucleoside. In some embodiments, the nucleoside analog is selected from methylene-substituted C-nucleotide mimics (E) or L-2'-deoxy-2'-fluororibonucleotides (CLV).

[0200] In some embodiments, the nucleoside analog is a methylene-substituted carbon glycoside nucleotide analog (E).

[0201] In some embodiments, the G-base nucleoside of the RNAi activator selected from the following positions is replaced by a nucleoside analog:

[0202] (i) One or two G base nucleosides at the 3' end of the antisense chain (e.g., the G base at position 20 or 21), preferably two G base nucleosides;

[0203] (ii) The G base nucleoside in the sense strand corresponding to the 9th position from the 5' end of the antisense strand;

[0204] (iii) The G base nucleoside in the sense strand corresponding to the 13th position from the 5' end of the antisense strand;

[0205] (iv) Any one or more G-base nucleotides at other positions in the positive chain; or

[0206] (v) Combinations of the above positions.

[0207] In some embodiments, the nucleoside analog is linked to its adjacent nucleotide via a phosphate thioester group. In some embodiments, the nucleoside analog is directly linked to its adjacent nucleotide.

[0208] In some preferred embodiments, the two G-nucleotides at the 3' end of the antisense strand of the RNAi activator are replaced by nucleoside analogs, optionally the two nucleoside analogs are linked by a thiophosphate group, and optionally the nucleoside analog is linked to the third nucleotide at the 3' end of the antisense strand via a thiophosphate group.

[0209] In some preferred embodiments, one or two of the sense strands corresponding to the 9th or 13th position from the 5' end of the antisense strand of the RNAi activator, preferably one G nucleoside, are replaced by a nucleoside analog.

[0210] In some embodiments, the nucleoside analog suitable for replacing the G-base nucleoside in the RNAi activator disclosed herein is a methylene-substituted carbon glycoside nucleotide mimic (E).

[0211] In some embodiments, any one of the G bases of the RNAi activator is replaced by a methylene-substituted C-nucleotide mimic (E); in some embodiments, one G base of the RNAi activator is replaced by a methylene-substituted C-nucleotide mimic (E). In some embodiments, the two G bases at the 3' end of the antisense strand of the RNAi activator are replaced by a methylene-substituted C-nucleotide mimic (E). In some embodiments, one or two, preferably one, G bases of the sense strand corresponding to the 9th or 13th position from the 5' end of the antisense strand of the RNAi activator are replaced by a methylene-substituted C-nucleotide mimic (E).

[0212] In some embodiments, the structure of the methylene-substituted carbon glycoside nucleotide mimic (E) is shown in A18.

[0213] Where X includes O, S, Se, CH2, CH-CH3, and R includes H, OH, OMe, F, OMOE, etc. Preferably, X represents CH2 and R represents H, and its structure is shown in A19:

[0214] In some embodiments, the nucleoside analog suitable for replacing the G base in the RNAi activator disclosed herein is L-2'-deoxy-2'-fluororibonucleotide (CLV).

[0215] In some embodiments, any one of the G bases of the RNAi activator is replaced by an L-2'-deoxy-2'-fluororibonucleotide (CLV); in some embodiments, one G base of the RNAi activator is replaced by an L-2'-deoxy-2'-fluororibonucleotide (CLV). In some embodiments, the two G bases at the 3' end of the antisense strand of the RNAi activator are replaced by an L-2'-deoxy-2'-fluororibonucleotide (CLV). In some embodiments, one or two, preferably one, G bases of the sense strand corresponding to the 9th or 13th position from the 5' end of the antisense strand of the RNAi activator are replaced by an L-2'-deoxy-2'-fluororibonucleotide (CLV).

[0216] In some embodiments, the structure of the L-2'-deoxy-2'-fluororibonucleotide (CLV) is shown as CLV-I.

[0217] Where X includes O, S, Se, CH2, CH-CH3, and R includes H, OH, OMe, F, OMOE, etc. Preferably, X represents O and R represents F, and its structure is shown in CLV-II.

[0218] Exemplary modified RNAi activators

[0219] In some embodiments, this disclosure provides modified RNAi activators. In some embodiments, the modified RNAi activator comprises at least one phosphate backbone modification and / or at least one nucleotide modification. In some embodiments, the RNAi activator comprises at least one thiophosphate or methylphosphate linker, preferably wherein the antisense strand and the sense strand each comprise one or two thiophosphate links at the 5' end and / or the 3' end. In some embodiments, the RNAi activator comprises at least one 2'-modified nucleotide, wherein the 2'-modification is selected from 2'-deoxy, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), and 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, the RNAi activator comprises at least one modified nucleotide selected from the following: 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-O-methoxyethyl modified nucleotide, deoxynucleoside nucleotide, methylene-substituted C-nucleotide analog (E), UNA, LNA, and TNA (threonucleotide). In some embodiments, the RNAi activator comprises a 5'-terminal phosphate ester modification, preferably wherein the modification is selected from: 5'-(E)-vinylphosphonate (5'-(E)-VP), 5'-methylphosphonate (5'-MP), (S)-5'-C-methyl analog, and 5'-thiophosphate (5'-PS), more preferably the RNAi activator comprises a 5'-(E)-vinylphosphonate modification at the 5' end of the antisense strand.

[0220] In some embodiments, the RNAi activator has one or more of the following modified nucleotides:

[0221] 2'-O-methyl (2'-methoxy) modified nucleotides are nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group, as shown in B1.

[0222] 2'-O-methoxyethyl modified nucleotides are nucleotides formed by replacing the hydrogen atom of the 2'-hydroxyl group of the ribosome with a methoxyethyl group, as shown in B2.

[0223] 2'-Fluorinated nucleotides are nucleotides formed by replacing the 2' hydroxyl group of the ribosome with fluorine, and their structure is shown in B3.

[0224] 2'-Deoxy-2'-Fluoro-arabinofuranose nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of arabinofuranose with fluorine, and its structure is shown in B4.

[0225] 2'-Deoxyribonucleotide (DNA) is a nucleotide formed by replacing the 2' hydroxyl group of the ribose with hydrogen, and its structure is shown in B5.

[0226] 2'-Amino modified nucleotides are nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with an amino group, and their structure is shown in B6.

[0227] 2'-alkoxy modified nucleotides are nucleotides formed by replacing the hydrogen atom of the 2'-hydroxyl group of the ribosome with an alkyl group, and their structure is shown in B7.

[0228] Locked nucleotides (also known as LNA nucleotides) are nucleotides in which an oxymethylene bridge is formed between the 2'-O and 4'-C positions of the ribosome, as shown in B8.

[0229] 2',3'-broken nucleotides (also known as UNA nucleotides) are nucleotides formed by the breaking of the carbon-carbon bonds at the 2' and 3' positions of the ribosome of a nucleotide, and their structure is shown in B9.

[0230] Glyceryl nucleotides (also known as GNA nucleotides) are acyclic nucleotide analogs formed by replacing the pentose sugar of a nucleotide with propylene glycol, and their structure is shown in B10.

[0231] L-2'-deoxyribonucleotide refers to a nucleotide in which the chirality of the 1', 3', and 4' positions of the ribosome is completely opposite to that of the natural nucleotide, and the hydroxyl group at the 2' position is replaced by hydrogen. Its structure is shown in B11.

[0232] Threononucleotides are nucleotides that have a threonucleotide structure, as shown in B12.

[0233] The structure of the 4'-modified threonine is shown in B13, where R represents a C1-C30 alkyl group, preferably a C10-C30 alkyl group;

[0234] L-nucleotide analogs, the structure of which is shown in B14, wherein Base is preferably T, X includes O, S, Se, CH2, CH-CH3, and R includes H, OH, OMe, F, OMOE, etc., preferably X represents O and R represents F, the structure of which is shown in B15.

[0235] The structure of the L-2'-O-methyl modified nucleotide is shown in B16:

[0236] Where Base represents natural bases and base analogues, with natural bases A, U, C, G and T being preferred.

[0237] In some preferred embodiments, the RNAi activator has nucleotides modified at one or more of the following positions:

[0238] The positive strand contains 2'-fluorinated nucleotides at positions 5, 7, 8, and 9 of its sequence motif;

[0239] The antisense strand contains 2'-fluorinated nucleotides at positions 2, 14, and 16 of its sequence motif;

[0240] The antisense strand contains 2'-deoxyribonucleotides at positions 5 and 7 of its sequence motif;

[0241] The positive strand contains a 2'-fluorinated nucleotide at positions 7 and 9 of its sequence motif;

[0242] The antisense strand contains 2'-fluorinated nucleotides at positions 2, 12, 14, and 16 of its sequence motif; the antisense strand contains 2'-deoxyribonucleotides at positions 5 and 7 of its sequence motif;

[0243] The antisense strand contains a 2'-fluorinated nucleotide at positions 2, 12, 14, and 16 of its sequence motif; the antisense strand contains a 2'-deoxyribonucleotide at positions 5 and 7 of its sequence motif; and the antisense strand contains a 2'-O-methoxyethyl nucleotide at position 15 of its sequence motif.

[0244] The positive strand contains 2'-fluorinated nucleotides at positions 7, 9, and 11 of its sequence motif;

[0245] Preferably, the sense strand and / or antisense strand contain nucleotides modified with 2'-O-methyl (2'-methoxy) at the remaining positions of their sequence motifs.

[0246] In some preferred embodiments, at least one G nucleoside (preferably one or two G nucleosides) of the RNAi activator is substituted with a nucleoside analog such as a methylene-substituted C-nucleotide mimic (E); for example, one G nucleoside of the RNAi activator is substituted with a nucleoside analog such as a methylene-substituted C-nucleotide mimic (E). In some more preferred embodiments, the two G nucleosides at the 3' end of the antisense strand of the RNAi activator are substituted with a nucleoside analog such as a methylene-substituted C-nucleotide mimic (E); or one or two, preferably one, G nucleosides of the sense strand corresponding to the 9th or 13th position from the 5' end of the antisense strand of the RNAi activator are substituted with a nucleoside analog such as a methylene-substituted C-nucleotide mimic (E). In some embodiments, the structure of the methylene-substituted C-nucleotide mimic (E) is shown in A18, preferably as shown in A19.

[0247] In some embodiments, the RNAi activator has modifications on one or more phosphate backbones, such as including phosphate thioester links at the following positions:

[0248] The positive chain contains two thiophosphate links at its 5' end;

[0249] The antisense strand contains two thiophosphate links at its 5' end and two thiophosphate links at its 3' end; and / or

[0250] The positive chain contains a thiophosphate linker at its 5' end.

[0251] In some embodiments, the RNAi activator comprises modified nucleotides, G-base nucleoside substitutions, and / or thiophosphate linkages.

[0252] In some embodiments, substantially all nucleotides of the antisense strand and / or the sense strand (e.g., at least 80%, 85%, 90%, 95%, or 96% of the nucleotides, or even 100% of the nucleotides) are modified nucleotides.

[0253] In some preferred embodiments, the RNAi activator has the following modification pattern:

[0254] Justice Chain (SS): NmsNmsNmNmNmNmNfNmNfNmNNmNmNmNmNmNmNmNm

[0255] Antisense strand (AS):NmsNfsNmNmdNNmdNNmNmNmNmNfNmNfN(moe)NfNmNmNmsNmsNm

[0256] or

[0257] Justice Chain (SS): NmsNmsNmNmNmNmNfNmNfNmNNmNmNmNmNmNmNmNm

[0258] Antisense strand (AS): NmsNfsNmNmdNNmdNNmNmNmNmNfNmNfNmNfNmNmNmsNmsNm;

[0259] The N here represents a ribonucleotide, such as G, C, A, or U;

[0260] m indicates that the nucleotide adjacent to the left of the letter m is a nucleotide modified with 2'-O-methyl;

[0261] f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluorinated nucleotide;

[0262] The letter 'd' indicates that the nucleotide adjacent to the letter to the right of 'd' is a 2'-deoxyribonucleotide.

[0263] "moe" indicates that the nucleotide adjacent to the left of the letter "moe" is a nucleotide modified with 2'-O-methoxyethyl; and

[0264] The letter 's' indicates that the connection between the two nucleotides adjacent to the letter 's' is a phosphate thioester group.

[0265] Preferably, the N in the sense strand corresponding to the 9th position from the 5' end of the antisense strand is replaced by a methylene-substituted carbon glycoside nucleotide analog (E).

[0266] In some preferred embodiments, the RNAi activator comprises a modified sense strand, a modified antisense strand, or preferably both a modified sense strand and a modified antisense strand of any of the compounds shown in Table 2.

[0267] In some preferred embodiments, the RNAi activator comprises a sense strand, a modified antisense strand, or preferably both a modified sense strand and a modified antisense strand selected from any of the compounds in Table B.

[0268] In some preferred embodiments, the RNAi activator comprises a modified sense strand and a modified antisense strand, wherein the modified sense strand and the modified antisense strand each comprise, or are composed of, the modified nucleic acid sequences shown in SEQ ID NO:, or are respectively composed of the modified nucleic acid sequences shown in SEQ ID NO:.

[0269] a) Justice Chain: SEQ ID NO:7; Antisense Chain: SEQ ID NO:8;

[0270] b) Justice Chain: SEQ ID NO:9; Antisense Chain: SEQ ID NO:10;

[0271] c) Justice Chain: SEQ ID NO:11; Antisense Chain: SEQ ID NO:12;

[0272] d) Justice chain: SEQ ID NO:13; Antisense chain: SEQ ID NO:14;

[0273] e) Justice Chain: SEQ ID NO:15; Antisense Chain: SEQ ID NO:8;

[0274] f) Justice Chain: SEQ ID NO:16; Antisense Chain: SEQ ID NO:17;

[0275] g) Justice Chain: SEQ ID NO:18; Antisense Chain: SEQ ID NO:19;

[0276] h) Justice chain: SEQ ID NO:20; Antisense chain: SEQ ID NO:21;

[0277] i) Justice Chain: SEQ ID NO:22; Antisense Chain: SEQ ID NO:23;

[0278] j) Justice Chain: SEQ ID NO:16; Antisense Chain: SEQ ID NO:24;

[0279] k) Justice Chain: SEQ ID NO:25; Antisense Chain: SEQ ID NO:19;

[0280] l) Justice Chain: SEQ ID NO:26; Antisense Chain: SEQ ID NO:27;

[0281] m) Justice Chain: SEQ ID NO:28; Antisense Chain: SEQ ID NO:29;

[0282] n) Justice Chain: SEQ ID NO:30; Antisense Chain: SEQ ID NO:31;

[0283] o) Justice Chain: SEQ ID NO:32; Antisense Chain: SEQ ID NO:33;

[0284] p) Justice Chain: SEQ ID NO:34; Antisense Chain: SEQ ID NO:33;

[0285] q) Justice chain: SEQ ID NO:35; Antisense chain: SEQ ID NO:8;

[0286] r) Justice Chain: SEQ ID NO:36; Antisense Chain: SEQ ID NO:8;

[0287] s) Justice chain: SEQ ID NO:37; Antisense chain: SEQ ID NO:17;

[0288] t) Justice chain: SEQ ID NO:36; Antisense chain: SEQ ID NO:19;

[0289] u) Justice Chain: SEQ ID NO:37; Antisense Chain: SEQ ID NO:24;

[0290] v) Justice Chain: SEQ ID NO:37; Antisense Chain: SEQ ID NO:38;

[0291] w) Justice Chain: SEQ ID NO:39; Antisense Chain: SEQ ID NO:40;

[0292] x) Justice Chain: SEQ ID NO:41; Antisense Chain: SEQ ID NO:42; or

[0293] y) Justice chain: SEQ ID NO:16; Antisense chain: SEQ ID NO:38.

[0294] In some preferred embodiments, the RNAi activator comprises a modified sense strand and a modified antisense strand, wherein the modified sense strand and the modified antisense strand each comprise the modified sense strand nucleic acid sequence and antisense strand nucleotide sequence shown in SEQ ID NO: in Table B below, or each comprises the modified sense strand nucleic acid sequence and antisense strand nucleotide sequence shown in SEQ ID NO: in Table B below:

[0295] Table B: Modified siRNA duplexes

[0296] RNAi conjugates

[0297] On the other hand, a conjugate formed by the covalent coupling of a nucleotide moiety and a non-nucleotide moiety in RNAi of this disclosure is provided. In this document, the conjugate may be referred to as an "RNAi conjugate" or "siRNA conjugate," and the non-nucleotide moiety may be referred to as the conjugated moiety.

[0298] In some embodiments, the nucleotide moiety of the RNAi disclosed herein is conjugated to one or more non-nucleotide moieties, which can improve the pharmacological properties of the RNAi disclosed herein, for example, by affecting the activity, cellular distribution, cellular uptake, or stability of the oligonucleotide. In some embodiments, the conjugated moiety can modulate or enhance the pharmacokinetic properties of the RNAi disclosed herein, for example, by improving the cellular distribution, bioavailability, metabolism, excretion, permeability, and / or cellular uptake of the oligonucleotide. In particular, the conjugate can direct the oligonucleotide to a specific organ, tissue, or cell type and thereby enhance the efficacy of the RNAi disclosed herein in that organ, tissue, or cell type. Simultaneously, the conjugate can reduce the activity of the RNAi disclosed herein in non-target cell types, tissues, or organs (e.g., off-target activity or activity in non-target cell types, tissues, or organs).

[0299] Regarding the conjugation sites and conjugation modifications applicable to RNAi, descriptions are provided in Vajinder Kumar, Targeted delivery of oligonucleotides using multivalent protein–carbohydrate interactions, Cite this: Chem. Soc. Rev., 2023, 52, 1273; Rosemary Kanasty, Delivery materials for siRNA therapeutics, NATURE MATERIALS, VOL 12, NOVEMBER 2013; Wanyi Tai, Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery, Molecules 2019, 24, 2211; doi:10.3390 / molecules24122211; and WO 93 / 07883 and WO 2013 / 033230, which are incorporated herein by reference in their entirety.

[0300] In some embodiments, the conjugation portion applicable to this disclosure may include portions selected from: antibodies, peptides, peptide mimics, aptamers, small chemical compounds, lipids, cell-penetrating peptide polymers, or nanoparticle conjugations.

[0301] In some embodiments, the conjugation portion applicable to this disclosure may comprise a binding molecule that recognizes a cell surface molecule. For example, as in International Patent Application WO 91 / 04753, an oligonucleotide may be conjugated to a binding molecule that recognizes a cell surface molecule for therapeutic administration. The binding molecule may comprise, for example, an antibody against a surface antigen, an antibody against a cell surface receptor, a ligand having a corresponding cell surface receptor, an antibody against such a ligand, or an antibody recognizing a complex of said ligand and its receptor. Methods for conjugating the binding molecule to an oligonucleotide are detailed in WO 91 / 04753. Additionally, conjugation methods and methods for improving cellular uptake that can be used are also described in the following International Patent Applications: WO 9640961, WO 9964449, WO 9902673, WO 9803533, WO 0015265, and U.S. Patents 5856438 and 5138045.

[0302] In some embodiments, the conjugation portion suitable for this disclosure may comprise peptides, such as poly(L-lysine) and antennal foot transport peptides that can significantly increase cell permeability. Such conjugations are described in Lemaitre et al., "Specific antiviral activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site," Proc. Natl. Acad. Sci. USA, 84:648-652, 1987; U.S. Patent Nos. 6,166,089 and 6,086,900.

[0303] In some embodiments, the conjugation portion applicable to this disclosure may comprise sugars, cell surface receptor ligands, drugs, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or any combination thereof.

[0304] In some embodiments, the conjugated portion may comprise a lipid moiety, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553); bile acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053); thioether, such as hexyl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533); or an aliphatic chain, such as dodecyl glycol or undecyl residue (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS). Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49); phospholipids, such as di-hexadecyl-racemic-glycerol or triethylammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-H-phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969) or adamantaneacetic acid (Manoharan et al., Tetrahedron ... Lett., 1995, 36:3651); palmitic moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229) or octadecylamine or hexano-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). In some embodiments, a cholesterol-containing conjugate moiety is preferred because it enhances targeting to hepatocytes.

[0305] In some embodiments, the conjugated portion is a sugar or contains a sugar. The sugar includes, but is not limited to, galactose, lactose, N-acetylgalactosamine (GalNAc), mannose, and mannose-6-phosphate. The sugar conjugated portion or the sugar-containing conjugated portion may enhance delivery or activity in tissues such as the liver and / or muscle. See, for example, EP 1495769, WO 99 / 65925, Yang et al., Bioconjug Chem (2009) 20(2):213-21; Zatsepin and Oretskaya Chem Biodivers. (2004) 1(10):1401-17.

[0306] In some embodiments, the conjugation moiety used in this disclosure is a chemical moiety capable of binding to the asialic acid glycoprotein receptor (ASGPR). For example, monovalent, divalent, and trivalent N-acetylgalactosamine (GalNAc) and their derivatives are suitable for binding to ASGPR and are therefore applicable to this disclosure. For examples of conjugations with ASGPR targeting, see WO 2014 / 076196, WO 2014 / 207232, and WO 2014 / 179620 (these documents are incorporated herein by reference in their entirety). Such conjugations can be used to enhance hepatic uptake of RNAi oligonucleotides while simultaneously reducing their presence in the kidneys, thus increasing the liver / kidney ratio of the conjugated oligonucleotide compared to the corresponding unconjugated form. For information on these oligonucleotide conjugates and their synthesis, see Manoharan’s review, cited in Antisense Drug Technology, Principles, Strategies, and Applications, ed. STCrooke, Chapter 16, Marcel Dekker, Inc., 2001, and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, each of which is incorporated herein by reference in its entirety.

[0307] In some embodiments, the conjugation portion is attached to the 5' and / or 3' terminal nucleotide of the sense strand of the RNAi disclosed herein, and optionally the 3' terminal nucleotide of the antisense strand, optionally attached to the nucleotide via a thiophosphate group (as shown in formula (A22)) or a phosphate group. In some embodiments, the conjugation portion may also be attached to the internal sequence of the RNAi oligonucleotide. In some embodiments, the conjugation portion may be attached to a phosphate group, a 2′-hydroxyl group, or a base of the nucleotide. In some embodiments, the conjugation portion may be attached to a 3′-hydroxyl group of the nucleotide, in which case the nucleotides are linked by a 2′-5′ phosphate ester bond. When the conjugation portion is attached to the end of an RNAi (such as siRNA) oligonucleotide chain, it is typically attached to a phosphate group of the nucleotide; when the conjugation portion is attached to the internal sequence of an RNAi (such as siRNA) oligonucleotide, it is typically attached to a sugar ring of the ribose or a base.

[0308] The conjugation portion applicable to this disclosure can be directly linked to the oligonucleotide duplex of the RNAi disclosed herein or linked via a linker group. In some embodiments, the RNAi conjugate of this disclosure may optionally contain a linker group located between the RNAi oligonucleotide duplex and the conjugation portion. In some embodiments, the linker group is a biocleavable linker group. Such linker groups typically contain or consist of physiologically unstable bonds that are cleavable under normal or similar in vivo conditions in mammals. These conditions include conditions present in mammalian cells or similar chemical conditions, such as pH, temperature, oxidative or reducing conditions, substance and salt concentrations. These conditions also include enzymatic activity normally present in mammalian cells, such as from proteases, hydrolases, or nucleases. In some embodiments, the linker group need not be biocleavable, but the conjugation portion may be covalently linked to the oligonucleotide. Such linker groups may contain repeating units, such as ethylene glycol units, amino acid units, or aminoalkyl chain structures or oligomers. For example, the linker group may be an aminoalkyl group, such as C 2-36 aminoalkyl, such as C 6-12 Aminoalkyl groups. In some embodiments, conjugates having such linker groups can be separated from RNAi oligonucleotides by processing with the Dicer enzyme.

[0309] In some embodiments, the linker group is used to covalently couple a conjugated moiety, such as a conjugated moiety containing N-acetylgalactosamine (GalNAc), to the RNAi of this disclosure. In some embodiments, the linker group may include a branching component. Hereinafter, the term "branching component" refers to a chemical moiety capable of covalently coupling three or more entities (e.g., conjugated moieties and / or the RNAi of this disclosure). In some embodiments, a linker group having a branching component can conjugate two or more conjugated moieties, such as conjugated moieties containing N-acetylgalactosamine (GalNAc), to an oligonucleotide of the RNAi of this disclosure. Linker groups containing branching components that can be used for this purpose are known in the art and include, but are not limited to, amino acids (including natural and non-natural amino acids), peptides and their derivatives, sugar units and their derivatives, aromatic-substituted compounds and their derivatives, substituted hydrocarbon groups and their derivatives, triazole-containing derivatives, etc. See, for example, CN104651408A, CN113286888A, WO2015 / 173208 and WO2023 / 076451, which are incorporated herein by reference in their entirety.

[0310] In this document, the term "ligand" used for RNAi conjugates refers to the portion covalently coupled to RNAi, including the conjugated portion as described above and (if any) a linker group. It is understood that a ligand may have a linker group (when the conjugated portion is linked to RNAi via a linker group) or may not have a linker group (when the conjugated portion is directly linked to RNAi).

[0311] ASAGPR ligand conjugate

[0312] The desialyl glycoprotein receptor (ASGPR) is a hepatocyte-specific endocytic receptor that specifically recognizes and binds to ligand molecules with galactose or galactose derivatives as terminal glycosyl groups. It has been shown that galactose derivative modifications can be used to construct drug carriers, thereby targeting and delivering drugs to hepatocytes via ASGPR-mediated endocytosis.

[0313] Therefore, in some embodiments, the RNAi conjugates of this disclosure comprise the RNAi oligonucleotide duplex of this disclosure and a targeting ligand, particularly an asialic acid glycoprotein receptor (ASGPR) ligand, linked thereto. The ASGPR ligand can increase the uptake of the oligonucleotide duplex by hepatocytes, targeting the drug to hepatocytes via ASGPR-mediated endocytosis. Specifically, the ASPGR ligand comprises a moiety selected from galactose or galactose derivatives (e.g., galactosamine, N-formylgalactosamine, N-acetylgalactosamine (GalNAc), N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, etc.). The ASPGR ligand may or may not have a linker group.

[0314] In this disclosure, "galactose derivatives" specifically refer to galactose derivatives with an affinity for ASGPR similar to or higher than that of galactose. Preferred galactose derivatives are N-acetylgalactosamine (GalNAc) or GalNAc derivatives. Other sugars with affinity for ASGPR may also be used, including but not limited to galactosamine, N-butyrylgalactosamine, and N-isobutyrylgalactosamine. The affinity of galactose derivatives for ASGPR can be determined by conventional methods in the art.

[0315] In some embodiments, the ASGPR ligand is monovalent, i.e., it contains a single targeting moiety capable of binding to ASGPR. In other embodiments, the ASGPR ligand is multivalent, i.e., it contains multiple targeting moieties capable of binding to ASGPR. In some embodiments, the ASGPR ligand is a monovalent galactose ligand, providing a single galactose derivative capable of serving as an ASGPR targeting moiety. In some embodiments, the ASGPR ligand is a multivalent galactose ligand, providing multiple galactose derivatives, such as galactose clusters, capable of serving as ASGPR targeting moieties.

[0316] In this document, when the galactose derivative in the ligand is N-acetylgalactosamine (GalNAc) or a GalNAc derivative, the ligand is also referred to as a GalNAc ligand, including monovalent, divalent, trivalent, or tetravalent GalNAc ligands capable of providing 1, 2, 3, or 4 structural moieties of GalNAc or GalNAc derivatives.

[0317] In some embodiments, in the RNAi conjugate formed by the RNAi molecule of this disclosure and a ligand containing a galactose derivative (e.g., GalNAc) as a targeting group, the molar ratio of the RNAi molecule to the galactose derivative (e.g., GalNAc) can be any suitable ratio, such as 1:1, 1:2, 1:3 or 1:4.

[0318] In this paper, a galactose cluster refers to a molecule having multiple, for example, two, three, or four terminal galactoses or galactose derivatives. For example, a preferred galactose cluster has three identical or different terminal galactoses or galactose derivatives that exhibit affinity for ASGPR, wherein the terminal galactoses or galactose derivatives can be linked to the molecule via their C-1 or C-6 carbons. Such galactose clusters are also referred to as triadic galactoses, trivalent galactoses, and galactose trimers. It is known that triadic galactose clusters can bind to ASGPR with a higher affinity than biadic or monoadic galactoses (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, Biol. Chem., 257, 939-945).

[0319] In some embodiments, the galactose cluster to be conjugated with the oligonucleotide may comprise two or three galactose derivatives respectively linked to a central branch point. In some embodiments, the galactose derivatives are preferably linked to the branch point via a spacer group. In some embodiments, the preferred spacer group is a flexible hydrophilic spacer group (US Patent 5,885,968; Biessen et al., J. Med. Chem. 1995, Vol. 39, pp. 1538-1546). A preferred flexible hydrophilic spacer group is a PEG spacer group, such as a PEG3 spacer group (three ethylene units). In some aspects, the branch point group can be any small molecule group that allows the conjugation of two or three galactose derivatives and further allows the branch point to conjugate to the oligonucleotide. An exemplary branch point group is dilysine. A dilysine molecule contains three amino groups that can thereby link three galactose derivatives and a carboxyl reactive group that can thereby link the oligonucleotide. Another exemplary branch point group is a tetravalent linker based on trihydroxyalkylmethane, such as a tetravalent linker based on trihydroxymethylmethane containing the following structure: The linking group comprises three oxygen groups that can be linked to the three galactose derivatives and a central branch point carbon atom that can be linked to the oligonucleotide. In some embodiments, each galactose derivative (e.g., GalNAc) in the galactose cluster can be linked to the oligonucleotide via a spacer group, such as a polyethylene glycol (PEG) spacer group, or such as di-, tri-, tetra-, penta-, or hexa-glycol spacer groups. In this case, the spacer group, such as the PEG portion, can form a spacer group between the sugar portion of the galactose derivative and the branch point group. See, for example, WO2015 / 173208.

[0320] In other embodiments, the sugar moiety (e.g., GalNAc) or sugar-spacer group moiety (e.g., sugar-PEG moiety) to be conjugated with the oligonucleotide may also be covalently linked (conjugated) to the oligonucleotide by means of a branching group or branching component, such as an amino acid or peptide with two or more amino groups (e.g., 3, 4, or 5), such as lysine, dilysine, trilysine, or tetralysine. In some embodiments, the trilysine molecule is optional, containing four amino groups that can be used to link the sugar conjugation moiety, such as a galactose derivative (e.g., GalNAc), and a carboxyl reactive group that can be used to link the trilysine to the oligonucleotide. In other embodiments, other conjugation moieties, such as lipophilic / hydrophobic moieties, may also be linked to the oligonucleotide via the lysine residues.

[0321] In other embodiments, the galactose cluster to be conjugated with the oligonucleotide comprises a peptide linker group connected to the oligonucleotide via a bifunctional linker group, such as a Tyr-Asp(Asp) tripeptide or an Asp(Asp) dipeptide. Additionally, other branched molecules may be selected from 1,3-bis-[5-(4,4'-dimethoxytriphenylmethoxy)pentylamino]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphorimide (Glen Research catalog number: 10-1920-xx), tri-2,2,2-[3-(4,4'-dimethoxytriphenylmethoxy)propoxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphorimide (Glen Research catalog number: 10-1922-xx), tri-2,2,2-[3-(4,4'-dimethoxytriphenylmethoxy)propoxymethyl]methyleneoxypropyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphorimide and 1-[5-(4,4'-dimethoxy-triphenylmethoxy)pentylamide]-3-[5-fluorenmethoxy-carbonyl-oxy-pentylamide]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphorimide (Glen Research catalog number: 10-1925-xx). WO 2014 / 179620 and European application number 14188444.5 describe the generation of various GalNac conjugates (the aforementioned documents are hereby incorporated by reference).

[0322] In some embodiments, the linking (conjugation) of the branch point to the oligonucleotide can be accomplished by a spacer group. In some embodiments, the spacer group can be a flexible hydrophilic spacer group. An example of such a flexible hydrophilic spacer group is a PEG spacer group or a C6 linker. For example, the spacer group is a PEG3 spacer group (three ethylene units). In other embodiments, the spacer group can be a spacer group containing an N-acylpyrrolidine, such as the following spacer groups:

[0323] In some embodiments, the conjugates of this disclosure comprise an ASGPR ligand comprising GalNAc (N-acetylgalactosamine) or a derivative thereof, such as a monovalent, divalent, trivalent, or tetravalent GalNAc ligand. Such GalNAc ligands can target the RNAi compounds of this disclosure to the liver.

[0324] In some embodiments, the RNAi conjugates of this disclosure comprise one or more GalNAc or GalNAc derivatives. The GalNAc or GalNAc derivative may be linked to the oligonucleotide of the RNAi via a linker group, such as a divalent, trivalent, or tetravalent branching point group. In some embodiments, the GalNAc ligand binds to the 3' end of the sense strand of the RNAi. In some embodiments, the GalNAc ligand is linked to the 3' end of the sense strand of the RNAi oligonucleotide via a linker group. In some embodiments, the GalNAc ligand binds to the 5' end of the sense strand. In some embodiments, the GalNAc ligand is linked to the 5' end of the sense strand of the RNAi oligonucleotide via a linker group. In some embodiments, the GalNAc ligand binds to the 3' end of the antisense strand. In some embodiments, the GalNAc ligand is linked to the 3' end of the antisense strand of the RNAi oligonucleotide via a linker group.

[0325] In some embodiments, GalNAc or a GalNAc derivative is linked to the RNAi of this disclosure via a divalent linker. In other embodiments, GalNAc or a GalNAc derivative is linked to the RNAi of this disclosure via a trivalent linker. In still other embodiments, GalNAc or a GalNAc derivative is linked to the RNAi of this disclosure via a tetravalent linker.

[0326] In some embodiments, the RNAi conjugates of this disclosure comprise a single GalNAc or GalNAc derivative linked to a double-stranded RNAi. Preferably, the single GalNAc or GalNAc derivative is linked via a linker group to the 5' or 3' end of the positive strand of the double-stranded RNAi.

[0327] In some embodiments, the RNAi conjugates of this disclosure comprise one or more (e.g., 1, 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives. In some embodiments, one or more of the GalNAc or GalNAc derivatives may be individually linked to the RNAi oligonucleotide via a linker group, independently of any other GalNAc or GalNAc derivative. In some embodiments, any two or more of the GalNAc or GalNAc derivatives may be linked to the RNAi oligonucleotide via a common linker group in a tandem cluster. Furthermore, RNAi conjugates comprising multiple GalNAc or GalNAc derivatives linked thereto in an independent form and / or in a tandem cluster are also included in this disclosure. In some embodiments, two GalNAc or GalNAc derivatives are tandemly linked to one end (5' or 3') of the positive strand of the double-stranded RNAi, and one GalNAc or GalNAc derivative is independently linked to the other end (3' or 5') of the positive strand of the double-stranded RNAi. In other embodiments, two GalNAc or GalNAc derivatives are independently linked to the two ends of the sense strand of the double-stranded RNAi, and optionally, the double-stranded RNAi may also include one GalNAc or GalNAc derivative linked to the 3' end of the antisense strand. In other embodiments, three GalNAc or GalNAc derivatives are linked in tandem to one end (5' or 3' end) of the sense strand of the double-stranded RNAi.

[0328] In some embodiments, the ligand portion, such as the ASPGR ligand portion, is conjugated to the RNAi of this disclosure via a phosphate ester group or a thiophosphate ester group.

[0329] In some embodiments, an RNAi conjugate comprising one or more (e.g., 1, 2, or 3) ligands linked to RNAi of the present disclosure is provided, wherein each ligand independently has the structure of formula (I):

[0330] in,

[0331] Gal represents terminal galactose derivatives independently;

[0332] L indicates a linking group;

[0333] n is an integer selected from 1, 2, 3, and 4; and

[0334] The wavy line indicates the connection to the RNAi disclosed herein via this valence bond. It will be understood that the ligand is connected to the 5' and / or 3' end of the sense strand and optionally the 3' end of the antisense strand of the RNAi disclosed herein, preferably via a phosphate ester bond or a thiophosphate bond as shown in formula (A22).

[0335] In some embodiments, the linker group L may or may not have branching components. In some embodiments, the branching components may be in the form of two-antenna, three-antenna, or other multi-branched shapes.

[0336] In some embodiments, each Gal is individually linked to the RNAi disclosed herein via a linker group L, independently of the other Gals. In some embodiments, Gals are linked to the RNAi disclosed herein via linker groups L in a tandem cluster.

[0337] Therefore, in some embodiments, the ligands of formula (I) of this disclosure each independently have the structure of formula (Ia):

[0338] in,

[0339] L A1 This indicates the connection used to link the Gal section to L. A2 Partial linker;

[0340] L A2 This indicates a divalent, trivalent, tetravalent, or pentavalent linking group, used to link n Gal-L... A1 - Partially linked to the RNAi disclosed herein;

[0341] Gal independently represents terminal galactose derivatives; and

[0342] n is an integer selected from 1, 2, 3, and 4; and

[0343] The wavy line indicates the connection to the RNAi disclosed herein via this valence bond. It will be understood that the ligand is connected to the 5' and / or 3' end of the sense strand and optionally the 3' end of the antisense strand of the RNAi disclosed herein, preferably via a phosphate ester bond or a thiophosphate bond as shown in formula (A22).

[0344] In other embodiments, the ligands of formula (I) of this disclosure each independently have the structure of formula (Ib):

[0345] in,

[0346] L B It is hydroxyethylidene;

[0347] L B1Each is independently represented as -(5 to 6-membered heteroaryl)-(C 1-6 (alkylene)-CO-;

[0348] L B2 Each independently represents the absence of or -NH-(C 1-8 (alkylene)-CO-;

[0349] L B3 Each can be independently represented as -(5 to 6 membered cycloalkylene or heteroalkylene)-C(O)-NH-(C 1-4 (alkylene)-;

[0350] L B4 Each of them can be represented independently when they exist. 1-20 Alkyl chain or -L B3 -L B2 -L B1 -Gal, where L represents B4 -L B3 -L B2 -L B1 -Gal section and connection to L B -L B3 -L B2 -L B1 - The Gal parts can be the same or different;

[0351] Gal represents terminal galactose derivatives independently;

[0352] X represents oxygen or sulfur;

[0353] s is 0, 1, 2, or 3; and

[0354] The wavy line indicates the connection to the RNAi disclosed herein via this valence bond. It will be understood that the ligand is connected to the 5' and / or 3' end of the sense strand and optionally the 3' end of the antisense strand of the RNAi disclosed herein, preferably via a phosphate ester bond or a thiophosphate bond as shown in formula (A22).

[0355] It is understandable that the connection to L B -L B3 -L B2 -L B1 -Gal part can be used as L B The hydroxyethylidene can be attached to either of the two carbon atoms, for example, to a carbon atom attached to O or to another carbon atom. For example, as L B The hydroxyethylidene can have the following structure: The wavy valence key indicates a connection to the rest of the part.

[0356] In the RNAi conjugates of the ligands comprising formula (I) or its subforms such as (Ia) or (Ib) of this disclosure, Gal independently represents GalNAc (N-acetylgalactosamine) or a GalNAc derivative. In some embodiments, Gal independently represents a galactose derivative moiety having the following structure:

[0357] in,

[0358] R1 is an H or hydroxyl protecting group,

[0359] R2 is selected from hydrogen, hydroxyl group, C 1-20 Alkyl, C 2-20 alkenyl, C 1-20 Alkoxy, C 1-20 Alkylthio, -NR a R b C 6-20 Aryl-C 0-8 Alkylene-O-, C 6-20 Aryl-C 0-8 Alkylene-S- and CH3O-(CH2CH2O) q -CH2CH2O-, where R a and R b Each is independently H or C 1-20 Alkyl, q represents an integer from 1 to 16, and wherein the aryl group is optionally surrounded by one or more C... 1-8 Alkyl substitution; and

[0360] A wavy valence bond indicates that the bond is connected to the rest of the molecule.

[0361] In this document, suitable hydroxyl protecting groups are known to those skilled in the art, including but not limited to acetyl (Ac), benzoyl (Bz), phenoxyacetyl, tertvalyl, monomethoxytriphenylmethyl (MMTr), dimethoxytriphenylmethyl (DMTr), isobutyryl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl and isopropyldimethylsilyl.

[0362] Therefore, in some embodiments, R1 is independently H, acetyl (Ac), benzoyl (Bz), monomethoxytriphenylmethyl (MMTr), dimethoxytriphenylmethyl (DMTr), or tert-butyldiphenylsilyl (TBDPS). Preferably, R1 is independently H, acetyl (Ac), benzoyl (Bz), dimethoxytriphenylmethyl (DMTr), or tert-butyldiphenylsilyl (TBDPS). In some embodiments, R1 is independently H or benzoyl (Bz). In some embodiments, R1 is independently H.

[0363] In some implementations, R2 is independently selected from: hydrogen, hydroxyl, C 1-6 Alkyl, C 2-6 alkenyl, C 1-16 Alkoxy groups (e.g., C) 1-6 alkoxy), C 1-6 Alkylthio, -NR a R b , phenyl-C 0-4 alkylene-O-, phenyl-C 0-4 Alkylene-S- and CH3O-(CH2CH2O) q -CH2CH2O-, where R a and R b Each is independently H or C 1-6 Alkyl groups such as C 1-4 Alkyl, q represents an integer from 1 to 12, and the phenyl group is optionally constituting one or more C atoms. 1-4 Alkyl substitution. In some embodiments, R2 is independently C10. 1-16 Alkoxy groups, such as C 1-6 Alkyl group.

[0364] In some implementations, R2 is independently H, OH,

[0365] Preferably, R2 is OH, More preferably, R2 is independently OH or in particular

[0366] In some implementations, Gal is represented independently. Preferred The variables are defined as described in this paper, and the wavy valence bond represents the connection to the rest of the molecule via this valence bond.

[0367] In some implementations, Gal is represented independently. Preferred The variables are defined as described in this paper, and the wavy valence bond represents the connection to the rest of the molecule via this valence bond.

[0368] In some implementations, Gal is represented independently. The wavy valence bond indicates that the valence bond is connected to the rest of the molecule.

[0369] The definition of Gal above can be applied equally to the variable Gal mentioned in other parts of this article.

[0370] In some implementations, in formula (Ia), L A1 Each can be independently represented as a linking base having the following structure:

[0371] -(CH2) m1 -C(O)-NH-(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0372] -(CH2) m1 -C(O)-NH-(CH2) m2 -C(O)-NH-(CH2) m3 -;

[0373] -(CH2) m1 -NH-C(O)-(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0374] -(CH2) m1 -NH-C(O)-(CH2) m2 -C(O)-NH-(CH2) m3 -;

[0375] -(CH2) m1 -O-(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0376] -(CH2CH2O) m4 -(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0377] Wherein, m1, m2, m3 and m4 are each independently 1, 2, 3, 4, 5, 6, 7 or 8; and the left side of the group is connected to Gal, and the right side is connected to the rest of the molecule.

[0378] In some implementations, m1 is preferably 3, 4, 5, 6, 7 or 8, more preferably 3, 4, 5 or 6, and most preferably 4.

[0379] In some implementations, m2 is preferably 1, 2, 3, 4, 5 or 6, more preferably 1, 2 or 3.

[0380] In some implementations, m3 is preferably 1, 2, 3, 4, 5 or 6, more preferably 1, 2 or 3, and most preferably 2.

[0381] In some implementations, m4 is preferably 1, 2, 3, 4, 5 or 6, more preferably 1, 2, 3 or 4, and most preferably 1 or 3.

[0382] In some implementations, L A1 Each of these independently represents a linker base selected from the following:

[0383] Wherein, m1, m2 and m4 are each independently 1, 2, 3, 4, 5, 6, 7 or 8; and wherein the 1 position of the group is connected to Gal, and the 2 position is connected to the rest of the molecule.

[0384] In some implementations, L A1 Each of these independently represents a linker base selected from the following:

[0385] Preferred is

[0386] The group is connected to Gal at position 1 and to the rest of the molecule at position 2.

[0387] In some implementations, L A2 This indicates a divalent, trivalent, or tetravalent linking group comprising a monohydroxymethylmethane, dihydroxymethylmethane, or trihydroxymethylmethane member, wherein the L... A2 via the oxygen atom in the hydroxymethyl group and L A1 Some are linked via ether bonds and are also linked to the RNAi disclosed herein via methane carbon atoms (directly or indirectly).

[0388] In some implementations, L A2 It indicates a linking group selected from the following structures:

[0389] Where L A3 This indicates the absence of a spacer group; the oxygen atom on the left side of the group is related to L. A1 Partially linked via ether bonds, the right side is linked to the RNAi disclosed herein. The carbon atom marked with an asterisk can be considered as an L... A2 Some branch points.

[0390] In some implementations, L A3 Represents spacer bases with the following structure:

[0391] Preferred

[0392] Wherein, q is an integer selected from 1 to 16, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16; preferably, q is an integer selected from 1 to 12, for example, an integer selected from 8 to 12; the 1 position of the group is related to L. A2The branch point in the part is connected, and the 2-position is connected to the RNAi disclosed herein, for example, by a phosphate ester bond or a thiophosphate bond as shown in formula (A22).

[0393] In some implementations, L A3 Represents spacer bases with the following structure:

[0394] Preferred Wherein, the 1 position of the group is related to L A2 The branch point in the part is connected, and the 2-position is connected to the RNAi disclosed herein, for example, by a phosphate ester bond or a thiophosphate bond as shown in formula (A22).

[0395] In some implementations, the ligands of formula (I) or (Ia) each independently have the structure of formula (Ia-1):

[0396] In this document, each variable is defined as such, for example, as defined in formula (I) or (Ia); the wavy line indicates a connection to RNAi via the valence bond. It is understood that the ligand is connected to the 5' and / or 3' end of the sense strand and optionally the 3' end of the antisense strand of the RNAi disclosed herein, preferably via a phosphate ester bond or a thiophosphate bond as shown in formula (A22). In a preferred embodiment, R1 is independently H; m1 and m2 are independently 1, 2, 3, 4, 5, 6, 7, or 8, preferably 3 or 4; q is an integer selected from 1 to 16, preferably an integer selected from 1 to 12, more preferably an integer from 8 to 12.

[0397] In particular, the ligands of formula (I) or (Ia) are each independently an L96 moiety having the following structure:

[0398] The wavy line indicates the connection to RNAi via this valence bond. It is understood that the ligand is connected to the 5' and / or 3' end of the sense strand of the RNAi disclosed herein, and optionally the 3' end of the antisense strand, preferably via a phosphate ester bond or a thiophosphate bond as shown in formula (A22). In some embodiments, the ligand is connected to the 3' end of the sense strand of the RNAi disclosed herein.

[0399] In some implementations, in formula (Ib), L B1 Preferred representation: -(5-membered heteroaryl)-(C 1-6 alkylene)-CO-, for example -triazolyl-(C 1-6 (alkylene)-CO-, wherein the group is connected to Gal on the left and to L on the right. B2 Connect or when L B2 When it does not exist and L B3 Connection. In some implementations, LB1 express Where x is 1, 2, 3, 4, 5, or 6, preferably 1, 2, 3, or 4, and the 1-position of the group is connected to Gal, and the 2-position is connected to L. B2 Connect or when L B2 When it does not exist and L B3 Connection. In some implementations, L B1 express The group is connected to Gal at position 1 and to L at position 2. B2 Connection or when L B2 When it does not exist and L B3 connect.

[0400] In some implementations, L B2 This indicates that it does not exist. In some implementations, L B2 It represents -NH-(C 1-8 (linear alkylene)-CO-, preferably -NH-(C 1-6 Straight-chain alkylene groups (-CO-), such as -NH-methylene-CO-, -NH-(CH2)2-CO-, -NH-(CH2)3-CO-, -NH-(CH2)4-CO-, -NH-(CH2)5-CO-, -NH-(CH2)6-CO-, -NH-(CH2)7-CO-, or -NH-(CH2)8-CO-.

[0401] In some implementations, L B3 Preferred representation: -(5-membered heterocyclic alkylene)-C(O)-NH-(C 1-2 alkylene)-, for example -(pyrrolidine)-C(O)-NH-(C 1-2 alkylene)-, wherein the left side of the group is adjacent to L B2 Connection or when L B2 When it does not exist and L B1 Connect, right side with L B Connection, for example, to L B Either of the two carbon atoms of the hydroxyethylidene group. In some embodiments, L B3 yes For example The 1 position of the group and L B2 Connect or when L B2 When it does not exist and L B1 Connect, 2 bits and L B Connection, for example, to L B Either of the two carbon atoms of the hydroxyethylidene.

[0402] In some implementations, L B4 Indicate C 1-20Alkyl chains, such as C 1-16 Alkyl chain, C 6-14 Alkyl chain, C 8-14 Alkyl chain, C 10-14 Alkyl chain or C 10-12 Alkyl chain. Preferably, L B4 It is a linear chain. In some implementations, L B4 Indicate C 6-14 Straight-chain alkyl chain, C 8-14 Straight-chain alkyl chain, C 10-14 Alkyl chain or C 10-12 Straight-chain alkyl chain.

[0403] In some implementations, L B4 Indicates -L B3 -L B2 -L B1 -Gal, where the variables are as defined herein, for example, in equation (Ib). In some implementations, it represents L. B4 -L B3 -L B2 -L B1 -Gal part is connected to L in equation (Ib) B -L B3 -L B2 -L B1 -The Gal parts are the same or different.

[0404] In some implementations, in formula (Ib), -L B3 -L B2 -L B1 -Gal part or L B4 Part of it is:

[0405] Where x is 1, 2, 3, 4, 5 or 6, preferably 1, 2, 3 or 4; y is 1, 2, 3, 4, 5, 6, 7 or 8; Gal is a terminal galactose derivative as defined herein; and the wavy line indicates that it is connected to the rest of the molecule via this valence bond.

[0406] In some implementations, -L B3 -L B2 -L B1 -Gal part or L B4 Partially selected from:

[0407] In some embodiments, the ligands of formula (I) or (Ib) each independently have a structure selected from the following:

[0408] in,

[0409] LB It is hydroxyethylidene;

[0410] L B4 Each is represented independently when it exists.

[0411] C 1-20 Alkyl chain, The wavy valence bond represents L. B4 It is connected to the rest of the molecule via this valence bond;

[0412] x can be 1, 2, 3, 4, 5 or 6 independently, preferably 1, 2, 3 or 4;

[0413] Each y can be independently 1, 2, 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6;

[0414] Each Gal is an independent terminal galactose derivative as defined herein;

[0415] X represents oxygen or sulfur;

[0416] s is 0, 1, 2, or 3; and

[0417] The wavy line in formula (Ib-1) or (Ib-2) indicates a connection to the disclosed RNAi via this valence bond. It is understood that the ligand is connected to the 5' and / or 3' end of the sense strand and optionally the 3' end of the antisense strand of the disclosed RNAi, preferably via a phosphate ester bond or a thiophosphate bond of formula (A22).

[0418] In some embodiments, the ligands of formula (Ib-1) have structures selected from the following:

[0419] The variables are as defined in this paper.

[0420] In some embodiments, the ligands of formula (Ib-2) have structures selected from the following:

[0421] The variables are as defined in this paper.

[0422] In some implementations, Gal is independently represented in formula (Ib) and its subformulas such as (Ib-1), (Ib-2), (Ic-1), (Ic-2), (Ic-3), or (Ic-4). Preferred The variables are defined as follows, and the wavy valence bond represents the connection to the rest of the molecule via this valence bond. Specifically, Gal is independently... The wavy valence bond indicates that the valence bond is connected to the rest of the molecule.

[0423] In some embodiments, s is 0 in formula (Ib) and its sub-formulas, such as in formula (Ic-1) or (Ic-2), particularly in formula (Ic-1). In other embodiments, s is 1, 2, or 3, for example, 1.

[0424] In some implementations, in formula (IIb) and its sub-formulas, such as in formula (Ic-1) or (Ic-2), particularly in formula (Ic-1), L B4 Indicate C 6-14 Straight-chain alkyl chain, C 8-14 Straight-chain alkyl chain, C 10-14 Straight-chain alkyl chain or C 10-12 Straight-chain alkyl chain. Preferably, L B4 Indicate C 10-14 Straight-chain alkyl chain or C 10-12 Straight-chain alkyl chain, more preferably C 10-12 Straight-chain alkyl chain. In some embodiments, in formula (Ib-1) and its subforms, such as in formula (Ic-1) or (Ic-2), particularly in formula (Ic-1), s is 1, and L B4 Indicate C 10-14 Straight-chain alkyl chain, preferably C 10-12 Straight-chain alkyl chain.

[0425] In some implementations, in formula (IIb) and its sub-formulas, such as in formulas (Ic-1) and (Ic-2), particularly in formula (Ic-1), L B4 express The variables are as defined herein. In some implementations, in equation (IIb) and its sub-equations, such as in equation (Ic-1) or (Ic-2), particularly in equation (Ic-1), s is 1, and L B4 express The variables are as defined herein. Preferably, in equation (Ic-1), L B4 Indicates connection to as L B The hydroxyethylidene portion is the same as the portion.

[0426] In some implementations, in formula (IIb) and its sub-formulas, such as in formulas (Ic-3) and (Ic-4), particularly in formula (Ic-3), L B4 express The variables are as defined herein. In some implementations, in equation (IIb) and its sub-equations, such as in equations (Ic-3) and (Ic-4), particularly in equation (Ic-3), s is 1, and L B4 express The variables are as defined herein. Preferably, in equation (Ic-3), L B4Indicates connection to as L B The hydroxyethylidene portion is the same as the portion.

[0427] In particular, the ligands of formula (I) or (Ib) are each independently a part having the following structure: The wavy valence indicates the connection to RNAi via this valence. It can be understood that the ligand is connected to the 5' and / or 3' end of the sense strand and optionally the 3' end of the antisense strand of the RNAi disclosed herein, preferably via a phosphate ester bond or a thiophosphate bond of formula (A22).

[0428] In some embodiments, the ligand of formula (Ia-1) is linked to the RNAi of this disclosure as follows:

[0429] in, The '3' represents the 3' end of the RNAi positive strand, X represents oxygen or sulfur, and other variables are as defined in this paper.

[0430] Specifically, the ligand with the L96 structure is linked to the RNAi disclosed herein as follows:

[0431] in, The '3' represents the 3' end of the RNAi oligonucleotide double strand, and 'X' represents oxygen or sulfur.

[0432] In some embodiments, the ligand having the p36 structure is linked to the RNAi disclosed herein as follows:

[0433] in, The oligonucleotide double strand represents RNAi, where 3' or 5' represents the 3' or 5' end of the positive strand of RNAi, and X represents oxygen or sulfur. In particular, when X in the above formula represents sulfur, the ligand having the P36 structure is linked to the RNAi disclosed herein via a thiophosphate group structure as shown in formula (A22), which can be represented as P36s or sP36.

[0434] In some embodiments, the ligand having the (P36P36) structure is linked to the RNAi of this disclosure as follows:

[0435] in, 5' represents the oligonucleotide double strand of RNAi, and 5' represents the 5' end of the positive strand of RNAi.

[0436] In some embodiments, the ligand having the (P36P67) structure is linked to the RNAi of this disclosure as follows:

[0437] in, 5' represents the oligonucleotide double strand of RNAi, and 5' represents the 5' end of the positive strand of RNAi.

[0438] In some embodiments, the ligand having the (P34P34) structure is linked to the RNAi of this disclosure as follows:

[0439] in, 5' represents the oligonucleotide double strand of RNAi, and 5' represents the 5' end of the positive strand of RNAi.

[0440] In some embodiments, an RNAi conjugate is provided comprising one or more, for example, 1, 2, or 3 ASGPR ligands linked together by the RNAi oligonucleotide double strand of the present disclosure, wherein each ASGPR ligand independently has any structure selected from formula (I) or any subform thereof, for example, a structure selected from any of (Ia), (Ia-1), (Ib), (Ib-1), (Ib-2), (Ic-1), (Ic-2), (Ic-3), (Ic-4), L96, P36, P36P36, P36P67, P34P34, and each ASGPR ligand is independently linked to the 5' end and / or 3' end of the sense strand of the RNAi and optionally the 3' end of the antisense strand, preferably via a phosphate ester bond or a thiophosphate bond of formula (A22).

[0441] In some embodiments, in the RNAi conjugates of this disclosure, the RNAi oligonucleotide double strand is linked to two ASGPR ligands, wherein each ASGPR ligand independently has any structure selected from formula (I) or any subform thereof, such as structures selected from (Ia), (Ia-1), (Ib), (Ib-1), (Ib-2), (Ic-1), (Ic-2), (Ic-3), (Ic-4), L96, P36, P36P36, P36P67, P34P34, and one of the ASGPR ligands is attached to the 5' end of the RNAi positive strand, and the other is attached to the 3' end of the RNAi positive strand. Preferably, the ligands are linked by a phosphate ester bond or a thiophosphate bond of formula (A22).

[0442] In some embodiments, in the RNAi conjugates of this disclosure, the RNAi oligonucleotide double strand is linked to two ASGPR ligands, one ASGPR ligand having a structure of (Ia), preferably (Ia-1), and linked to the 3' end of the RNAi positive strand, and the other ASGPR ligand having a structure of (Ib), preferably selected from any of (Ib-1), (Ib-2), (Ic-1), (Ic-2), (Ic-3), and (Ic-4), and linked to the 5' end of the RNAi positive strand. Preferably, the ligands are linked via a phosphate ester bond or a thiophosphate bond of formula (A22).

[0443] In some embodiments, in the RNAi conjugates of this disclosure, the RNAi oligonucleotide double strand is linked to two ASGPR ligands, one ASGPR ligand having an L96 structure and attached to the 3' end of the positive strand of the RNAi of this disclosure, and the other ASGPR ligand having a structure selected from any one of P36, P36P36, P36P67, and P34P34 and attached to the 5' end of the positive strand of the RNAi of this disclosure. Preferably, the ligands are linked by a phosphate ester bond or a thiophosphate bond of formula (A22).

[0444] In some embodiments, in the RNAi conjugates of this disclosure, the RNAi oligonucleotide double strand is linked to two ASGPR ligands, one ASGPR ligand having an L96 structure and attached to the 3' end of the positive strand of the RNAi of this disclosure, and the other ASGPR ligand having a P36P67 structure and attached to the 5' end of the positive strand of the RNAi of this disclosure. Preferably, the ligands are linked by a phosphate ester bond or a thiophosphate ester bond of formula (A22), more preferably by a phosphate ester bond.

[0445] In some embodiments, in the RNAi conjugates of this disclosure, the RNAi oligonucleotide double strand is linked to two ASGPR ligands, wherein each of the two ASGPR ligands independently has a structure of (Ib), preferably selected from any one of (Ib-1), (Ib-2), (Ic-1), (Ic-2), (Ic-3), and (Ic-4), and is respectively linked to the 3' and 5' ends of the positive strand of the RNAi of this disclosure. Preferably, the ligands are linked by a phosphate ester bond or a thiophosphate bond of formula (A22).

[0446] In some embodiments, in the RNAi conjugates of this disclosure, the RNAi oligonucleotide double strand is linked to two ASGPR ligands, wherein each of the two ASGPR ligands independently has a structure selected from any one of P36, P36P36, P36P67, and P34P34, and is respectively linked to the 3' and 5' ends of the positive strand of the RNAi of this disclosure. Preferably, the ligands are linked by a phosphate ester bond or a thiophosphate bond of formula (A22).

[0447] In some embodiments, in the RNAi conjugates of this disclosure, the RNAi oligonucleotide double strand is linked to two ASGPR ligands, each having a p36 structure and respectively attached to the 3' and 5' ends of the positive strand of the RNAi of this disclosure. Preferably, the ligands are linked via phosphate ester bonds or thiophosphate bonds of formula (A22), more preferably via thiophosphate bonds of formula (A22).

[0448] In some embodiments, in the RNAi conjugates of this disclosure, the RNAi oligonucleotide double strand is linked to an ASGPR ligand, wherein the ASGPR ligand has any structure selected from formula (I) or any subform thereof, for example, a structure selected from (Ia), (Ia-1), (Ib), (Ib-1), (Ib-2), (Ic-1), (Ic-2), (Ic-3), (Ic-4), L96, P36, P36P36, P36P67, P34P34, and the ASGPR ligand is linked to the 5' or 3' end of the positive strand of the RNAi of this disclosure. Preferably, the ligand is linked via a phosphate ester bond or a thiophosphate bond of formula (A22).

[0449] In some embodiments, in the RNAi conjugates of this disclosure, the RNAi oligonucleotide double strand is linked to an ASGPR ligand, wherein the ASGPR ligand has a structure of formula (Ia), preferably (Ia-1), more preferably L96, and the ASGPR ligand is linked to the 3' end of the positive strand of the RNAi of this disclosure. Preferably, the ligand is linked via a phosphate ester bond or a thiophosphate ester bond of formula (A22), more preferably via a phosphate ester bond.

[0450] In some embodiments, in the RNAi conjugates of this disclosure, the RNAi oligonucleotide double strand is linked to an ASGPR ligand, wherein the ASGPR ligand has a structure of formula (Ib), preferably a structure selected from (Ib-1), (Ib-2), (Ic-1), (Ic-2), (Ic-3), and (Ic-4), more preferably a structure selected from P36, P36P36, P36P67, and P34P34, and the ASGPR ligand is linked to the 5' or 3' end of the positive strand of the RNAi of this disclosure, preferably the 5' end. Preferably, the ligand is linked via a phosphate ester bond or a thiophosphate bond of formula (A22).

[0451] In some embodiments, in the RNAi conjugate of this disclosure, the RNAi oligonucleotide double strand is linked to an ASGPR ligand having a P36P36 structure and being linked to the 5' end of the positive strand of the RNAi of this disclosure, preferably via a phosphate ester bond or a thiophosphate bond of formula (A22), more preferably via a phosphate ester bond.

[0452] In some embodiments, in the RNAi conjugate of this disclosure, the RNAi oligonucleotide double strand is linked to an ASGPR ligand having a P34P34 structure and attached to the 5' end of the positive strand of the RNAi of this disclosure, preferably via a phosphate ester bond or a thiophosphate bond of formula (A22), more preferably via a phosphate ester bond.

[0453] In some preferred embodiments, the RNAi conjugate comprises a modified sense strand conjugated with a non-nucleotide moiety and a modified antisense strand, wherein the modified sense strand conjugated with the non-nucleotide moiety and the modified antisense strand each comprise, or are composed of, the modified nucleic acid sequences shown in SEQ ID NO:, or are respectively composed of the modified nucleic acid sequences shown in SEQ ID NO:.

[0454] a) Justice Chain: SEQ ID NO:43; Antisense Chain: SEQ ID NO:8;

[0455] b) Justice Chain: SEQ ID NO:44; Antisense Chain: SEQ ID NO:10;

[0456] c) Justice Chain: SEQ ID NO:45; Antisense Chain: SEQ ID NO:12;

[0457] d) Justice chain: SEQ ID NO:46; Antisense chain: SEQ ID NO:14;

[0458] e) Justice Chain: SEQ ID NO:47; Antisense Chain: SEQ ID NO:8;

[0459] f) Justice Chain: SEQ ID NO:48; Antisense Chain: SEQ ID NO:17;

[0460] g) Justice Chain: SEQ ID NO:49; Antisense Chain: SEQ ID NO:19;

[0461] h) Justice chain: SEQ ID NO:50; Antisense chain: SEQ ID NO:21;

[0462] i) Justice Chain: SEQ ID NO:51; Antisense Chain: SEQ ID NO:23;

[0463] j) Justice Chain: SEQ ID NO:48; Antisense Chain: SEQ ID NO:24;

[0464] k) Justice Chain: SEQ ID NO:52; Antisense Chain: SEQ ID NO:19;

[0465] l) Justice Chain: SEQ ID NO:53; Antisense Chain: SEQ ID NO:27;

[0466] m) Justice Chain: SEQ ID NO:54; Antisense Chain: SEQ ID NO:29;

[0467] n) Justice chain: SEQ ID NO:55; Antisense chain: SEQ ID NO:31;

[0468] o) Justice chain: SEQ ID NO:56; Antisense chain: SEQ ID NO:33;

[0469] p) Justice chain: SEQ ID NO:57; Antisense chain: SEQ ID NO:33;

[0470] q) Justice chain: SEQ ID NO:58; Antisense chain: SEQ ID NO:8;

[0471] r) Justice Chain: SEQ ID NO:59; Antisense Chain: SEQ ID NO:8;

[0472] s) Justice chain: SEQ ID NO:48; Antisense chain: SEQ ID NO:38;

[0473] t) Justice chain: SEQ ID NO:59; Antisense chain: SEQ ID NO:8;

[0474] u) Justice Chain: SEQ ID NO:60; Antisense Chain: SEQ ID NO:17;

[0475] v) Justice Chain: SEQ ID NO:59; Antisense Chain: SEQ ID NO:19;

[0476] w) Justice Chain: SEQ ID NO:60; Antisense Chain: SEQ ID NO:24;

[0477] x) Justice Chain: SEQ ID NO:60; Antisense Chain: SEQ ID NO:38;

[0478] y) Justice chain: SEQ ID NO:61; Antisense chain: SEQ ID NO:40; or

[0479] z) Justice chain: SEQ ID NO:62; Antisense chain: SEQ ID NO:42.

[0480] In some preferred embodiments, the RNAi conjugate comprises a modified sense strand conjugated with a non-nucleotide moiety and a modified antisense strand, wherein the modified sense strand conjugated with a non-nucleotide moiety and the modified antisense strand each comprise the modified sense strand nucleic acid sequence and antisense strand nucleotide sequence conjugated with a non-nucleotide moiety as shown in SEQ ID NO: in Table C below, or each comprises the modified sense strand nucleic acid sequence and antisense strand nucleotide sequence conjugated with a non-nucleotide moiety as shown in SEQ ID NO: in Table C below:

[0481] The various embodiments and examples described herein, and any combination thereof, are equally applicable to the RNAi conjugates of this disclosure. For example, the variable definitions described in formula (I) (e.g., Gal) are equally applicable to other RNAi activators, including RNAi conjugates. Moreover, the various aspects and / or embodiments and / or examples and / or features described herein can be combined arbitrarily, and the resulting technical solutions are also included within the scope of this disclosure.

[0482] Delivery of RNAi activators

[0483] The RNAi active agents and RNAi conjugates of this disclosure can be delivered or introduced by any means known in the art (e.g., to cells in vitro, to test animals, or to humans). In some embodiments, the RNAi active agents or RNAi conjugates of this disclosure are delivered to a subject, the delivery of which enables the RNAi to hybridize with HBV mRNA within cells containing HBV and to suppress expression by inhibiting transcription. Examples of routes of delivery of RNAi active agents or RNAi conjugates include direct injection at a tissue site. Optionally, the RNAi active agent can be modified to target selected cells and then administered systemically. For example, for systemic administration, the RNAi molecule can be modified to specifically bind to a receptor or antigen expressed on the surface of selected cells, for example, by forming the aforementioned RNAi conjugates according to this disclosure that target hepatocytes, for example, by conjugating an oligonucleotide duplex of RNAi to a peptide or antibody that binds to a receptor on the surface of hepatocytes, for example, by conjugating an ASGPR ligand according to this disclosure, for example, forming an RNAi conjugate as defined herein. The nucleic acid molecules disclosed herein can also be delivered to cells using vectors commonly known in the art and, for example, those described in US20070111230 (the full contents of which are incorporated herein by reference).

[0484] In this document, when referring to RNAi active agents, “introduced into cells” means to facilitate or achieve uptake or absorption into cells, as understood by those skilled in the art. Absorption or uptake of RNAi active agents can occur through unassisted diffusive or active cellular processes, or through the use of an auxiliary active agent or device. The term is not limited to cells in vitro; RNAi active agents can also be “introduced into cells,” where the cells are part of a living organism. In such cases, introduction into cells will include delivery to the organism. For example, for in vivo delivery, RNAi active agents can be injected into tissue sites or administered systemically. In vivo delivery can also be performed using β-glucan delivery systems, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Publication No. 2005 / 0281781. In vitro introduction into cells includes methods known in the art, such as electroporation and lipid transfection. Other means are described herein or are known in the art. Mechanisms for delivering RNAi active agents to target cells have been proposed. Methods known in the art include, but are not limited to: viral delivery (retroviruses, adenoviruses, lentiviruses, baculoviruses, AAVs); liposomes (Lipofectamine, cationic DOTAP, neutral DOPC) or nanoparticles (cationic polymers, PEI); bacterial delivery (tkRNAi); and chemical modification of siRNA (LNA) to increase stability. Xia et al., 2002 Nat. Biotechnol. 20 and Devroe et al., 2002 BMC Biotechnol. 2 1:15 disclose the incorporation of siRNA into viral vectors. Other systems for delivering RNAi active agents are considered, and the RNAi active agents disclosed herein can be delivered by a variety of methods pending discovery and / or approval by the FDA or other regulatory agencies. The RNAi active agents of this disclosure can be delivered in suitable pharmaceutical compositions.

[0485] Pharmaceutical compositions of RNAi activators

[0486] As used herein, “pharmaceutical composition” includes pharmaceutically effective amounts of one or more RNAi active agents or RNAi conjugates, pharmaceutically acceptable carriers, and optionally other therapeutic agents that synergize with RNAi active agents.

[0487] As used herein, "pharmaceutically effective amount," "therapeuticly effective amount," or simply "effective amount" refers to an amount of RNAi active agent or RNAi conjugate that is effective in producing the intended pharmacological, therapeutic, or preventative outcome. For example, a given clinical treatment is considered effective if a measurable parameter associated with a disease or condition is reduced by at least 10%, and the therapeutically effective amount of the drug used to treat said disease or condition is the amount necessary to produce a reduction of at least 10% in said parameter. In this embodiment, a therapeutically effective amount of an HBV-targeting RNAi active agent or RNAi conjugate can reduce serum HBV protein levels by at least 10%. In other embodiments, a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition exhibits a reduction of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%, and the therapeutically effective amount of the drug used to treat the disease or condition is the amount necessary to produce a reduction of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%, respectively.

[0488] The term "medicinal carrier" refers to a carrier for administering a therapeutically active agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof.

[0489] In some embodiments, this disclosure provides pharmaceutical compositions comprising the RNAi active agent or RNAi conjugate of this disclosure (especially modified siRNA or siRNA conjugates conjugated to a GalNAc ligand) as the active ingredient and pharmaceutically acceptable diluents, carriers, and / or excipients (e.g., PBS buffer, physiological saline, water). The pharmaceutical compositions are intended to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity. Pharmaceutically acceptable diluents, carriers, and / or excipients as used in this disclosure include any suitable pharmaceutically acceptable diluents, carriers, and / or excipients known in the art.

[0490] In some embodiments, the RNAi active agent or RNAi conjugate according to this disclosure may be present in a non-buffered solution, preferably saline or water. In other embodiments, the RNAi active agent or RNAi conjugate according to this disclosure may be present in a buffered solution, preferably comprising acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof; more preferably, the buffered solution is phosphate-buffered saline (PBS). In some embodiments, the RNAi active agent or RNAi conjugate according to this disclosure is formulated as a subcutaneous formulation. In other embodiments, the RNAi active agent or RNAi conjugate according to this disclosure is formulated as an intravenous formulation.

[0491] Application of RNAi activators

[0492] Pharmaceutical compositions comprising the HBV RNAi active agent or RNAi conjugate of this disclosure targeting HBV can be administered via a variety of suitable routes of administration, including but not limited to buccal, inhalation (including blowing or deep inhalation), nasal, oral, parenteral, implantation, injection, or infusion (via epidural, intra-articular, intra-articular, intracapsular, intracardiac, intracranial, intradermal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intrathecal, intravenous, subarachnoid, subcapsular, subcutaneous, subepidermal, transendothelial, transtracheal, transvascular, rectal, sublingual, local, and / or vaginal routes). Administration may be performed by injection, infusion, skin patch, or any other method known in the art. Formulations for administration may be powdered, atomized, aerosolized, granulated, or suitably prepared for delivery.

[0493] Pharmaceutical compositions comprising the HBV RNAi active agent or RNAi conjugate disclosed herein can be administered using medical devices known in the art. For example, in certain embodiments, the RNAi active agent can be administered using a needle-free subcutaneous injection device, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of known implants and modules that can be used in the disclosure herein include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medication via the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable-flow-rate implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses a permeable drug delivery system with multiple compartments; and U.S. Patent No. 4,475,196, which discloses a permeable drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art. In some embodiments, pharmaceutical compositions comprising the HBV RNAi active agent or RNAi conjugate of the present disclosure can be formulated to ensure proper distribution in vivo.

[0494] Treatment and prevention of HBV-related diseases or conditions

[0495] This disclosure provides the use of the RNAi active agent or RNAi conjugate of this disclosure in the treatment or prevention of HBV-related diseases or conditions, wherein a therapeutically effective amount or a preventively effective amount of the RNAi active agent or RNAi conjugate of this disclosure is administered to a human or non-human animal subject in need.

[0496] This disclosure provides a method for preventing or treating HBV-related diseases or conditions, including administering the RNAi active agent or RNAi conjugate of this disclosure to a subject in need, such as a human or a non-human animal.

[0497] This disclosure provides RNAi active agents or RNAi conjugates for use in therapies, such as for the treatment or prevention of HBV-related diseases or conditions.

[0498] This disclosure also provides the use of the RNAi active agent or RNAi conjugate disclosed herein for the treatment or prevention of HBV-related diseases or conditions.

[0499] This disclosure also provides the use of the RNAi active agent or RNAi conjugate of this disclosure in the preparation of a medicament for the treatment or prevention of HBV-related diseases or conditions.

[0500] In this article, “HBV-related disease or condition” means any disease involving HBV infection or HBV-mediated disease, and / or any disease that can be treated and / or alleviated by inhibiting the level, expression and / or activity of HBV (e.g., mRNA level or protein expression or protein activity), such as hepatitis B virus infection or related disease, acute or chronic hepatitis, hepatitis B (e.g., acute or chronic hepatitis B) or hepatitis D (e.g., acute or chronic hepatitis D), cirrhosis or liver cancer such as hepatocellular carcinoma.

[0501] The RNAi active agents or RNAi conjugates against HBV described herein can be formulated into pharmaceutical compositions that can be administered to humans and / or non-human animals. These compositions may include one or more of the RNAi active agents or RNAi conjugates disclosed herein, and optionally other therapeutic agents that can be used to treat HBV-related diseases. They may be administered as part of early / preventive treatment or may be administered at a therapeutically effective dose to treat individuals who have already presented with symptoms related to the disease or condition. The administerable pharmaceutical compositions include those of any of the embodiments described above.

[0502] In some embodiments, the RNAi activator or RNAi conjugate used for this purpose may be any RNAi activator or RNAi conjugate described in this disclosure. In some embodiments, the HBV-related disease is HBV infection or HBV infection-mediated disease, including, for example, acute or chronic hepatitis, such as hepatitis B (e.g., acute or chronic hepatitis B) or hepatitis D (e.g., acute or chronic hepatitis D), cirrhosis, or liver cancer such as hepatocellular carcinoma.

[0503] In some embodiments, this disclosure also provides a method for treating pathological conditions at least partially mediated or associated with HBV infection or HBV expression, the method comprising administering to a subject in need a therapeutically effective amount of the disclosed RNAi active agent or RNAi conjugate against HBV.

[0504] In some embodiments, the subject is a human being, and the RNAi active agent or RNAi conjugate is administered subcutaneously or intravenously.

[0505] In some embodiments, the RNAi activator according to this disclosure can be used to reduce or inhibit (e.g., completely inhibit) HBV expression levels in cells (e.g., mammalian cells, such as human cells).

[0506] In some embodiments, the RNAi activator according to this disclosure comprises at least 15, at least 16, at least 17, or at least 18 or more consecutive nucleotides targeting HBV mRNA. In some embodiments, the RNAi activator according to this disclosure comprises two oligonucleotide chains capable of forming a double-stranded region of at least 19 or more nucleotides. In some embodiments, the two oligonucleotide chains each comprise a sequence motif differing by 0, 1, 2, or 3 nucleotides from at least 18 consecutive nucleotides in the sense and antisense strand sequences of any RNAi molecule provided in Tables 1, 2, and 3 or Table AC. In some embodiments, each strand of the RNAi activator according to this disclosure has a length of less than 30 nucleotides, for example, 18-23 nucleotides and / or 19-21 nucleotides. In some embodiments, the double-stranded RNAi activator according to this disclosure may have one or two blunt ends or one or two overhangs, such as 3' and / or 5' overhangs of 1, 2, 3, or 4 nucleotides (i.e., 1-4 nt). The double-stranded RNAi activator according to this disclosure may optionally contain one or two 5' and / or 3' end modifications (i.e., 5' caps and / or 3' caps) and / or one or more modified nucleotides. In some embodiments, the double-stranded RNAi activator according to this disclosure is an RNAi conjugate according to this disclosure, said conjugate, for example, containing a GalNAc ligand according to this disclosure, particularly a monovalent, divalent, or trivalent GalNAc ligand having a P36 and / or L96 and / or P34 and / or P67 structure.

[0507] In some embodiments, the RNAi active agents or RNAi conjugates of this disclosure (especially modified siRNAs or siRNA conjugates conjugated to GalNAc ligands) and their pharmaceutical compositions are particularly useful for the treatment and / or prevention of HBV infection. The RNAi active agents or RNAi conjugates are preferably any RNAi molecules selected from those provided in Tables 3 and C, especially the RNAi conjugates provided in Table C. In this document, RNAi conjugates containing GalNAc ligands are also referred to as GalNAc-siRNA of this disclosure.

[0508] In some embodiments of the treatment and prevention provided in this disclosure, the subject is a mammal, such as a primate, rodent, or human. In some embodiments, administration of the RNAi conjugate of this disclosure or a pharmaceutical composition thereof results in a decrease in the subject's blood, such as serum, HBV protein. It has been demonstrated that reducing serum HBV protein levels can effectively treat and / or prevent HBV-related diseases or conditions, such as hepatitis B virus infection or related diseases, including, for example, acute or chronic hepatitis, such as hepatitis B (e.g., acute or chronic hepatitis B) or hepatitis D (e.g., acute or chronic hepatitis D), cirrhosis, or liver cancer such as hepatocellular carcinoma.

[0509] The siRNA can be administered to the patient via any suitable route known in the art, including but not limited to: subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and / or cerebrospinal fluid administration. The dosage of the RNAi reagents and compositions disclosed herein can be determined based on the patient's weight, age, sex, disease severity, etc. Subjects can be given therapeutic doses of siRNA, such as 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg body weight, etc. Dosing frequency can be based on regularity, such as daily, weekly, every two weeks, every three weeks, every one month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, annually, or longer, with repeated administration. After an initial treatment regimen, treatment can be given at a lower frequency, for example, after monthly administration for three months, administration can be continued for six months, one year, or longer. Administration of an RNAi active agent or RNAi conjugate can reduce the level of any mRNA (other than HBx mRNA) or any protein (other than HBx) in HBV in a patient's cells, tissues, blood, urine, or other compartments by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0510] Example

[0511] The following embodiments are provided to further illustrate this disclosure. It should be understood that they are merely for the purpose of better understanding this disclosure and are not intended to limit the scope of this disclosure in any way.

[0512] Unless otherwise specified, all experimental materials and reagents used in the examples are commercially available or can be readily prepared using methods known to those skilled in the art. Unless otherwise stated, solvent ratios or percentages herein are volume ratios. Abbreviations used herein generally have meanings known in the art, unless otherwise indicated.

[0513] Specifically, the meanings of the abbreviations used in this article are shown in the table below:

[0514] Unless otherwise specified, all raw materials or reagents used in the embodiments and comparative examples of this disclosure are commercially available products.

[0515] The specific meanings of each symbol in this public sequence are shown in the table below.

[0516] Example 1: Synthesis and Preparation of siRNA Conjugates

[0517] Example 1.1: Synthesis of compound 211 (TU0)

[0518] Synthesis of compound 201:

[0519] Compound 200 (30.0 g, 157.73 mmol, 1.0 eq.) was dissolved in ultra-dry DCM (300 mL). Imidazole (26.8 g, 394.33 mmol, 2.5 eq.) was added to the reaction system, and the reaction was cooled to 0 °C and stirred continuously for 30 minutes. Then, tert-butyldiphenylchlorosilane (47.7 g, 173.50 mmol, 1.1 eq.) was slowly added to the reaction system. After the addition was complete, the ice bath was removed, and the reaction was gradually brought to room temperature and allowed to proceed overnight at room temperature. TLC analysis showed that compound 201 reacted completely. Water was added to the reaction system, followed by extraction twice with ethyl acetate. The organic phases were combined and washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and slurryed to obtain compound 201 (52.0 g). ESI-MS: m / z 429.6 [M+H] +

[0520] Synthesis of compound 202:

[0521] Compound 201 (52.0 g, 121.33 mmol, 1.0 eq.) was dissolved in DMF (500 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. 60% NaH (5.82 g, 145.60 mmol, 1.2 eq.) was slowly added dropwise to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought to room temperature and reacted overnight at room temperature. TLC analysis showed that the reaction was complete, and compound 201 reacted completely. The reaction system was slowly poured into a saturated ammonium chloride aqueous solution at approximately 0 °C. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 202 (78.3 g), which was directly used in the next reaction step. ESI-MS: m / z 519.7 [M+H] +

[0522] Synthesis of compound 203:

[0523] Crude compound 202 (78.3 g) was added to a 500 mL round-bottom flask, followed by 200 mL of THF and then 1.2 eq. of tetrabutylammonium fluoride (1 M in THF, 145.5 mL). After addition, the reaction mixture was stirred at room temperature for 2 hours. TLC analysis confirmed complete reaction of compound 202. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 203. Crude compound 203 was then subjected to column chromatography (PE / EA = 4 / 1) to obtain compound 203 (45.0 g, 160.53 mmol). ESI-MS: m / z 281.3 [M+H] +

[0524] Synthesis of compound 204:

[0525] Compound 203 (45.0 g, 160.53 mmol, 1.00 eq.) was added to a 500 mL round-bottom flask, and toluene (400 mL) was added to the flask and stirred to dissolve. Then, imidazole (38.25 g, 561.86 mmol, 3.5 eq.) and triphenylphosphine (50.53 g, 192.64 mmol, 1.2 eq.) were added, followed by the slow addition of iodine (48.9 g, 192.64 mmol, 1.2 eq.). After the addition was complete, the reaction system was placed in an oil bath and heated to 80 °C. The mixture was stirred for 3 hours. TLC and LCMS analysis showed that compound 203 had reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding an aqueous sodium sulfite solution. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 204. The crude product was subjected to column chromatography (PE / EA = 1 / 4) to give compound 204 (54.4 g, 139.41 mmol, 86.8% yield). ESI-MS: m / z 391.23 [M+H] +

[0526] Synthesis of compound 205:

[0527] Compound 204 (54.4 g, 139.42 mmol, 1.0 eq.) was dissolved in MeOH (500 mL). Potassium carbonate (19.34 g, 139.42 mmol, 1.0 eq.) and palladium on carbon (10% W, 5.4 g) were then added to the reaction solution, and the mixture was stirred at room temperature until compound 204 was completely reacted. The system was filtered through diatomaceous earth, concentrated, extracted twice with ethyl acetate, filtered to remove salt, and concentrated under reduced pressure to obtain crude product 205. Crude product 205 was subjected to column chromatography (PE / EA = 4 / 1) to obtain compound 205 (33.2 g, 125.61 mmol, 90.1% yield). 1 H NMR (400MHz, DMSO-d6) δ7.51–7.20(m,5H),5.81(d,J=4.0Hz,1H),4.72–4.64(m,2H),4.49(d,J=12.0Hz,1 H),4.25–4.16(m,1H),3.71(d,J=3.1Hz,1H),1.38(s,3H),1.25(s,3H),1.20(d,J=6.4Hz,3H).ESI-MS:m / z 265.31[M+H] +

[0528] Synthesis of compound 206:

[0529] Compound 205 (33.2 g, 125.61 mmol, 1.0 eq) was added to a 500 mL round-bottom three-necked flask, and acetic acid (300 mL) and acetic anhydride (64.12 g, 628.05 mmol, 5.0 eq.) were added to the reaction system, and the mixture was stirred until completely dissolved. The reaction mixture was cooled to 0 °C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (10 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5 °C. After the addition was complete, the reaction system was allowed to return to room temperature and stirred for 8 hours until compound 205 reacted completely. The reaction system was cooled to approximately -5 °C and neutralized with ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 206. Crude product 206 was subjected to column chromatography (PE / EA = 4 / 1) to give compound 206 (21.2 g, 68.76 mmol, 54.7% yield). ESI-MS: m / z 309.33 [M+H]+

[0530] Synthesis of compound 207:

[0531] Compound 206 (10.0 g, 32.43 mmol, 1.00 eq.) and uracil (7.27 g, 64.86 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (100 mL) was added and stirred to dissolve. Then, BSA (19.79 g, 97.29 mmol, 3.0 eq.) was added. The reaction system was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the reaction system was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (7.21 g, 32.43 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 206 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. A saturated aqueous sodium bicarbonate solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 207. The crude product was subjected to column chromatography (PE / EA = 1 / 3) to give compound 207 (9.6 g, 26.64 mmol, 82.1% yield). 1HNMR(400MHz,DMSO-d6)δ11.36(s,1H),7.46–7.21(m,6H),6.32(d,J=8.0Hz,1H),5.90(t,J=9.0Hz,1H),5.18(s,1H),4.77(d,J =11.5Hz,1H),4.58(t,J=11.0Hz,1H),4.31–4.18(m,1H),3.94(d,J=3.4Hz,1H),2.11(s,3H),1.33(d,J=6.3Hz,3H).ESI-MS:m / z 361.37[M+H]+

[0532] Synthesis of compound 208:

[0533] The dried compound 207 (9.6 g, 26.64 mmol, 1.0 eq) was added to a 500 mL three-necked flask, and ultra-dry dichloromethane (100 mL) was added to the reaction flask. The reaction system was cooled to -40 °C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 80 mL, 79.92 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 207 was completely reacted. The reaction was quenched at -10 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 208. The crude compound was subjected to column chromatography (PE / EA = 1 / 2) to obtain compound 208 (5.0 g, 18.5 mol, 69.4% yield). 1 H NMR (400MHz, DMSO-d6) δ11.36(s,1H),7.59(s,1H),6.32(d,J=8.0Hz,1H),5.90(t,J=9.0Hz,1H),5.18(s,1H),4 .49(d,J=7.8Hz,1H),4.31–4.18(m,1H),3.94(d,J=3.4Hz,1H),2.11(s,3H),1.33(d,J=6.3Hz,3H).ESI-MS:m / z 271.24[M+H]

[0534] Synthesis of compound 209:

[0535] Compound 208 (5.0 g, 18.5 mmol, 1.0 eq) was added to a 100 mL round-bottom flask, and ultra-dry DCE (100 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (12.54 g, 37.0 mmol, 2.0 eq.), silver nitrate (3.14 g, 18.5 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (11.21 g, 92.5 mmol, 5.0 eq.) were added to the reaction mixture at room temperature, and the mixture was stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 208 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 209. The crude product was subjected to column chromatography (PE / EA = 1 / 1) to give compound 209 (7.3 g, 12.75 mmol, 70% yield). 1H NMR (400MHz, DMSO-d6) δ11.36(s,1H),7.46(d,J=7.5Hz,3H),7.39–7.22(m,7H),6.92(dd,J=8.9,7.3Hz,4H),6.32(d,J=8.0Hz,1H),5.9 0(t,J=9.0Hz,1H),5.18(s,1H),4.31–4.18(m,1H),3.94(d,J=3.4Hz,1H),3.81(s,6H),2.11(s,3H),1.33(d,J=6.3Hz,3H).ESI-MS:m / z 573.6[M+H] -

[0536] Synthesis of compound 210:

[0537] Compound 209 (7.3 g, 12.75 mmol, 1.0 eq.) was dissolved in THF (100 mL), followed by the addition of sodium methoxide (826.51 mg, 15.3 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 10 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 210. Crude product 210 was subjected to column chromatography (PE / EA = 1 / 2) to obtain compound 210 (6.2 g, 11.68 mmol, 91.61% yield). 1 H NMR (400MHz, DMSO-d6) δ11.36(s,1H),7.46(d,J=7.5Hz,3H),7.39–7.22(m,7H),6.92(dd,J=8.9,7.3Hz,4H),6.32(d,J=8.0Hz,1H),5.90(t, J=9.0Hz,1H),5.30(d,J=3.8Hz,1H),5.18(s,1H),4.31–4.18(m,1H),3.94(d,J=3.4Hz,1H),3.81(s,6H),1.33(d,J=6.3Hz,3H).ESI-MS:m / z 531.58[M+H] -

[0538] Synthesis of compound 211:

[0539] The dried compound 210 (2.0 g, 3.77 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (20 mL) was added and stirred until completely dissolved. DIPEA (974.5 mg, 7.54 mmol, 2.0 eq.) and DMAP (92.12 mg, 0.754 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.34 g, 5.66 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 210 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 211. The crude product was purified by column chromatography (PE / EA = 1 / 1) to give compound 211 (2.3 g, 3.15 mmol, 83.55% yield). 1 H NMR (400MHz, DMSO-d6) δ11.36(s,1H),7.46(d,J=7.5Hz,3H),7.39–7.22(m,7H),6.92(dd,J=8.9,7.3Hz,4H),6.32(d,J=8.0Hz,1H),5.90(t,J=9 .0Hz,1H),5.18(s,1H),4.31–4.18(m,1H),3.99–3.80(m,9H),2.95–2.7 0(m,2H),2.60–2.49(m,2H),1.33(d,J=6.3Hz,3H),1.12–0.92(m,12H). 31 P NMR(162MHz,DMSO-d6)δ150.02(s),149.73(s).ESI-MS:m / z 731.8[M+H] -

[0540] Example 1.2: Preparation of the double strand

[0541] We analyzed 9,000 HBV genome sequences in the HBVdb database and designed a positive strand in the conserved region of the genome. The design reference sequence was NC_003977.2. Through bioinformatics design and previous experimental results, candidate sequences BPR-991404 and BPR-991203 were obtained.

[0542] HBV mRNA sequence:

[0543] Table 1 provides the sequence motifs of the designed siRNA duplexes; Table 2 provides the modified forms of the siRNA duplexes shown in Table 1. In Tables 1 and 2, "SS" represents the sense strand of the siRNA, and "AS" represents the antisense strand of the siRNA. Table 1 also provides the start position of the target gene sequence (the mRNA sequence corresponding to NC_003977.2) targeted by the siRNA duplexes.

[0544] Table 1: Unmodified siRNA

[0545] Table 2: Modified siRNAs

[0546] *The corresponding sequences in the sequence list are the raw sequences used for retrieval; see this table for modifications.

[0547] E represents A19.

[0548] The oligonucleotide synthesis process in the RNAi reagent shown in Table 2 of this disclosure uses an insoluble polymer as a carrier and phosphoramidite nucleoside monomers as starting materials. Through solid-phase synthesis and cleavage / deprotection reactions, crude single-stranded oligonucleotides are obtained. After purification and ultrafiltration, two single-stranded intermediates are obtained. These two intermediates are annealed to form a double-stranded siRNA with inversely complementary base pairing. The siRNA is then obtained by freeze-drying. The specific reaction process is as follows:

[0549] a. Deprotection reaction: Under room temperature conditions (20-25℃), the protecting group DMTr (dimethoxytriphenylmethyl) on the carrier / nucleotide is removed with dichloroacetic acid to obtain an active hydroxyl group that can undergo coupling reaction. The deprotecting agent is a dichloromethane solution of dichloroacetic acid (3% v / v).

[0550] b. Coupling reaction: The nucleotide phosphoramide monomer and the activator are simultaneously fed into a solid-phase synthesis column. The phosphoramide group is activated and undergoes a coupling condensation reaction with the active hydroxyl group to generate a phosphite trimer. The activator is a 0.6 M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT).

[0551] c. Oxidation or thiolation. This depends on whether the phosphate ester bond or the thiophosphate bond is linked. The oxidation reaction proceeds as follows: under the action of the oxidizing agent iodine water, the triphosphite formed in the previous coupling condensation reaction is converted into a stable triphosphite. The oxidizing agent is a 0.04M iodine / water / pyridine solution, v(water):v(pyridine) = 1:9. The thiolation reaction proceeds as follows: under the action of the thioreagent ADTT (hydrogenated yellow element), the triphosphite formed in the previous coupling condensation reaction is converted into a stable triphosphite.

[0552] d. Capping reaction: The active hydroxyl groups that have not fully reacted during the coupling reaction are capped to prevent them from participating in subsequent reactions. The capping reagents are Cap A (acetic anhydride / acetonitrile) and Cap B (N-methylimidazolium:Py:acetonitrile = 2:3:5).

[0553] The above four-step reaction constitutes a cycle, which is repeated until a complete oligonucleotide sequence is synthesized. After completing the four-step cycle reaction for all nucleoside bases, the 5'-DMTr protecting group at the end of the nucleotide is removed by cleavage, and then the cyanoethyl protecting group is removed with 20% diethylamine, completing the solid-phase reaction. The solid-phase support is washed with acetonitrile and dried with an inert gas. The solid-phase support is collected and subjected to a cleavage and deprotection reaction using an ammonia deprotection reagent (25%–28% concentrated ammonia aqueous solution), cleaving and separating the oligonucleotide from the solid-phase support, while removing various protecting groups on the nucleoside bases. The solid-phase support is removed by filtration and washed with 50% ethanol. The mother liquors are combined and collected, and the resulting solution is concentrated to obtain a crude solution containing the desired oligonucleotide single strand.

[0554] The crude sample was sent for testing, and the target molecular weight was determined using high-resolution liquid chromatography-mass spectrometry (LC-MS).

[0555] The crude oligonucleotides were purified by anion exchange chromatography, followed by desalting using a gel column (HiTrap™ Desalting). The desalted sense and antisense strands were mixed in an equimolar ratio, heated to 65°C, held for 30 minutes, and then allowed to cool naturally to room temperature. The two single strands formed a double-stranded structure, siRNA, through hydrogen bonding. Molecular weight was determined using ion-pair reversed-phase chromatography (IPRP-HPLC) and high-resolution liquid chromatography-mass spectrometry (LC-MS), confirming the successful preparation of the HBV siRNA shown in Table 2.

[0556] Example 1.3: Synthesis of P67 precursor

[0557] The P67 precursor used in the synthesis of siRNA conjugates is synthesized as described below.

[0558] Compound 181 (2.3 g, 10.0 mmol, 1.0 eq) was weighed and dissolved in 20 mL of pyridine, then purged with nitrogen. The mixture was cooled to 0 °C in an ice-water bath. 4,4'-bismethoxytriphenylmethyl chloride (3.38 g, 10.0 mmol, 1.0 eq) was added in portions. After the addition was complete, the mixture was allowed to react at room temperature overnight. The reaction was quenched with water after TLC monitoring. The mixture was extracted twice with ethyl acetate, and the organic phases were combined, dried, concentrated, and purified by column chromatography to give compound 182 (4.4 g, yield: 82.6%).

[0559] Compound 182 (4.4 g, 8.3 mmol, 1.0 eq) and 4-dimethylaminopyridine (195.2 mg, 1.6 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purging protection, anhydrous dichloromethane (20 mL) and N,N-diisopropylethylamine (2.13 g, 16.5 mmol, 2.0 eq) were added. Then, 2-cyanoethyl N,N-diisopropylphosphoramide (2.89 g, 4.3 mmol, 1.5 eq) was added dropwise using a syringe. After the addition was complete, the reaction was allowed to proceed at room temperature for 1 h. The reaction was monitored by TLC until it was complete. After removing dichloromethane by concentration under reduced pressure at room temperature, the product was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase: 0.01% ammonium bicarbonate) to obtain the precursor of compound P67 (4.3 g, yield: 70.2%). 1 H NMR(400MHz, DMSO-d6)δ7.41(d,J=7.5Hz,2H),7.33–7.24(m,6H),7.24–7.17(m,1H),6.90–6.83(m,4H),3. 90(ddd,J=16.3,10.7,5.6Hz,1H),3.73(d,J=2.8Hz,6H),3.60(dddd,J=34.2,20.9,16.9,10.6,6.4Hz,4H ),3.08(dd,J=9.5,5.4Hz,1H),3.00(dd,J=9.5,5.0Hz,1H),2.73(t,J=5.9Hz,1H),2.65–2.54(m,1H),1.70 –1.42(m,2H),1.21(d,J=8.3Hz,20H),1.13(t,J=6.7Hz,10H),1.02(d,J=6.7Hz,2H),0.84(t,J=6.7Hz,3H). 31 P NMR (162MHz, DMSO) δ 147.61, 147.27. MS (ESI): m / z Calculated C 44 H 65 N₂O₅P[M+H] + :733.47, Actual measurement:733.21.

[0560] Example 1.4: Synthesis of P36 precursor

[0561] 1. Synthesis of Compound 57

[0562] Compound 100 (30 g, 77.09 mmol) was dissolved in 300 mL of dry 1,2-DCE. The system was placed at 0 °C, and trimethylsilyl trifluoromethanesulfonate (21 mL, 115.64 mmol, 1.5 eq.) was added. The reaction system was stirred at 0 °C for 10 min under nitrogen protection, and then placed in an oil bath at 50 °C for 12 h. The reaction of the starting material was monitored by TLC and LC-MS to ensure complete reaction. The system was then placed at room temperature, and... The mixture was stirred with a molecular sieve for 30 min, then anhydrous 3-pentanol (12.7 mL, 115.64 mmol, 1.5 eq.) was added. The system was stirred at room temperature under nitrogen protection for 12 h. The reaction was monitored by TLC and LC-MS. After the reaction was complete, triethylamine was added until the system was neutral. The mixture was then extracted successively with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1 to 1:2) to give a white solid compound 105 (19 g, two-step yield: 60%). MS (ESI): calculated m / z C19H31NO9[M+H]+: 418.20, actual: 418.21

[0563] Compound 105 (58 g, 139.568 mmol) was dissolved in 300 mL of dry NH3 / MeOH solution. The system was stirred at room temperature for 12 h under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was directly concentrated under reduced pressure to obtain the crude product compound 106. The crude product was not separated and purified and was directly used for subsequent reactions. MS (ESI): calculated m / z C13H25NO6[M+H]+: 292.17, measured: 292.19.

[0564] Compound 106 (40.6 g, 139.57 mmol) was dissolved in dry pyridine (800 mL), and TBDPSCl (47 mL, 181.44 mmol, 2 eq.) and 4-dimethylaminopyridine (5.12 g, 41.87 mmol, 0.3 eq.) were added. The system was stirred at room temperature for 12 h under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, compound 107 was obtained, which was directly used for subsequent reactions without post-treatment. MS (ESI): calculated m / z C29H43NO6Si[M+H]+: 530.29, measured: 530.26.

[0565] The reaction system of compound 107 was placed at 0 °C, and benzoyl chloride (48 mL, 418.8 mmol, 2 eq.) was added dropwise, followed by the addition of 4-dimethylaminopyridine (6.8 g, 55.8 mmol, 0.4 eq.). The system was stirred at room temperature for 12 h under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the reaction system was diluted with ethyl acetate and extracted successively with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 to 2:1) to obtain a white solid compound 108, which was directly used for the next step. MS (ESI): calculated m / z C43H51NO8Si[M+H]+: 738.34, actual: 738.33.

[0566] Compound 108 (103 g, 139.57 mmol) was dissolved in anhydrous tetrahydrofuran (800 mL), and triethylamine trihydrofluoric acid (114 mL, 697.85 mmol, 5.0 eq.) was added. The reaction system was stirred at 50 °C under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After about 12 h at room temperature, the reaction was complete. The reaction system was directly concentrated under reduced pressure to remove most of the solvent, diluted with ethyl acetate, and extracted successively with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1 to 1:1) to give a white solid compound 109 (147 g, yield from compound 105 in four steps: 65%). MS (ESI): calculated m / z C27H33NO8[M+H]+: 500.22, actual: 500.23.

[0567] Compound 109 (10 g, 20.031 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL). The system was placed at 0 °C, and PPh3 (10.5 g, 40.062 mmol, 2 eq.) and DPPA (8.6 mL, 40.062 mmol, 2 eq.) were added. DIAD (7.9 mL, 40.062 mmol, 2 eq.) was added dropwise. The system was stirred at 0 °C under nitrogen protection for 4 h, and then the system was placed at room temperature for about 12 h. The reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was directly concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1 to 3:1) to give a white solid compound 57 (4.7 g, 44%). 1H NMR (400MHz, DMSO-d6) δ8.01–7.91(m,3H),7.75–7.68(m,3H),7.63–7.53(m,3H),7.40(t, J=7.8Hz,2H),5.62(d,J=3.4Hz,1H),5.34(dd,J=11.1,3.4Hz,1H),4.77(d,J=8.5Hz,1H), 4.26–4.15(m,2H), 3.60–3.49(m,2H), 3.32(d,J=3.7Hz,1H), 1.70(s,3H), 1.61–1.42(m,4H), 0.87(dt,J=17.7,7.4Hz,6H). MS(ESI): m / z Calculated C27H32N4O7[M+H]+: 525.23, Measured: 525.24.

[0568] 2. Synthesis of the precursor of compound P36

[0569] Compound 101 (50 g, 0.55 mol, 1.0 eq.) was weighed and dissolved in 500 mL of acetonitrile. After purging with nitrogen, triethylamine (167 g, 1.65 mol, 3.0 eq.) and ethyl trifluoroacetate (160 g, 1.1 mol, 2.0 eq.) were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the solvent was removed by direct concentration under reduced pressure to obtain an oily crude product.

[0570] The crude oily product was dissolved in 400 mL of pyridine, purged with nitrogen, and cooled to 0 °C in an ice-water bath. 4,4'-bismethoxytriphenylmethyl chloride (186 g, 0.55 mol, 1.0 eq.) was added in portions, and the mixture was allowed to react overnight at room temperature. After TLC monitoring, the reaction was quenched with water, extracted twice with ethyl acetate, and the organic phases were combined, dried, concentrated, and purified by column chromatography to give 102,233 g of the compound. The two-step yield was 86.9%.

[0571] Compound 102 (233 g, 0.47 mol, 1.0 eq.) was dissolved in 1.5 L of methanol, and potassium hydroxide (53.4 g, 0.95 mol, 2.0 eq.) (prepared as a 3.0 mol / L aqueous solution) was added. The reaction was carried out at room temperature for 2 h. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, followed by rapid column chromatography purification to obtain compound 103, 174 g, yield: 92.8%.

[0572] Compound 104 (5.0 g, 43.5 mmol, 1.0 eq.) was weighed and dissolved in 50 mL of acetonitrile. After purging with nitrogen, triethylamine (15.0 g, 130.5 mmol, 3.0 eq.) and ethyl trifluoroacetate (14.1 g, 87.0 mmol, 2.0 eq.) were added, and the mixture was reacted overnight at room temperature. After the reaction was completed by TLC monitoring, the product was directly concentrated under reduced pressure to obtain a crude oily product (100% yield was directly used for the next reaction).

[0573] The crude oily product and compound 103 (17.1 g, 43.5 mmol, 1.0 eq.) were dissolved in 100 mL of N,N-dimethylformamide. Under nitrogen protection, N,N-diisopropylethylamine (11.2 g, 87.0 mmol, 2.0 eq.) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) (19.8 g, 52.2 mmol, 1.2 eq.) were added, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by TLC, the reaction was quenched with water, extracted with ethyl acetate, and the organic phase was washed with saturated brine. The mixture was separated, dried, concentrated, and purified by column chromatography to give compound 110, 17.5 g. The two-step yield was 68.2%.

[0574] Compound 110 (17.5 g, 29.8 mmol, 1.0 eq.) was dissolved in 200 mL of methanol, and potassium hydroxide (3.4 g, 59.7 mmol, 2.0 eq.) (prepared as a 3.0 mol / L aqueous solution) was added. The reaction was carried out at room temperature for 2 h. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, followed by rapid column purification to obtain compound 111, 13.8 g, yield: 95.1%.

[0575] Compound 111 (1.7 g, 3.5 mmol, 1.0 eq.) and acetylacetic acid (0.4 g, 4.2 mmol, 1.2 eq.) were weighed and dissolved in 10 mL of N,N-dimethylformamide. Under nitrogen protection, N,N-diisopropylethylamine (0.9 g, 7.0 mmol, 2.0 eq.) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (1.6 g, 4.2 mmol, 1.2 eq.) were added, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by TLC until it was complete. The mixture was quenched with water, extracted with ethyl acetate, and the organic phase was washed with saturated brine. The mixture was separated, dried, concentrated, and purified by reverse-phase preparative column chromatography (5%-95% acetonitrile / water, aqueous phase: 0.01% ammonium bicarbonate) to give compound 113, 1.2 g of white solid, yield: 60.9%.

[0576] Compound 113 (570 mg, 1.0 mmol, 1.0 eq.) and compound 57 (524 mg, 1.0 mmol, 1.0 eq.) were weighed and dissolved in 10 mL of methanol. Copper sulfate pentahydrate (500 mg, 2.0 mmol, 2.0 eq.) and sodium ascorbate (400 mg, 2.0 mmol, 2.0 eq.) were dissolved in 10 mL of water and added to the system. The reaction was monitored by TLC until it ended. After concentration, the mixture was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase: 0.01% ammonium bicarbonate) to obtain compound 114 (560 mg, yield: 51.2%).

[0577] Compound 114 (560 g, 0.51 mmol, 1.0 eq.) and 4-dimethylaminopyridine (12 mg, 0.1 mmol, 0.2 eq.) were weighed into a 100 mL single-necked flask. After nitrogen purging protection, anhydrous dichloromethane (10 mL) and N,N-diisopropylethylamine (132 mg, 1.02 mmol, 2.0 eq.) were added dropwise using a syringe. Then, 2-cyanoethyl N,N-diisopropylphosphonamide chloroform (CEP-Cl) (181 mg, 0.77 mmol, 1.5 eq.) was added dropwise. After the addition was complete, the reaction was allowed to proceed at room temperature for 1 h. The reaction was monitored by TLC until completion. After removing dichloromethane by concentration under reduced pressure at room temperature, the product was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase: 0.01% ammonium bicarbonate) to obtain the precursor of compound P36, a white solid, 430 mg, yield: 65.2%. NMR(400MHz,DMSO-d6)δ7.98–7.91(m,3H),7.91–7.80(m,1H),7.78–7.63(m,4H),7 .59(t,J=7.5Hz,3H),7.46–7.36(m,4H),7.36–7.16(m,7H),6.93–6.82(m,4H),5.6 6(d,J=3.5Hz,1H),5.34(dt,J=11.3,3.7Hz,1H),4.59(dt,J=9.8,6.3Hz,2H),4.52 –4.41(m,2H),4.22(tq,J=16.2,7.4,6.9Hz,2H),4.05(dt,J=10.3,5.2Hz,1H),3.8 3(dq,J=12.9,6.3Hz,1H),3.73(d,J=2.4Hz,7H),3.57(ddt,J=23.6,10.2,6.3Hz,2 H),3.38(s,1H),3.27(dt,J=16.0,5.8Hz,3H),3.09–2.98(m,1H),2.92–2.68(m,4H ), 2.68–2.51(m,2H), 2.07(s,3H), 1.90–1.77(m,1H), 1.68(d,J=1.7Hz,5H), 1.48–1.22(m,5H), 1.21–1.10(m,8H), 1.03(dd,J=6.8,4.4Hz,3H), 0.83–0.62(m,6H). 31P NMR (162MHz,DMSO) δ 148.36, 148.32, 148.22, 148.16, 148.05, 147.87. MS (ESI): m / z Calculated C70H86N8O14P[MH]-: 1293.60, Measured: 1293.63.

[0578] Example 1.5: Synthesis of P34 precursor

[0579] Weigh out acetylenic acid (10.0 g, 102 mmol, 1.0 eq) and N-hydroxysuccinimide (12.3 g, 107.1 mmol, 1.05 eq) and dissolve them in 20 mL of acetonitrile. Dissolve N,N'-dicyclohexylcarbodiimide (21.1 g, 102 mmol, 1.0 eq) in acetonitrile and add it dropwise to the system. React at room temperature overnight. Filter and dry under reduced pressure to give 20.0 g of white solid. Dissolve 20.0 g of white solid in 150 mL of tetrahydrofuran, add compound 15 (13.3 g, 102 mmol, 1.0 eq), and react at room temperature. After mass spectrometry analysis, filter and dry under reduced pressure to give compound 95, 19.75 g, yield: 92%.

[0580] Compound 111 (1.9 g, 3.9 mmol, 1.0 eq) and compound 95 (1.0 g, 4.7 mmol, 1.2 eq) were weighed and dissolved in 10 mL of dichloromethane. Under nitrogen protection, N,N-diisopropylethylamine (1.0 g, 7.7 mmol, 2.0 eq) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) (1.8 g, 4.7 mmol, 1.2 eq) were added, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by TLC, the reaction was quenched with water, extracted with ethyl acetate, and the organic phase was washed with saturated brine. The mixture was separated, dried, concentrated, and purified by reverse-phase preparative column chromatography (5%-95% acetonitrile / water, aqueous phase: 0.01% ammonium bicarbonate) to obtain compound 96, 1.5 g, yield: 46.8%.

[0581] Compound 96 (846 mg, 1.24 mmol, 1.0 eq) and compound 57 (650 mg, 1.24 mmol, 1.0 eq) were weighed and dissolved in 10 mL of methanol. Copper sulfate pentahydrate (620 mg, 2.5 mmol, 2.0 eq) and sodium ascorbate (496 mg, 2.5 mmol, 2.0 eq) were dissolved in 10 mL of water and added to the system. The reaction was monitored by TLC until it ended. After concentration, the product was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase 0.01% ammonium bicarbonate) to obtain 590 mg of product 93.

[0582] 590 mg (0.49 mmol, 1.0 eq) of compound 93 and 12 mg (0.1 mmol, 0.2 eq) of 4-dimethylaminopyridine were weighed into a 100 mL single-necked flask. After nitrogen purging protection, anhydrous dichloromethane (10 mL) and N,N-diisopropylethylamine (132 mg, 1.02 mmol, 2.0 eq) were added. Then, 2-cyanoethyl N,N-diisopropylphosphoramide (181 mg, 0.77 mmol, 1.5 eq) was added dropwise using a syringe. After the addition was complete, the reaction was allowed to proceed at room temperature for 1 h. The reaction was monitored by TLC until it was complete. After removing dichloromethane by concentration under reduced pressure at room temperature, the mixture was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase: 0.01% ammonium bicarbonate) to obtain compound 97, the P34 precursor (380 mg, yield: 56.7%). 1 H NMR (400MHz, DMSO) δ7.95(d,J=7.1Hz,3H),7.86–7.80(m,1H),7.79(t,J=4.5Hz,1H),7.72(t,J=7.9Hz,3 H),7.59(t,J=7.7Hz,3H),7.39(t,J=7.8Hz,4H),7.33–7.18(m,7H),6.87(t,J=7.2Hz,4H),5.66(d,J=3. 2Hz,1H),5.34(dd,J=11.1,3.2Hz,1H),4.59(dd,J=12.4,8.2Hz,2H),4.51–4.38(m,2H),4.28–4.12(m,2 H),4.09–3.96(m,1H),3.87–3.78(m,1H),3.73(d,J=2.1Hz,7H),3.66–3.58(m,1H),3.58–3.50(m,1H),3 .46–3.36(m,2H),3.25(dd,J=11.4,5.8Hz,2H),3.02(dd,J=12.4,6.2Hz,3H),2.91–2.84(m,1H),2.83–2 .74(m,3H),2.64(t,J=4.9Hz,1H),2.38(t,J=7.3Hz,2H),2.20(dd,J=11.7,6.5Hz,1H),2.13–1.98(m,1H ),1.84(dt,J=18.1,8.0Hz,1H),1.77–1.55(m,6H),1.52–1.27(m,9H),1.25(d,J=7.0Hz,3H),1.15(dd,J =12.7, 6.7Hz, 9H), 1.04 (t, J = 6.8Hz, 3H), 0.79 (t, J = 7.3Hz, 3H), 0.73 (t, J = 7.4Hz, 3H). MS (ESI): m / z calculation C 76 H 98N9O 15 P[MH] - :1406.68, Actual measurement:1406.69.

[0583] Example 1.6: Solid-phase carrier loading

[0584] 1. P36 solid support loading

[0585] Compound 114 (420 mg, 0.35 mmol) and 4-dimethylaminopyridine (8.5 mg, 0.07 mmol) were added to a 100 mL single-necked flask. After nitrogen purging protection, anhydrous dichloromethane (30 mL) and succinic anhydride (70 mg, 0.7 mmol) were added, and the reaction was allowed to proceed overnight. The reaction was monitored by TLC until completion. After removing dichloromethane by concentration under reduced pressure at room temperature, the mixture was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase 0.01% ammonium bicarbonate) to obtain 350 mg of compound (P36)a.

[0586] Compound (P36)a was coupled with an amino support (polystyrene resin (PS) or controlled-pore glass (CPG) powder) through a coupling reaction and a capping reaction to prepare a solid-phase synthesis support pre-loaded with P36. After the support loading was determined by analysis, it was used as a support for subsequent solid-phase synthesis to synthesize oligonucleotides with 3'-terminal conjugated ligand P36.

[0587] 2. L96 solid support load

[0588] Similar to the process steps in WO2014025805A1, a solid-phase synthesis support pre-loaded with L96 was prepared. After analyzing and determining the support loading, it was used as a support for subsequent solid-phase synthesis to perform base monomer coupling to synthesize oligonucleotides with 3'-terminal conjugated ligand L96.

[0589] Example 1.7: Synthesis of GalNAc-RNAi activator

[0590] 1. Following the above process steps, L96 (GalNAc3) or P36 (ASGPR monovalent ligand) is coupled with an amino carrier (polystyrene resin or CPG powder) through a coupling reaction and a capping reaction to prepare a solid-phase synthetic carrier preloaded with L96 or P36. The carrier loading was measured by analysis and testing.

[0591] 2. Load a commercially available general-purpose solid-phase support (Tianjin Nankai Hecheng Technology Co., Ltd.) or the above-mentioned solid-phase synthesis support into a solid-phase reaction column. In a nucleic acid synthesizer, following the standard oligonucleotide solid-phase synthesis method, using modified nucleotide monomers, apply the dedimethoxytriphenylmethylation reaction, coupling reaction, oxidation reaction, and capping reaction as described in Example 1.3, and synthesize the coupled-delivered oligonucleotide sequence (modified SS chain) by cyclically repeating these four steps. If the target product has a thiophosphate modification, a thiolation reaction is used instead of an oxidation reaction; that is, the oxidation reaction in Example 1.3 is replaced by the following thiolation reaction: under the action of the thiolation reagent PADS / ADTT, the triphosphite generated in the previous coupling condensation step is converted into a stable triphosphite. After synthesizing the desired sequence, the cyanoethyl protecting group is removed with 20% diethylamine, and the solid-phase support is dried by argon gas; the support is then deprotected by ammonolysis in concentrated ammonia at 55°C for 5 to 16 hours. After ammonia desorption and protection are completed, the carrier is removed by filtration; the ammonia hydrolysate is concentrated to remove ammonia water, and the remaining concentrated sample is sent for LC-MS analysis to confirm that the sample molecular weight is consistent with the theoretical molecular weight.

[0592] 3. Oligonucleotide conjugate samples coupled with L96 or P67, P36, P34 monomer combinations were purified by ion exchange on an AKTA Pure 150, followed by gel column desalting to obtain single-chain samples that met the requirements.

[0593] 4. Using the general-purpose solid-phase synthesis support Nitto Phase HLUny Linker, antisense chains (modified AS chains) were synthesized according to the standard solid-phase synthesis method.

[0594] 5. Add the modified SS chain and the modified AS chain to the annealing container in an equimolar ratio, heat to 55°C, hold for about 30 minutes, and then allow to cool naturally to room temperature.

[0595] 6. The annealed double-stranded samples were dispensed into freeze-drying containers according to the required amount and then freeze-dried to obtain GalNAc-siRNA conjugates.

[0596] The synthesized oligonucleotide conjugates are shown in Table 3 below:

[0597] *The corresponding sequences in the sequence list are the raw sequences used for retrieval; see this table for modifications.

[0598] Example 2: Transgenic Mouse Animal Experiment

[0599] This embodiment uses a transgenic HBV mouse model to evaluate the in vivo efficacy of the HBV siRNA reagent. The transgenic mouse strain was C57B / 6N-Tg(1.28HBV) / Vst, purchased from Beijing Vitonda Biotechnology Co., Ltd. Mice were grouped according to serum HBsAg levels and body weight, ensuring no statistically significant differences in serum HBsAg and body weight. Mice with lower HBsAg levels and lighter body weight were removed from the experiment. Each group consisted of 4 males, and the dosage was 3 mg / kg, administered subcutaneously as a single dose. The saline group served as the negative control (NC). Blood samples were collected from the orbital venous plexus of mice before administration and on day 7 after administration. The collected blood samples were incubated at room temperature for 3 minutes, centrifuged at 5 rpm for 1 minute, and the supernatant was used for HBV DNA and HBsAg detection. The reagents used were the Hepatitis B Virus Nucleic Acid Assay Kit (Sansure) and the Hepatitis B Virus Surface Antigen Assay Kit (Mike), and the detection methods were performed according to the corresponding instructions. Serum HBV DNA and HBsAg expression values ​​on day 7 post-administration were compared with the corresponding Log10 values ​​before autologous administration. The reduction effect compared to before autologous administration was used to evaluate the efficacy of the conjugate. The corresponding efficacy results are shown in Table 4.

[0600] Analysis of the knockdown effect on day 7 after drug administration revealed that, compared with the corresponding parental compounds BPR-99120354 and BPR-99140454 without E modification, the E-modified compounds BPR-99120357 and BPR-99140457 did not improve the knockdown effect on HBsAg, but significantly improved the knockdown effect on HBV DNA.

[0601] Table 4: Inhibitory effects of compounds on various viral components in transgenic mice

[0602] Example 3: Transgenic Mouse Animal Experiment

[0603] This embodiment uses a transgenic HBV mouse model to evaluate the in vivo efficacy of the HBV siRNA reagent. The transgenic mouse strain was C57B / 6N-Tg(1.28HBV) / Vst, purchased from Beijing Vitonda Biotechnology Co., Ltd. Mice were grouped according to serum HBsAg levels and body weight, ensuring no statistically significant differences in serum HBsAg and body weight. Mice with lower HBsAg levels and lighter body weight were removed from the experiment. Each group consisted of 4 males, and the dosage was 3 mg / kg, administered subcutaneously as a single dose. The saline group served as the negative control (NC). Blood samples were collected from the orbital venous plexus of mice before administration and on days 7, 14, and 21 after administration. The collected blood samples were incubated at room temperature for 3 minutes, centrifuged at 5 rpm for 1 minute, and the supernatant was used for HBsAg and / or HBV DNA detection. The reagents used were a Hepatitis B Virus Nucleic Acid Assay Kit (Sansure) and a Hepatitis B Virus Surface Antigen Assay Kit (Mike), and the detection methods were performed according to the corresponding instructions. The Log10 values ​​of serum HBV DNA and HBsAg expression after drug administration were compared with the corresponding Log10 expression values ​​before drug administration. The reduction effect compared to the pre-drug administration was used to evaluate the efficacy of the conjugate.

[0604] In some embodiments, an E modification was introduced at the 3' end of AS, and this modification was found to significantly enhance the knockdown effect of HBV DNA. In this embodiment, E modifications were introduced at positions in the SS chain (the complementary SS pairing positions at AS13 and 9, corresponding to positions 11 and 7 of the 19-21 siRNA SS chain), such as BPR-99140425 at position 7 of the SS chain, and BPR-99140412, BPR-99140417, BPR-99140411, BPR-99140413, BPR-99140418, BPR-99140414, BPR-99140423, BPR-99140422, BPR-99140415, BPR-99140424, BPR-99140432, and BPR-99140421 at position 11 of the SS chain. Compared to the unmodified compound BPR-99140454, this significantly increased HBV levels. The DNA knockdown effect was significant, and the effect was noticeable at least 21 days after administration (see Table 5).

[0605] This embodiment optimized the parent compound BPR-99140454. Besides the additional HBV DNA knockdown effect brought about by E modification in the SS chain, BPR-99140417, BPR-99140412, BPR-99140411, and BPR-99140413 showed a significant improvement in HBsAg knockdown. In particular, BPR-99140417 increased the knockdown of parental HBsAg from 2.47 Log10 to 3.22 after 21 days of administration.

[0606] Table 5: Inhibitory effects of compounds on HBV virus components in transgenic mice

[0607] Example 4: AAV-Mouse Animal Experiment

[0608] This embodiment uses an AAV-HBV mouse model to evaluate the in vivo efficacy of the HBV siRNA reagent. Five-week-old male C57BL / 6 mice (from Shanghai Silex Laboratory Animal Co., Ltd.) were infected with 1x1^11 v.g. rAAV8-1.3HBV (type D) virus via tail vein injection to establish the model. Each group consisted of five animals, with a positive control and a negative control group. The negative control was an equal volume of physiological saline, and the positive control was AD-66810 from CN11913898A. The day of the first administration was designated as day 1, the day before administration as day -1, the day after administration as day 1, and so on. On day -2 (27 days after virus injection), plasma was collected from all mice injected with rAAV8-1.3HBV virus via the submandibular vein. Whole blood was collected in EDTA-K2 coated tubes, centrifuged at 7g for 1 min at 4°C, and the supernatant was collected. Plasma was used to detect HBV DNA and HBsAg levels. Grouping and baseline determination were performed based on plasma HBV DNA and HBsAg levels and body weight. Mice with abnormal parameters were removed to ensure no statistically significant differences in HBV DNA, HBsAg levels, and body weight among groups. The mean plasma HBsAg expression level of enrolled mice was not lower than 4.5 log IU / mL. The dosage was 3 mg / kg, administered subcutaneously as a single dose. Physiological saline was used as the solvent and negative control. Blood samples were collected on days 7, 14, 21, 28, 35, 42, and 56 after administration. Detection methods followed the instructions and reagents provided with the Hepatitis B Virus Nucleic Acid Assay Kit (Sansure) and the Hepatitis B Virus Surface Antigen Assay Kit (Mike). The effects of siRNA duplex expression are summarized in Figures 1 and 2. The Log10 values ​​of serum HBV DNA and HBsAg expression after drug administration were compared with the corresponding Log10 expression values ​​before drug administration. The reduction effect compared to the pre-drug administration was used to evaluate the efficacy of the conjugate.

[0609] In some embodiments, the E modification was evaluated to show significantly stronger knockdown of HBV DNA in HBV transgenic mice in both the SS and AS chains compared to compounds without this modification. In this embodiment, the effect of E modification in an AAV-HBV mouse model was evaluated. Compared to the unmodified parental compound BPR-9140482, the introduction of E modification in BPR-99140483 (at position 11 of the SS chain and the para position 9 of the AS chain) significantly enhanced the HBV DNA reduction effect, maintaining a 2 Log10 level of inhibition even after 56 days of administration. Simultaneously, the knockdown effect on HBsAg was slightly enhanced compared to the parental compound. This embodiment suggests that E modification in siRNA not only has a strong inhibitory effect on HBV DNA but may also slightly increase the inhibitory effect on HBsAg.

[0610] Example 5: AAV-Mouse Animal Experiment

[0611] This embodiment uses an AAV-HBV mouse model to evaluate the in vivo efficacy of the HBV siRNA reagent. Five-week-old male C57BL / 6 mice (from Shanghai Silex Laboratory Animal Co., Ltd.) were infected with 1x1^11 v.g. rAAV8-1.3HBV (type D) virus via tail vein injection to establish the model. Each group consisted of five animals, with a positive control and a negative control. The negative control was an equal volume of physiological saline, and the positive control was AD-66810 from CN11913898A. The day of the first administration was designated as day 1, the day before administration as day -1, the day after administration as day 1, and so on. On day -2 (27 days after virus injection), plasma was collected from all mice injected with rAAV8-1.3HBV virus via the submandibular vein. Whole blood was collected in EDTA-K2 coated tubes, centrifuged at 7g for 1 min at 4°C, and the supernatant was collected. Plasma was used to detect the levels of HBV DNA, HBsAg, and HBeAg. Grouping and baseline determination were performed based on plasma HBV DNA, HBsAg, and HBeAg levels and body weight. Mice with abnormal parameters were removed to ensure no statistically significant differences in HBV DNA, HBsAg, HBeAg levels, and body weight among groups. The mean plasma HBsAg expression level of enrolled mice was not lower than 4.5 log IU / mL. The dosage was 3 mg / kg, administered subcutaneously as a single dose. Physiological saline was used as the solvent and negative control. Blood samples were collected on days 7, 14, 21, 28, 35, 42, and 56 after administration. Detection methods were performed according to the instructions and reagents provided with the Hepatitis B Virus Nucleic Acid Assay Kit (Sansure) and the Hepatitis B Virus Surface Antigen Assay Kit (Mike). The Log10 values ​​of serum HBV DNA, HBsAg, and HBeAg expression after drug administration were compared with the corresponding Log10 expression values ​​before drug administration. The reduction effect compared to the pre-drug administration was used to evaluate the efficacy of the conjugate.

[0612] Figures 3-5 show that BPR-99120322, BPR-99140447, BPR-99140417, and BPR-99140412 have good inhibitory effects on the virus group.

[0613] Example 6: Transgenic Mouse Animal Experiment

[0614] This embodiment uses a transgenic HBV mouse model to evaluate HBV siRNA reagents, comparing the HBsAg inhibitory effects of the siRNA compounds BPR-99140417 and the positive control (PC) HBV-21-11#mod(WO2022172083A2). The transgenic mouse strain was C57B / 6N-Tg(1.28HBV) / Vst, purchased from Beijing Vitonda Biotechnology Co., Ltd. Mice were grouped according to serum HBsAg levels and body weight, ensuring no statistically significant differences in serum HBsAg levels and body weight. Mice with low HBsAg levels and low body weight were removed from the experiment. Each group consisted of 4 males, and the drug was administered at a dose of 3 mg / kg via a single subcutaneous dose. The group using physiological saline as the drug solvent served as the negative control (NC). Blood samples were collected from the orbital venous plexus of mice before administration (day 0) and on days 7, 14, and 21 after administration. The collected blood samples were incubated at room temperature for 3 minutes, centrifuged at 5 rpm for 1 minute, and the supernatant was used for HBsAg detection. The reagent used for detection was a Hepatitis B virus surface antigen assay kit (Mike), and the detection method was performed according to the corresponding instructions. The relative expression values ​​of Log10 HBsAg in mouse serum after administration are shown in Figure 6. Analysis of the HBsAg knockdown effect after administration revealed that BPR-99140417 was significantly superior to PC.

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