Nucleic acid, composition containing nucleic acid, conjugate, preparation method and application

Abstract

The invention provides siRNA (small interfering Ribonucleic Acid) for inhibiting RNA (Ribonucleic Acid) of hepatitis A virus (HAV), a pharmaceutical composition containing the siRNA and a conjugate. The siRNA contains a positive-sense strand and an antisense strand, each nucleotide in the siRNA is independently modified or unmodified nucleotide, the positive-sense strand contains a nucleotide sequence I, the length of the nucleotide sequence I is equal to that of a nucleotide sequence shown in SEQ ID NO: 1, the difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 is not more than 3 nucleotides, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence II is a nucleotide sequence shown in SEQ ID NO: 2. The length of the nucleotide sequence II is equal to that of the nucleotide sequence shown in SEQ ID NO: 2, and the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 is not more than 3 nucleotides The siRNA, the pharmaceutical composition and the conjugate provided by the invention can be used for effectively treating hepatitis A related diseases.

Description

Technical Field

[0001] The present disclosure relates to a nucleic acid capable of inhibiting hepatitis A virus (HAV) RNA, a pharmaceutical composition and an siRNA conjugate containing the nucleic acid. The present disclosure also relates to methods for preparing and uses of these nucleic acids, pharmaceutical compositions and siRNA conjugates. Background Art

[0002] Hepatitis A, abbreviated as hepatitis A, is an acute infectious disease caused by hepatitis A virus (HAV). Hepatitis A is one of the six viral hepatitis that have been found to seriously affect human health and has the highest infection rate. Hepatitis A virus belongs to the family Picornaviridae and the genus Hepatovirus. After human beings are infected with hepatitis A virus, it first proliferates in the digestive tract. During the short viremia, the virus can continue to proliferate in blood leukocytes and then enter the liver and replicate in hepatocytes. HAV can be excreted from hepatocytes into the intestine through bile and is mainly transmitted through the fecal-oral route, with a relatively fast transmission speed and strong infectivity.

[0003] After HAV infection, the symptoms recover slowly, which may cause liver damage and, in severe cases, lead to death, especially for the elderly or those with other health problems. Vaccination is so far the most effective measure for humans to control hepatitis A, and there is currently no specific drug for treating HAV infection.

[0004] There is a significant need in the art for the development of new drugs to inhibit HAV viral RNA and thereby treat HAV-related diseases or symptoms. Summary of the Invention

[0005] The inventors of the present disclosure unexpectedly found that the following siRNAs and their modified sequences provided by the present disclosure can specifically inhibit HAV viral RNA in cells, and the pharmaceutical compositions and siRNA conjugates containing the siRNAs of the present disclosure can effectively deliver the siRNAs of the present disclosure to target tissues and / or cells, thereby showing high drug development potential in treating pathological conditions or diseases caused by hepatitis A virus.

[0006] In one aspect, the present disclosure provides an siRNA capable of inhibiting HAV viral RNA. The siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide. Wherein, the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II form a double-stranded region at least partially in reverse complementarity. Wherein, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:1 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO:2 and has no more than 3 nucleotide differences:

[0007] 5'-CCAGUUUGGGAAUUGACAZ a1 -3'(SEQ ID NO:1);

[0008] 5'-Z a2 UGUCAAUUCCCAAACUGG-3'(SEQ ID NO:2),

[0009] Wherein, Z a1 is A, Z a2 is U, and the nucleotide sequence I contains a nucleotide Z a1 corresponding to the position of Z a3 , and the nucleotide sequence II contains a nucleotide Z a2 corresponding to the position of Z a4 , and the Z a4 is the first nucleotide at the 5' end of the antisense strand;

[0010] Alternatively, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:33 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO:34 and has no more than 3 nucleotide differences:

[0011] 5'-GAUAUGUCUUGUGUUGAUZ b1 -3'(SEQ ID NO:33);

[0012] 5'-Z b2 AUCAACACAAGACAUAUC-3'(SEQ ID NO:34),

[0013] Where Z b1 is U, Z b2 is A, and the nucleotide sequence I contains a nucleotide Z b1 corresponding to the position of Z b3, the nucleotide sequence II contains a nucleotide Z whose position corresponds to Z b2 nucleotide Z b4 , said Z b4 is the first nucleotide at the 5'-end of the antisense strand;

[0014] Alternatively, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:65, with no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO:66, with no more than 3 nucleotide differences:

[0015] 5'-GGUGGAACUUACUAUUCAZ c1 -3'(SEQ ID NO:65);

[0016] 5'-Z c2 UGAAUAGUAAGUUCCACC-3'(SEQ ID NO:66),

[0017] where Z c1 is A, Z c2 is U, the nucleotide sequence I contains a nucleotide Z whose position corresponds to Z c1 nucleotide Z c3 , the nucleotide sequence II contains a nucleotide Z whose position corresponds to Z c2 nucleotide Z c4 , said Z c4 is the first nucleotide at the 5'-end of the antisense strand;

[0018] Alternatively, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:97, with no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO:98, with no more than 3 nucleotide differences:

[0019] 5'-GGUACAACAGUUGAUUUAZ d1 -3'(SEQ ID NO:97);

[0020] 5'-Z d2 UAAAUCAACUGUUGUACC-3'(SEQ ID NO:98),

[0021] where Z d1 is A, Z d2 is U, the nucleotide sequence I contains a nucleotide Z whose position corresponds to Z d1 nucleotide Z d3 , the nucleotide sequence II contains a nucleotide Z whose position corresponds to Z d2 nucleotide Z d4, said Z d4 is the first nucleotide at the 5'-end of the antisense strand.

[0022] In yet another aspect, the present disclosure provides an siRNA conjugate, which comprises the siRNA provided by the present disclosure and a conjugating group conjugated to the siRNA.

[0023] In another aspect, the present disclosure provides a pharmaceutical composition, which comprises the siRNA of the present disclosure or the siRNA conjugate, and a pharmaceutically acceptable carrier.

[0024] In yet another aspect, the present disclosure provides the use of the siRNA and / or siRNA conjugate and / or pharmaceutical composition of the present disclosure in the preparation of a medicament for treating a pathological condition or disease caused by hepatitis A virus.

[0025] In yet another aspect, the present disclosure provides a method for treating a disease or symptom associated with HAV infection, the method comprising administering to a subject in need thereof an effective amount of the siRNA or siRNA conjugate and / or pharmaceutical composition.

[0026] In yet another aspect, the present disclosure provides a method for inhibiting HAV viral RNA in cells, the method comprising contacting the cells with an effective amount of the siRNA and / or siRNA conjugate and / or pharmaceutical composition of the present disclosure.

[0027] In addition, the present disclosure also provides a kit, which comprises the siRNA and / or siRNA conjugate and / or pharmaceutical composition of the present disclosure.

[0028] Incorporated by reference

[0029] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application was specifically and individually incorporated herein by reference.

[0030] Beneficial effects

[0031] The siRNA, siRNA conjugate and pharmaceutical composition provided by the present disclosure have good stability and high HAV viral RNA inhibitory activity, and can significantly treat diseases or symptoms caused by HAV virus. The specific description is as follows.

[0032] In one aspect, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure shows excellent inhibitory activity against HAV viral RNA in in vitro cell experiments. For example, in in vitro Huh7.5.1 human hepatoma cells, at a low concentration of 5 nM, the siRNA conjugate of the present disclosure shows an inhibition rate of greater than 75%, and even greater than 90%, against the RNA copy number of HM175 / 18f hepatitis A virus. For another example, in in vitro primary monkey hepatocytes, the siRNA conjugate of the present disclosure shows an inhibition rate of at least 57% against the HAV viral RNA copy number of HM175 / 18f hepatitis A virus, and the highest inhibition rate can reach 82%. For another example, in in vitro Huh7.5.1 human hepatoma cells, the siRNA conjugate of the present disclosure shows an inhibition rate of at least 84% against the viral RNA copy number of the live attenuated hepatitis A vaccine 18F strain and an inhibition rate of greater than 60% against the viral RNA copy number of the live attenuated hepatitis A vaccine H2 strain. For another example, in in vitro Huh7.5.1 human hepatoma cells, the siRNA conjugate of the present disclosure shows good inhibitory effects on HAV virus proliferation from day 1 to day 4, and the relative inhibition rate of virus proliferation on day 4 reaches more than 86%, showing good and stable inhibitory effects on HAV virus. For another example, in the in vivo activity test in mice, on day 14, compared with the average copy number of HAV virus in the blank control group of 1×10 5.47 GE / ml, the intravenous injection group and the subcutaneous injection group are reduced to 1×10 3.94 GE / ml and 1×10 4.26 GE / ml respectively; in the intravenous injection group, the average inhibition rate of conjugate 3 against HAV virus is 95.5%, and in the subcutaneous injection group, the average inhibition rate of conjugate 3 against HAV virus is 93.5%, indicating that the siRNA conjugate of the present disclosure has a significant inhibitory effect on HAV virus. For the HAV virus in mouse liver cells, on day 21, compared with the blank control group, the inhibition rate of HAV virus in the intravenous injection group is 61.5%, and the inhibition rate of HAV virus in the subcutaneous injection group is 55.1%, which indicates that conjugate 3 of the present disclosure has a significant and long-lasting inhibitory effect on HAV virus

[0033] In another aspect, the siRNA conjugate provided by the present disclosure has high stability in vivo.

[0034] In another aspect, the siRNA conjugate provided by the present disclosure has excellent inhibitory activity against HAV viral RNA in vivo. In some embodiments, the siRNA conjugate provided by the present disclosure shows an inhibition rate of HAV viral RNA in hepatocytes in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of human subjects.

[0035] This shows that the siRNA, siRNA conjugate, and pharmaceutical composition provided by the present disclosure can inhibit HAV viral RNA and effectively treat diseases or symptoms related to HAV function regulation, and have good application prospects.

[0036] Other features and advantages of the present disclosure will be described in detail in the following specific implementation section. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings are used to provide a further understanding of the present disclosure, and constitute a part of the specification. Together with the following specific implementation, they are used to explain the present disclosure, but do not constitute a limitation to the present disclosure. In the accompanying drawings:

[0038] Figure 1A-1D is a dot plot showing the relative copy number of HAV viral RNA in Huh7.5.1 human hepatoma cells after transfection with different concentrations of siRNA conjugate. The IC 50 value calculated using the fitted curve is also shown in the figure.

[0039] Figure 2 is a bar chart showing the relative copy number of HAV virus in primary monkey cells in vitro after transfection with 50 nM concentration of the siRNA conjugate or the control conjugate of the present disclosure.

[0040] Figure 3 is a bar chart showing the relative copy number of different strains of HAV virus in Huh7.5.1 human hepatoma cells after transfection with 50 nM concentration of the siRNA conjugate or the control conjugate of the present disclosure.

[0041] Figure 4 is a line chart showing the proliferation degree of HAV virus in Huh7.5.1 human hepatoma cells from day 0 to day 4 after transfection with the siRNA conjugate or the control conjugate of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0042] The following provides a detailed description of the specific implementation of the present disclosure. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0043] Definition

[0044] In the present disclosure, the HAV viral RNA refers to the sequence shown in Genbank accession number NC_001489.1.

[0045] In the foregoing and the following, unless otherwise specified, the capital letters C, G, U, and A represent the base composition of nucleotides; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a nucleotide modified with a methoxy group; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a nucleotide modified with a fluoro group; the lowercase letter s indicates that a phosphorothioate subunit linkage exists between the two nucleotides adjacent to the left and right of the letter s; P1 indicates that the nucleotide adjacent to the right of the P1 is a nucleotide modified with a 5'-phosphate nucleotide or a 5'-phosphate analog. In some embodiments, P1 is VP, Ps, or P representing specific modifications. Among them, the letter combination VP indicates that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinyl phosphate (5'-(E)-vinylphosphonate, E-VP); the letter combination Ps indicates that the nucleotide adjacent to the right of the letter combination Ps is a nucleotide modified with a phosphorothioate group; the capital letter P indicates that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0046] In the foregoing and the following, the "fluoro-modified nucleotide" refers to a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose group of the nucleotide with a fluorine atom, and the "non-fluoro-modified nucleotide" refers to a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose group of the nucleotide with a non-fluorine group or a nucleotide analog. A "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenosine ribonucleotide, guanosine ribonucleotide, cytosine ribonucleotide, uridine ribonucleotide, or thymidine deoxyribonucleotide. Such as a nucleotide analog, a bridged nucleic acid (abbreviated as BNA), or an acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide formed by substituting the 2'-hydroxyl group of the ribose group with a methoxy group.

[0047] In the context of this article, the expressions "complementary" or "reverse complementary" can be used interchangeably and have the meanings well-known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When the adenine on one strand always pairs with the thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be deduced from the sequence of its complementary strand. Correspondingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions do not pair in a complementary form.

[0048] As used above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between two nucleotide sequences involved; "essentially reverse complementary" means that there is no more than 1 base mismatch between two nucleotide sequences; "fully reverse complementary" means that there is no base mismatch between two nucleotide sequences.

[0049] As used above and below, when a nucleotide sequence has a "nucleotide difference" from another nucleotide sequence, it means that, compared with the latter, the base type of the nucleotide at the same position has changed. For example, when a nucleobase in the latter is A, if the corresponding nucleobase at the same position in the former is U, C, G, or T, it is determined that there is a nucleotide difference between the two nucleotide sequences at this position. In some embodiments, when a nucleotide at the original position is replaced by a abasic nucleotide or its equivalent, a nucleotide difference can also be considered to have occurred at this position. An abasic nucleotide refers to a monomeric compound formed after the nucleic acid base in a nucleotide is replaced by other groups or hydrogen atoms, and the other groups include, but are not limited to, substituted or unsubstituted aryl or heteroaryl groups.

[0050] As used above and below, especially when describing the preparation methods of the siRNAs, pharmaceutical compositions, or siRNA conjugates of the present disclosure, unless otherwise specified, the "nucleoside monomer" refers to the modified or unmodified RNA phosphoramidite monomers (sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites) used in solid-phase phosphoramidite synthesis according to the types and sequences of nucleotides in the siRNA or siRNA conjugate to be prepared. Solid-phase phosphoramidite synthesis is a method used in RNA synthesis well-known to those skilled in the art. The nucleoside monomers used in the present disclosure are all commercially available.

[0051] In the context of the present disclosure, unless otherwise specified, "conjugation" means that two or more chemical moieties each having a specific function are connected to each other in a covalent linkage manner; correspondingly, a "conjugate" means a compound formed by covalent connection between these individual chemical moieties. Further, an "siRNA conjugate" means a compound formed by covalently connecting one or more chemical moieties each having a specific function to an siRNA. The siRNA conjugate should be understood, according to the context, as the general term for multiple siRNA conjugates or an siRNA conjugate represented by a certain chemical formula. In the context of the present disclosure, a "conjugating molecule" should be understood as a specific compound that can be conjugated to an siRNA through a reaction to ultimately form the siRNA conjugate of the present disclosure.

[0052] Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitutions or substitution patterns that are spatially impracticable, synthetically infeasible and / or inherently unstable.

[0053] As used herein, "alkyl" refers to straight-chain and branched-chain groups having a specified number of carbon atoms, typically from 1 to 20 carbon atoms, such as from 1 to 10 carbon atoms, such as from 1 to 8 or from 1 to 6 carbon atoms. For example, C1-C6 alkyl includes straight-chain and branched-chain alkyl groups having 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbons, all branched-chain and straight-chain forms having that number of carbons are intended to be encompassed; thus, for example, "butyl" means including n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. An alkylene is a subset of alkyl and refers to a divalent moiety identical to alkyl but having two attachment points.

[0054] As used herein, "alkenyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond, which is obtained by removing one molecule of hydrogen from adjacent carbon atoms of the parent alkyl group. The group can be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyl, for example but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, and the like. In certain embodiments, the alkenyl group has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. An alkenylene is a subset of alkenyl and refers to a residue identical to alkenyl but having two attachment points.

[0055] As used herein, "alkoxy" refers to an alkyl group having a specified number of carbon atoms connected by an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, and the like. An alkoxy group typically has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by an oxygen bridge.

[0056] A variety of hydroxyl protecting groups can be used in the present disclosure. Generally, a protecting group renders a chemical functional group insensitive to specific reaction conditions and can be added to and removed from the functional group in the molecule without substantially damaging the remainder of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2nd ed, John Wiley & Sons, New York, 1991, which are incorporated herein by reference in their entireties. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenyloxanth-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanth-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4''-trimethoxytrityl).

[0057] As used herein, the term "subject" refers to any animal, such as a mammal or a marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, rabbits, sheep, rats, and any type of poultry.

[0058] As used herein, "treatment" refers to a method of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradicating or ameliorating the underlying disorder being treated. In addition, a therapeutic benefit is obtained by observing an improvement in the subject through eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, although the subject may still be afflicted with the underlying disorder.

[0059] In the siRNA or siRNA conjugate described in the present disclosure, each adjacent nucleotide is linked by a phosphodiester bond or a phosphorothioate bond, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond bears a negative charge, which may exist in the form of a hydroxyl group or a mercapto group, and the hydrogen ion in the hydroxyl group or mercapto group may also be partially or completely replaced by a cation. The cation can be any cation, such as a metal cation, ammonium ion NH4 +, one of the organic ammonium cations, for reasons of improving solubility, in some embodiments, the cation is selected from one or more of alkali metal ions, ammonium cations formed by tertiary amines, and quaternary ammonium cations. The alkali metal cation can be K + and / or Na + , and the cation formed by the tertiary amine can be the ammonium ion formed by triethylamine and / or the ammonium ion formed by N,N-diisopropylethylamine. Therefore, the siRNA or siRNA conjugate described in the present disclosure can exist at least partially in the form of a salt. In some embodiments, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond is at least partially bound to a sodium ion, and the siRNA or siRNA conjugate described in the present disclosure exists in the form of a sodium salt or a partial sodium salt. Therefore, when referring to the siRNA or siRNA conjugate described in the present disclosure, including but not limited to the siRNA conjugate represented by any structural formula described in the present disclosure, it is intended to cover the sodium salt or partial sodium salt form of the siRNA or siRNA conjugate.

[0060] siRNA of the present disclosure

[0061] In one aspect, the present disclosure provides an siRNA capable of inhibiting HAV viral RNA.

[0062] The siRNA of the present disclosure contains nucleotide groups as basic structural units. As is well known to those skilled in the art, the nucleotide groups contain phosphate groups, ribose groups, and bases, which will not be elaborated here.

[0063] The siRNA of the present disclosure contains a sense strand and an antisense strand. The lengths of the sense strand and the antisense strand are the same or different. The length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21, 21 / 21, 21 / 23 or 23 / 25.

[0064] According to the present disclosure, the siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide. Wherein, the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region.

[0065] In some embodiments, the siRNA with stabilized modified nucleotides of the present disclosure can be the following first to fourth siRNAs, and each siRNA will be described below.

[0066] The first siRNA

[0067] In some embodiments, for the siRNA of the present disclosure, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:1 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO:2 and has no more than 3 nucleotide differences:

[0068] 5'-CCAGUUUGGGAAUUGACAZ a1 -3'(SEQ ID NO:1);

[0069] 5'-Z a2 UGUCAAUUCCCAAACUGG-3'(SEQ ID NO:2),

[0070] wherein, Z a1 is A, Z a2 is U, the nucleotide sequence I contains a nucleotide Z a1 corresponding to the position of Z a3 , the nucleotide sequence II contains a nucleotide Z a2 corresponding to the position of Z a4 , and the Z a4 is the first nucleotide at the 5' end of the antisense strand.

[0071] In the above and below of the present disclosure, "position corresponding" means starting from the same end of the nucleotide sequence, being in the same position in the nucleotide sequence. For example, the first nucleotide at the 3' end of the nucleotide sequence I is the nucleotide corresponding to the 1st nucleotide at the 3' end of SEQ ID NO:1.

[0072] In some embodiments, the sense strand contains only nucleotide sequence I, and the antisense strand contains only nucleotide sequence II. In some embodiments, there are no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1, and / or there are no more than 1 nucleotide sequence difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2.

[0073] In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 includes the difference at position Z a4 , and Z a4 is selected from A, C, or G. In some embodiments, the Z a3 is a nucleotide complementary to Z a4 .

[0074] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary; substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; essentially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; completely reverse complementary means that there are no base mismatches between the two nucleotide sequences.

[0075] In some embodiments, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:4:

[0076] 5'-CCAGUUUGGGAAUUGACAZ a3 -3'(SEQ ID NO:3);

[0077] 5'-Z a4 UGUCAAUUCCCAAACUGG-3'(SEQ ID NO:4),

[0078] wherein the Z a4 is the first nucleotide at the 5' end of the antisense strand, Z a3 is selected from A, U, G, or C, and Z a4 is a nucleotide complementary to Z a3 ;

[0079] In some embodiments, Z a3 is A, and Z a4 is U.

[0080] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. The lengths of nucleotide sequence III and nucleotide sequence IV are each 1 to 4 nucleotides; nucleotide sequence III and nucleotide sequence IV are of equal length and are substantially reverse complementary or completely reverse complementary; nucleotide sequence III is linked to the 5'-end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3'-end of nucleotide sequence II.

[0081] In some embodiments, nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, where the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5'-end of the nucleotide sequence represented by SEQ ID NO:1 in HAV viral RNA and having the same length as nucleotide sequence IV.

[0082] In some embodiments, in the 5'-3' direction, the lengths of both nucleotide sequence III and nucleotide sequence IV are 1 nucleotide, the base of nucleotide sequence III is C, and the base of nucleotide sequence IV is G; at this time, the length ratio of the sense strand to the antisense strand is 20 / 20; or, the lengths of both nucleotide sequence III and IV are 2 nucleotides. In the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is AC, and the base composition of nucleotide sequence IV is GU; at this time, the length ratio of the sense strand to the antisense strand is 21 / 21; or, the lengths of both nucleotide sequence III and IV are 3 nucleotides. In the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is UAC, and the base composition of nucleotide sequence IV is GUA; at this time, the length ratio of the sense strand to the antisense strand is 22 / 22; or, the lengths of both nucleotide sequence III and IV are 4 nucleotides. In the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is AUAC, and the base composition of nucleotide sequence IV is GUAU; at this time, the length ratio of the sense strand to the antisense strand is 23 / 23.

[0083] In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Therefore, given the base of nucleotide sequence III, the base of nucleotide sequence IV is determined.

[0084] The second siRNA

[0085] In some embodiments, for the siRNA of the present disclosure, the length of nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO:33, with no more than 3 nucleotide differences, and the length of nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO:34, with no more than 3 nucleotide differences:

[0086] 5'-GAUAUGUCUUGUGUUGAUZ b1 -3'(SEQ ID NO:33);

[0087] 5'-Z b2 AUCAACACAAGACAUAUC-3'(SEQ ID NO:34),

[0088] wherein Z b1 is U, Z b2 is A, and the nucleotide sequence I contains a nucleotide Z at a position corresponding to Z b1 , and the nucleotide sequence II contains a nucleotide Z at a position corresponding to Z b3 , and the Z b2 is the first nucleotide at the 5'-end of the antisense strand. b4 The Z b4 is the first nucleotide at the 5'-end of the antisense strand.

[0089] In some embodiments, there are no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:33, and / or no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:34.

[0090] In some embodiments, the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:34 includes the difference at the position of Z b4 , and Z b4 is selected from U, G or C.

[0091] In some embodiments, the Z b3 is a nucleotide complementary to Z b4 . In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; the essentially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; and the completely reverse complementary means that there is no base mismatch between the two nucleotide sequences.

[0092] In some embodiments, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:35, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:36:

[0093] 5'-GAUAUGUCUUGUGUUGAUZ b3 -3'(SEQ ID NO:35);

[0094] 5'-Z b4 AUCAACACAAGACAUAUC-3'(SEQ ID NO:36),

[0095] wherein said Z b4 is the first nucleotide at the 5'-end of the antisense strand, and Z b3 is selected from A, U, G or C, and Z b4 is a nucleotide complementary to Z b3 .

[0096] In some embodiments, wherein Z b3 is U and Z b4 is A.

[0097] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. The lengths of nucleotide sequence III and nucleotide sequence IV are each 1 to 4 nucleotides; the lengths of nucleotide sequence III and nucleotide sequence IV are equal and substantially reverse complementary or completely reverse complementary; nucleotide sequence III is linked to the 5'-end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3'-end of nucleotide sequence II.

[0098] In some embodiments, nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, and this second nucleotide sequence refers to the nucleotide sequence adjacent to the 5'-end of the nucleotide sequence represented by SEQ ID NO:33 in HAV viral RNA and having the same length as nucleotide sequence IV.

[0099] In some embodiments, in the 5'-3' direction, the lengths of nucleotide sequence III and nucleotide sequence IV are both 1 nucleotide, the base of nucleotide sequence III is A, and the base of nucleotide sequence IV is U; at this time, the length ratio of the sense strand to the antisense strand is 20 / 20; or, the lengths of nucleotide sequence III and IV are both 2 nucleotides. In the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is AA, and the base composition of nucleotide sequence IV is UU; at this time, the length ratio of the sense strand to the antisense strand is 21 / 21; or, the lengths of nucleotide sequence III and IV are both 3 nucleotides. In the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is AAA, and the base composition of nucleotide sequence IV is UUU; at this time, the length ratio of the sense strand to the antisense strand is 22 / 22; or, the lengths of nucleotide sequence III and IV are both 4 nucleotides. In the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is CAAA, and the base composition of nucleotide sequence IV is UUUG; at this time, the length ratio of the sense strand to the antisense strand is 23 / 23.

[0100] In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Therefore, given the bases of nucleotide sequence III, the bases of nucleotide sequence IV are determined.

[0101] The third siRNA

[0102] In some embodiments, for the siRNA of the present disclosure, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO: 65 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO: 66 and has no more than 3 nucleotide differences:

[0103] 5'-GGUGGAACUUACUAUUCAZ c1 -3'(SEQ ID NO: 65);

[0104] 5'-Z c2 UGAAUAGUAAGUUCCACC-3'(SEQ ID NO: 66),

[0105] wherein Z c1 is A, Z c2 is U, the nucleotide sequence I contains the nucleotide Z c1 corresponding to the position of Z c3 and the nucleotide sequence II contains the nucleotide Z c2 corresponding to the position of Z c4 , and the Z c4 is the first nucleotide at the 5'-end of the antisense strand.

[0106] In some embodiments, there are no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 65, and / or no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 66;

[0107] In some embodiments, the differences between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 66 include the differences at the position of Z c4 and Z c4 is selected from A, G or C.

[0108] In some embodiments, the Z c3 is a nucleotide complementary to Z c4 .

[0109] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary; substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; essentially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; completely reverse complementary means that there are no base mismatches between the two nucleotide sequences.

[0110] In some embodiments, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:67, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:68:

[0111] 5'-GGUGGAACUUACUAUUCA Z c3 -3'(SEQ ID NO:67);

[0112] 5'-Z c4 UGAAUAGUAAGUUCCACC-3'(SEQ ID NO:68),

[0113] wherein the Z c4 is the first nucleotide at the 5'-end of the antisense strand, and Z c3 is selected from A, U, G, or C, and Z c4 is a nucleotide complementary to Z c3 .

[0114] In some embodiments, wherein Z c3 is A, and Z c4 is U.

[0115] In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV. The lengths of the nucleotide sequence III and the nucleotide sequence IV are each 1 to 4 nucleotides; the nucleotide sequence III and the nucleotide sequence IV are of equal length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is linked to the 5'-end of the nucleotide sequence I, and the nucleotide sequence IV is linked to the 3'-end of the nucleotide sequence II.

[0116] In some embodiments, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, and this second nucleotide sequence refers to the nucleotide sequence adjacent to the 5'-end of the nucleotide sequence represented by SEQ ID NO:65 in the HAV viral RNA and having the same length as the nucleotide sequence IV.

[0117] In some embodiments, in the 5'-3' direction, the lengths of both nucleotide sequence III and nucleotide sequence IV are 1 nucleotide, the base of nucleotide sequence III is A, and the base of nucleotide sequence IV is U; at this time, the length ratio of the sense strand to the antisense strand is 20 / 20; alternatively, the lengths of both nucleotide sequences III and IV are 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the nucleotide composition of nucleotide sequence III is GA, and the nucleotide composition of nucleotide sequence IV is UC; at this time, the length ratio of the sense strand to the antisense strand is 21 / 21; alternatively, the lengths of both nucleotide sequences III and IV are 3 nucleotides, and in the direction from the 5'-end to the 3'-end, the nucleotide composition of nucleotide sequence III is AGA, and the nucleotide composition of nucleotide sequence IV is UCU; at this time, the length ratio of the sense strand to the antisense strand is 22 / 22; alternatively, the lengths of both nucleotide sequences III and IV are 4 nucleotides, and in the direction from the 5'-end to the 3'-end, the nucleotide composition of nucleotide sequence III is UAGA, and the nucleotide composition of nucleotide sequence IV is UCUA; at this time, the length ratio of the sense strand to the antisense strand is 23 / 23.

[0118] In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Therefore, given the base of nucleotide sequence III, the base of nucleotide sequence IV is determined.

[0119] The fourth siRNA

[0120] In some embodiments, for the siRNA of the present disclosure, the length of nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO: 97, and there are no more than 3 nucleotide differences, and the length of nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO: 98, and there are no more than 3 nucleotide differences:

[0121] 5'-GGUACAACAGUUGAUUUAZ d1 -3'(SEQ ID NO:97);

[0122] 5'-Z d2 UAAAUCAACUGUUGUACC-3'(SEQ ID NO:98),

[0123] where Z d1 is A, Z d2 is U, nucleotide sequence I contains the nucleotide Z d1 corresponding to the position of Z d3 , nucleotide sequence II contains the nucleotide Z d2 corresponding to the position of Z d4 , and the Z d4is the first nucleotide at the 5'-end of the antisense strand.

[0124] In some embodiments, there are no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:97, and / or there are no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:98.

[0125] In some embodiments, the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:98 includes the difference at position Z d4 , and Z d4 is selected from A, G or C.

[0126] In some embodiments, the Z d3 is a nucleotide complementary to Z d4 . In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; the essentially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; the completely reverse complementary means that there is no base mismatch between the two nucleotide sequences.

[0127] In some embodiments, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:99, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:100:

[0128] 5'-GGUACAACAGUUGAUUUAZ d3 -3'(SEQ ID NO:99);

[0129] 5'-Z d4 UAAAUCAACUGUUGUACC-3'(SEQ ID NO:100),

[0130] wherein, the Z d4 is the first nucleotide at the 5'-end of the antisense strand, Z d3 is selected from A, U, G or C, and Z d4 is a nucleotide complementary to Z d3 .

[0131] In some embodiments, Z d3 is A, and Z d4 is U.

[0132] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV, and each of nucleotide sequence III and nucleotide sequence IV has a length of 1 to 4 nucleotides; nucleotide sequence III and nucleotide sequence IV have equal lengths and are substantially reverse complementary or completely reverse complementary; nucleotide sequence III is linked to the 5'-end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3'-end of nucleotide sequence II.

[0133] In some embodiments, nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5'-end of the nucleotide sequence represented by SEQ ID NO:97 in HAV viral RNA and having the same length as nucleotide sequence IV. In some embodiments, in the 5'-3' direction, both nucleotide sequence III and nucleotide sequence IV have a length of 1 nucleotide, the base of nucleotide sequence III is U, and the base of nucleotide sequence IV is A; at this time, the length ratio of the sense strand to the antisense strand is 20 / 20; alternatively, both nucleotide sequence III and IV have a length of 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is AU, and the base composition of nucleotide sequence IV is AU; at this time, the length ratio of the sense strand to the antisense strand is 21 / 21; alternatively, both nucleotide sequence III and IV have a length of 3 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is GAU, and the base composition of nucleotide sequence IV is AUC; at this time, the length ratio of the sense strand to the antisense strand is 22 / 22; alternatively, both nucleotide sequence III and IV have a length of 4 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is UGAU;, the base composition of nucleotide sequence IV is AUCA; at this time, the length ratio of the sense strand to the antisense strand is 23 / 23.

[0134] In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary, and thus, given the base of nucleotide sequence III, the base of nucleotide sequence IV is determined. In some embodiments, each U or T in the nucleotide sequence of the siRNA of the present disclosure can be arbitrarily replaced with each other. These nucleotide differences do not significantly reduce the HAV viral RNA inhibitory ability of the siRNA or increase the off-target effect of the siRNA. And these siRNAs containing nucleotide differences are also within the protection scope of the present disclosure.

[0135] Hereinafter, the descriptions of the nucleotide sequence V, nucleotide modifications in the siRNA, and the modified sequences are applicable to the siRNAs of the present disclosure above, such as the first siRNA, the second siRNA, the third siRNA, and the fourth siRNA. That is, if not specified otherwise, the following descriptions of the siRNA should be regarded as descriptions of the four siRNAs of the present disclosure above. For example, if not specifically indicating a particular siRNA, the meaning of "the siRNA further contains the nucleotide sequence V" is that "the siRNAs of the present disclosure, such as the first siRNA, the second siRNA, the third siRNA, or the fourth siRNA above, further contain the nucleotide sequence V". In some embodiments, the lengths of the sense strand and the antisense strand are different, and the antisense strand further contains the nucleotide sequence V, and the length of the nucleotide sequence V is 1 to 3 nucleotides, which is connected to the 3'-end of the antisense strand to form a 3'-overhang of the antisense strand.

[0136] In some embodiments, the sense strand further contains the nucleotide sequence VI, and the length of the nucleotide sequence VI is 1 to 3 nucleotides, which is connected to the 3'-end of the sense strand to form a 3'-overhang of the sense strand.

[0137] In some embodiments, the siRNAs provided by the present disclosure include the nucleotide sequence V but do not include the nucleotide sequence VI. Thus, the length ratio of the sense strand to the antisense strand of the siRNAs provided by the present disclosure can be 19 / 20, 19 / 21, 19 / 22, 20 / 21, 20 / 22, 20 / 23, 21 / 22, 21 / 23, 21 / 24, 22 / 23, 22 / 24, 22 / 25, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the siRNAs provided by the present disclosure include the nucleotide sequences V and VI. In some embodiments, the length of the nucleotide sequence V is the same as or different from the length of the nucleotide sequence VI. Thus, the length ratio of the sense strand to the antisense strand of the siRNAs provided by the present disclosure can be (19-26):(19-26). In some embodiments, the length of the nucleotide sequence V and / or VI is 2 nucleotides. Thus, the length ratio of the sense strand to the antisense strand of the siRNAs provided by the present disclosure can be 19 / 21, 21 / 21, 21 / 23, 23 / 23, 23 / 25, or 25 / 25.

[0138] Each nucleotide in the nucleotide sequence V can be any nucleotide. For the convenience of synthesis and cost savings, in some embodiments, the nucleotide sequence V is two consecutive thymidine deoxynucleotides (dTdT) or two consecutive uridine ribonucleotides (UU); or, in order to improve the affinity between the siRNA antisense strand and the HAV viral RNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the HAV viral RNA. In some embodiments, the corresponding position refers to the position of the third nucleotide sequence or the fourth nucleotide sequence. Therefore, in some embodiments, the ratio of the length of the sense strand to the antisense strand of the siRNA of the present disclosure is 19 / 21 or 21 / 23. At this time, the siRNA of the present disclosure has better mRNA silencing activity.

[0139] The third nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'-end of SEQ ID NO: 1 or SEQ ID NO: 33 or SEQ ID NO: 65 or SEQ ID NO: 97 or the second nucleotide sequence and has the same length as the nucleotide sequence V.

[0140] Each nucleotide in the nucleotide sequence VI can be any nucleotide. For the convenience of synthesis and cost savings in synthesis, in some embodiments, the nucleotide sequence VI is two consecutive thymidine deoxynucleotides (dTdT) or two consecutive uridine ribonucleotides (UU); or, in order to improve the affinity between the siRNA sense strand and the antisense strand, the nucleotide sequence VI is the same as the nucleotide at the corresponding position of the HAV viral RNA. In some embodiments, the corresponding position refers to the position of the fourth nucleotide sequence. Therefore, in some embodiments, the siRNA of the present disclosure contains the nucleotide sequences V and VI, and the ratio of the length of the sense strand to the antisense strand of the siRNA is 21 / 21 or 23 / 23. At this time, the siRNA of the present disclosure has better mRMA silencing activity.

[0141] The fourth nucleotide sequence refers to a nucleotide sequence that is adjacent to the 3'-end of SEQ ID NO: 1 or SEQ ID NO: 33 or SEQ ID NO: 65 or SEQ ID NO: 97 and has the same length as the nucleotide sequence VI.

[0142] In some embodiments, for the first type of siRNA, the sense strand of the siRNA contains the nucleotide shown in SEQ ID NO: 5, and the base composition of the third nucleotide sequence is AC; the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 6:

[0143] 5'-CCAGUUUGGGAAUUGACAZ a3-3'(SEQ ID NO:5);

[0144] 5'-Z a4 UGUCAAUUCCCAAACUGGGU-3'(SEQ ID NO:6);

[0145] Wherein, said Z a4 is the first nucleotide at the 5'-end of the antisense strand, and Z a3 is selected from A, U, G or C, and Z a4 is a nucleotide complementary to Z a3 .

[0146] Alternatively, the sense strand of said siRNA contains a nucleotide sequence as shown in SEQ ID NO:7, and the antisense strand of said siRNA contains a nucleotide sequence as shown in SEQ ID NO:8:

[0147] 5'-ACCCAGUUUGGGAAUUGACAZ a3 -3'(SEQ ID NO:7);

[0148] 5'-Z a4 UGUCAAUUCCCAAACUGGGUAU-3'(SEQ ID NO:8);

[0149] Wherein, said Z a4 is the first nucleotide at the 5'-end of the antisense strand, and Z a3 is selected from A, U, G or C, and Z a4 is a nucleotide complementary to Z a3 .

[0150] In some embodiments, for said second siRNA, the sense strand of said siRNA contains a nucleotide as shown in SEQ ID NO:37, and the base composition of the third nucleotide sequence is AA; the antisense strand contains a nucleotide sequence as shown in SEQ ID NO:38:

[0151] 5'-GAUAUGUCUUGUGUUGAUZ b3 -3'(SEQ ID NO:37);

[0152] 5'-Z b4 AUCAACACAAGACAUAUCUU-3'(SEQ ID NO:38);

[0153] Wherein, said Z b4 is the first nucleotide at the 5'-end of the antisense strand, and Z b3 is selected from A, U, G or C, and Z b4 is a nucleotide complementary to Zb3 Complementary nucleotides;

[0154] Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 39, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 40:

[0155] 5'-AAGAUAUGUCUUGUGUUGAUZ b3 -3' (SEQ ID NO: 39);

[0156] 5'-Z b4 AUCAACACAAGACAUAUCUUUG-3' (SEQ ID NO: 40);

[0157] wherein the Z b4 is the first nucleotide at the 5'-end of the antisense strand, and Z b3 is selected from A, U, G or C, and Z b4 is a nucleotide complementary to Z b3 ;

[0158] In some embodiments, for the third siRNA, the sense strand of the siRNA contains the nucleotides shown in SEQ ID NO: 69, and the base composition of the third nucleotide sequence is GA; the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 70:

[0159] 5'-GGUGGAACUUACUAUUCAZ c3 -3' (SEQ ID NO: 69);

[0160] 5'-Z c4 UGAAUAGUAAGUUCCACCUC-3' (SEQ ID NO: 70);

[0161] wherein the Z c4 is the first nucleotide at the 5'-end of the antisense strand, and Z c3 is selected from A, U, G or C, and Z c4 is a nucleotide complementary to Z c3 ;

[0162] Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 71, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 72:

[0163] 5'-GAGGUGGAACUUACUAUUCAZ c3 -3' (SEQ ID NO: 71);

[0164] 5'-Z c4 UGAAUAGUAAGUUCCACCUCUA-3'(SEQ ID NO:72);

[0165] wherein said Z c4 is the first nucleotide at the 5'-end of the antisense strand, Z c3 is selected from A, U, G or C, and Z c4 is a nucleotide complementary to Z c3 .

[0166] In some embodiments, for the fourth siRNA, the sense strand of the siRNA contains the nucleotides shown in SEQ ID NO:101, and the base composition of the third nucleotide sequence is UA; the antisense strand contains the nucleotide sequence shown in SEQ ID NO:102:

[0167] 5'-GGUACAACAGUUGAUUUAZ d3 -3'(SEQ ID NO:101);

[0168] 5'-Z d4 UAAAUCAACUGUUGUACCAU-3'(SEQ ID NO:102);

[0169] wherein said Z d4 is the first nucleotide at the 5'-end of the antisense strand, Z d3 is selected from A, U, G or C, and Z c4 is a nucleotide complementary to Z d3 ;

[0170] Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:103, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:104:

[0171] 5'-AUGGUACAACAGUUGAUUUAZ d3 -3'(SEQ ID NO:103);

[0172] 5'-Z d4 UAAAUCAACUGUUGUACCAUCA-3'(SEQ ID NO:104);

[0173] wherein said Z d4 is the first nucleotide at the 5'-end of the antisense strand, Z d3 is selected from A, U, G or C, and Z d4 is a nucleotide complementary to Zd3 Complementary nucleotides.

[0174] In the present disclosure, unless otherwise specified, all nucleotide sequences appear in the 5'-3' direction.

[0175] In some embodiments, the siRNAs of the present disclosure are siHAVa1, siHAVa2, siHAVa3, siHAVb1, siHAVb2, siHAVb3, siHAVc1, siHAVc2, siHAVc3, siHAVd1, siHAVd2, and siHAVd3 listed in Table 1.

[0176] As mentioned above, the nucleotides in the siRNAs of the present disclosure are each independently modified or unmodified nucleotides. In some embodiments, the nucleotides in the siRNAs of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNAs of the present disclosure are modified nucleotides, and these modifications on the nucleotide groups do not cause a significant weakening or loss of the function of the siRNAs of the present disclosure in inhibiting HAV viral RNA.

[0177] In some embodiments, the siRNAs of the present disclosure contain at least one modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" refers to a nucleotide or nucleotide analogue formed by replacing the 2'-hydroxyl group of the ribose moiety of a nucleotide with another group, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide does not cause a significant weakening or loss of the function of the siRNA in inhibiting HAV viral RNA. For example, the modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55 can be selected.

[0178] In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present disclosure is a modified nucleotide, and / or at least one phosphate group is a phosphate group with a modifying group; in other words, at least a part of the phosphate groups and / or ribose moieties in the phospho-sugar backbone of at least one single strand of the sense strand and the antisense strand are phosphate groups with modifying groups and / or ribose moieties with modifying groups.

[0179] In some embodiments, all nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluorine-modified nucleotide or a non-fluorine-modified nucleotide.

[0180] In some embodiments, the fluorine-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II. The number of fluorine-modified nucleotides in nucleotide sequence I is no more than 5, and, in the direction from the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of nucleotide sequence I are fluorine-modified nucleotides; the number of fluorine-modified nucleotides in nucleotide sequence II is no more than 7, and the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence II are fluorine-modified nucleotides.

[0181] In some embodiments, in the sense strand, in the direction from the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides or the 5th, 7th, 8th, and 9th nucleotides of nucleotide sequence I are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorine-modified nucleotides; in the antisense strand, in the direction from the 5'-end to the 3'-end, the 2nd, 6th, 14th, and 16th nucleotides or the 2nd, 6th, 8th, 9th, 14th, and 16th nucleotides of nucleotide sequence II are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorine-modified nucleotides.

[0182] In the context of the present disclosure, "fluorine-modified nucleotide" refers to a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose of the nucleotide with fluorine, and it has the structure shown in the following formula (7). "Non-fluorine-modified nucleotide" refers to a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose of the nucleotide with a non-fluorine group, or a nucleotide analog. In some embodiments, each non-fluorine-modified nucleotide is independently selected from a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose of the nucleotide with a non-fluorine group or a nucleotide analog.

[0183] These nucleotides formed by substituting the hydroxyl group at the 2'-position of the ribose with a non-fluorine group are well-known to those skilled in the art, and these nucleotides can be selected from one of 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxynucleotides.

[0184] In some embodiments, the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in formula (8). In some embodiments, the 2'-substituted alkoxy-modified nucleotide can be, for example, a 2'-O-methoxyethyl-modified nucleotide (2'-MOE), as shown in formula (9). In some embodiments, the 2'-amino-modified nucleotide (2'-NH2) is as shown in formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is as shown in formula (11):

[0185]

[0186] A nucleotide analogue refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from that of adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide, or thymidine deoxyribonucleotide. In some embodiments, the nucleotide analogue can be a non-natural nucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide.

[0187] BNA refers to a constrained or inaccessible nucleotide. BNA can contain a bridged structure with a "fixed" C3'-endo sugar puckering in a five-membered ring, six-membered ring, or seven-membered ring. Typically, this bridge is incorporated at the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, BNA can be LNA, ENA, cET BNA, etc., where LNA is as shown in formula (12), ENA is as shown in formula (13), and cET BNA is as shown in formula (14):

[0188]

[0189] An acyclic nucleotide is a type of nucleotide formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide can be unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), where UNA is as shown in formula (15) and GNA is as shown in formula (16):

[0190]

[0191] In the above formulas (15) and (16), R is selected from H, OH, or alkoxy (O-alkyl).

[0192] A non-natural nucleotide refers to a compound formed by changing the position of the base on the ribose ring in a nucleotide. In some embodiments, the non-natural nucleotide can be a compound formed by moving the base from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (17) or (18).

[0193]

[0194] In the compounds of the above formulas (17)-(18), Base represents a nucleic acid base, such as A, U, G, C, or T; R is selected from H, OH, F, or the non-fluorine groups as described above.

[0195] In some embodiments, the nucleotide analog is selected from the group consisting of a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), a cyclohexenyl nucleic acid (cET), an unlocked nucleic acid (UNA), and a glycol nucleic acid (GNA). In some embodiments, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide. As used herein and hereinafter, the methoxy-modified nucleotide refers to a nucleotide formed by substituting the 2'-hydroxyl group of the ribose group with a methoxy group.

[0196] As used herein and hereinafter, the "fluorinated modified nucleotide" refers to a compound having the structure shown in formula (7) formed by substituting the 2'-hydroxyl group of the nucleotide with a fluorine atom; the "methoxy-modified nucleotide" refers to a compound having the structure shown in formula (8) formed by substituting the 2'-hydroxyl group of the ribose group of the nucleotide with a methoxy group.

[0197] In some embodiments, the siRNA of the present disclosure is an siRNA having the following modifications: in the sense strand, in the direction from the 5'-end to the 3'-end, the 7th, 8th, 9th or 5th, 7th, 8th, 9th nucleotides of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand, the 2nd, 6th, 14th, 16th or 2nd, 6th, 8th, 9th, 14th, 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides.

[0198] In some embodiments, the siRNA of the present disclosure is an siRNA having the following modifications: in the sense strand of the siRNA, in the direction from the 5'-end to the 3'-end, the 5th, 7th, 8th, and 9th nucleotides of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; and in the antisense strand of the siRNA, in the direction from the 5'-end to the 3'-end, the 2nd, 6th, 8th, 9th, 14th, and 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides;

[0199] Alternatively, in the 5'-to-3' direction, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxylated nucleotides. Moreover, in the 5'-to-3' direction, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxylated nucleotides;

[0200] Alternatively, in the 5'-to-3' direction, the nucleotides at positions 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxylated nucleotides. Moreover, in the 5'-to-3' direction, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxylated nucleotides.

[0201] In some embodiments, the siRNA provided by the present disclosure is arbitrarily selected from one of the following siRNAs: siHAVa1-M1, siHAVa2-M1, siHAVa3-M1, siHAVb1-M1, siHAVb2-M1, siHAVb3-M1, siHAVc1-M1, siHAVc2-M1, siHAVc3-M1, siHAVd1-M1, siHAVd2-M1, and siHAVd3-M1.

[0202] The siRNA with the above modifications not only has a low cost but also makes ribonucleases in the blood less likely to cleave nucleic acids, thereby increasing the stability of nucleic acids and endowing nucleic acids with stronger resistance to nuclease hydrolysis. At the same time, the siRNA with the above modifications has a high activity of inhibiting HAV viral RNA.

[0203] In some embodiments, at least a part of the phosphate ester groups in the phospho-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA provided by the present disclosure are phosphate ester groups with modifying groups. In some embodiments, the phosphate ester group with a modifying group is a phosphorothioate subunit formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom. In some embodiments, the phosphate ester group with a modifying group is a phosphorothioate subunit having the structure shown in formula (1):

[0204]

[0205] This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.

[0206] In some embodiments, in the siRNA provided by the present disclosure, phosphorothioate subunit linkages are present at least at one location selected from the group consisting of: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the foregoing. In some embodiments, phosphorothioate subunit linkages are present at all of the foregoing locations except at the 5'-end of the sense strand. In some embodiments, phosphorothioate subunit linkages are present at all of the foregoing locations except at the 3'-end of the sense strand. In some embodiments, phosphorothioate subunit linkages are present at least at one of the following locations:

[0207] between the first and second nucleotides at the 5'-end of the sense strand;

[0208] between the second and third nucleotides at the 5'-end of the sense strand;

[0209] between the first and second nucleotides at the 3'-end of the sense strand;

[0210] between the second and third nucleotides at the 3'-end of the sense strand;

[0211] between the first and second nucleotides at the 5'-end of the antisense strand;

[0212] between the second and third nucleotides at the 5'-end of the antisense strand;

[0213] between the first and second nucleotides at the 3'-end of the antisense strand; and

[0214] between the second and third nucleotides at the 3'-end of the antisense strand.

[0215] In some embodiments, the siRNA provided by the present disclosure is arbitrarily selected from one of the following siRNAs: siHAVa1-M1S, siHAVa1-M1X, siHAVa2-M1S, siHAVa2-M1X, siHAVa3-M1S, siHAVa3-M1X, siHAVb1-M1S, siHAVb1-M1X, siHAVb2-M1S, siHAVb2-M1X, siHAVb3-M1S, siHAVb3-M1X, siHAVc1-M1S, siHAVc1-M1X, siHAVc2-M1S, siHAVc2-M1X, siHAVc3-M1S, siHAVc3-M1X, siHAVd1-M1S, siHAVd1-M1X, siHAVd2-M1S, siHAVd2-M1X, siHAVd3-M1S, and siHAVd3-M1X.

[0216] In some embodiments, the nucleotide at the 5'-end of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analog.

[0217] The commonly used 5'-phosphate nucleotide or nucleotide modified with a 5'-phosphate analog is well-known to those skilled in the art. For example, the 5'-phosphate nucleotide may have the following structure:

[0218]

[0219] Again, four 5'-phosphate analog-modified nucleotides are disclosed in Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3):  238-48 as follows:

[0220]

[0221] Among them, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base and is selected from A, U, C, G, or T.

[0222] In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (2), and the nucleotide modified with a 5'-phosphate analog is a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modification as shown in formula (3), or a phosphorothioate-modified nucleotide as shown in formula (5).

[0223] In some embodiments, the siRNA provided by the present disclosure is arbitrarily selected from one of the following groups: siHAVa1-M1P1, siHAVa2-M1P1, siHAVa3-M1P1, siHAVa1-M1SP1, siHAVa2-M1SP1, siHAVa3-M1SP1, siHAVa1-M1XP1, siHAVa2-M1XP1, siHAVa3-M1XP1, siHAVb1-M1P1, siHAVb2-M1P1, siHAVb3-M1P1, siHAVb1-M1SP1, siHAVb2-M1SP1, siHAVb3-M1SP1, siHAVb1-M1XP1, siHAVb2-M1XP1, siHAVb3-M1XP1, siHAVc1-M1P1, siHAVc2-M1P1, siHAVc3-M1P1, siHAVc1-M1SP1, siHAVc2-M1SP1, siHAVc3-M1SP1, siHAVc1-M1XP1, siHAVc2-M1XP1, siHAVc3-M1XP1, siHAVd1-M1P1, siHAVd2-M1P1, siHAVd3-M1P1, siHAVd1-M1SP1, siHAVd2-M1SP1, siHAVd3-M1SP1, siHAVd1-M1XP1, siHAVd2-M1XP1, and siHAVd3-M1XP1.

[0224] The inventors of the present disclosure surprisingly found that the siRNA provided by the present disclosure achieved a high balance of plasma stability and RNAi efficiency in animal experiments.

[0225] The inventors of the present disclosure unexpectedly found that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains a high viral RNA inhibitory activity.

[0226] The siRNA provided by the present disclosure can be obtained by conventional siRNA preparation methods in the art (such as solid-phase synthesis and liquid-phase synthesis methods). Among them, solid-phase synthesis already has commercial customization services. Modifying nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. The methods for preparing nucleoside monomers with corresponding modifications and the methods for introducing modified nucleotide groups into siRNA are also well-known to those skilled in the art.

[0227] siRNA conjugate

[0228] On the other hand, the present disclosure provides an siRNA conjugate, which contains the above siRNA and a conjugation group conjugated to the siRNA.

[0229] Generally speaking, the conjugation group includes at least one pharmaceutically acceptable targeting group and / or delivery assisting group. In some embodiments, the conjugation group further includes a linker, and the linker and / or the targeting group or the delivery assisting group are connected in sequence.

[0230] In some embodiments, the conjugation group includes a linker and a pharmaceutically acceptable targeting group and / or delivery assisting group, and the siRNA, the linker, and the targeting group or the delivery assisting group are connected covalently or non-covalently in sequence. Each targeting group is selected from ligands capable of binding to cell surface receptors, and each delivery assisting group is selected from groups capable of increasing the biocompatibility of the siRNA conjugate in the target organ or tissue to be delivered.

[0231] In some embodiments, the number of the targeting groups is 1 to 6. In some embodiments, the number of the targeting groups is 2 to 4. The siRNA molecule can be conjugated to the conjugation group non-covalently or covalently, for example, it can be conjugated to the conjugation group covalently. The conjugation site of the siRNA and the conjugation group can be at the 3'-end or 5'-end of the sense strand of the siRNA, or at the 5'-end of the antisense strand, or also in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and the conjugation group is at the 3'-terminus of the sense strand of the siRNA.

[0232] In some embodiments, the conjugating group can be linked to the phosphate group, 2'-hydroxyl group, or base of the nucleotide. In some embodiments, the conjugating group can also be linked to the 3'-hydroxyl group, in which case the nucleotides are linked by 2'-5' phosphodiester bonds. When the conjugating group is linked to the end of the siRNA strand, the conjugating group is usually linked to the phosphate group of the nucleotide; when the conjugating group is linked to the internal sequence of the siRNA, the conjugating group is usually linked to the ribose ring or the base. For various linking methods, reference can be made to the literature: Muthiah Manoharan et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.

[0233] The targeting group can be linked to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group. The types of these linkers, targeting groups, and the linking methods to the siRNA can be seen in the disclosure of WO2015006740A2, and its entire content is incorporated herein by reference. In some embodiments, the siRNA and the conjugating group can be linked by acid-labile or reducible chemical bonds, and these chemical bonds can be degraded in the acidic environment of the endosome in the cell, so that the siRNA becomes free. For non-degradable conjugation methods, the conjugating group can be linked to the sense strand of the siRNA to minimize the impact of conjugation on the activity of the siRNA.

[0234] In some embodiments, the targeting group can be a ligand commonly used in the field of siRNA administration, such as various ligands described in WO2009082607A2, and its entire disclosure is incorporated herein by reference.

[0235] In some embodiments, at least one or each of the targeting groups is selected from ligands capable of binding to the cell surface receptor containing the HAV viral RNA.

[0236] In some embodiments, at least one or each of the targeting groups is selected from ligands capable of binding to the mammalian hepatocyte surface receptor (ASGPR). In some embodiments, each of the targeting groups is independently a ligand that binds to the asialoglycoprotein receptor on the surface of mammalian hepatocytes. In some embodiments, each of the targeting groups is independently an asialoglycoprotein or a sugar. In some embodiments, each of the targeting groups is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments, each of the targeting groups is independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose. In some embodiments, at least one or each of the targeting groups is galactose or N-acetylgalactosamine.

[0237] In some embodiments, the linker in the siRNA conjugate of the present disclosure has the structure shown in formula (301):

[0238]

[0239] wherein k is an integer from 1 to 3;

[0240] LA has a structure containing an amide bond as shown in formula (302), L B has a structure containing N-acylpyrrolidine as shown in formula (303), containing a carbonyl group and an oxygen atom, L C is a linking group based on hydroxymethylaminomethane, bis(hydroxymethyl)aminomethane or tris(hydroxymethyl)aminomethane;

[0241]

[0242] wherein, n 302 、q 302 and p 302 are each independently an integer from 2 to 6, optionally, n 302 、q 302 and p 302 are each independently 2 or 3; n 303 is an integer from 4 to 16, optionally, n 303 is an integer from 8 to 12, represents the site where the groups are covalently linked.

[0243] In the linker, each L A is respectively connected to one of the targeting groups through an ether bond, and through the oxygen atom of the hydroxyl group in the L C portion to form an ether bond with the L C portion; L B is connected to the L C portion by forming an amide bond between the carbonyl group in formula (303) and the nitrogen atom of the amino group in the L C portion, and is connected to the siRNA through the oxygen atom in formula (303) to form a phosphodiester bond or a phosphorothioate bond through the oxygen atom.

[0244] In some embodiments, the siRNA conjugate provided by the present disclosure has a structure as shown in formula (305):

[0245]

[0246] wherein, Nu represents the siRNA provided by the present disclosure.

[0247] In some embodiments, the linker in the siRNA conjugate of the present disclosure has a structure as shown in formula (306):

[0248]

[0249] wherein, n 306 is an integer from 0 to 3, and each p 306 is independently an integer from 1 to 6, Indicates the site of covalent attachment of a group; the linking group is connected to the targeting group through an ether bond formed by the oxygen atom marked by *; the linking group is connected to the siRNA by forming a phosphate bond or a phosphorothioate bond by at least one of the oxygen atoms marked by #, and the remaining oxygen atoms marked by # are connected to hydrogen atoms to form hydroxyl groups, or are connected to C1-C3 alkyl groups to form C1-C3 alkoxy groups;

[0250] In some embodiments, the siRNA conjugates of the present disclosure have a structure as shown in formula (307):

[0251]

[0252] Wherein, Nu represents the siRNA provided by the present disclosure.

[0253] In some embodiments, the siRNA conjugates disclosed herein have the structure shown in formula (308):

[0254]

[0255] in,

[0256] n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;

[0257] Each m1, m2 or m3 is independently an integer selected from 2-10;

[0258] R 10 、R 11 、R 12 、R 13 、R 14 or R 15 are each independently H, or selected from the group consisting of: C1-C 10 Alkyl, C1-C 10 Halogenated alkyl and C1-C 10 alkoxy;

[0259] R3 has the structure shown in Formula A59:

[0260]

[0261] wherein E1 is OH, SH or BH2, and Nu represents the siRNA provided by the present disclosure;

[0262] R2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10Alkynylene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 Heteroarylene; and wherein R2 may optionally have substituents selected from any one or more of the following groups: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Haloalkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 Alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -CONH(C1-C 10 Alkylphenyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C10 alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0263] Each L1 is independently a straight-chain alkylene group having 1 to 70 carbon atoms, wherein one or more carbon atoms may optionally be replaced by any one or more of the groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18 heteroalkylene and C5-C 10 heteroarylene; and wherein L1 optionally has a substituent of any one or more of the groups consisting of: C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10 alkyl, -OC1-C 10 alkylphenyl, -C1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C 10 alkyl, -SC1-C 10 alkylphenyl, -C1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 alkyl), -CON(C1-C 10 alkyl)(C1-C 10 alkyl), -CONH(C1-C 10(alkyl), -CONH2, -NHC(O)(C1-C 10 (alkyl), -NHC(O)(phenyl), -N(C1-C 10 (alkyl)C(O)(C1-C 10 (alkyl), -N(C1-C 10 (alkyl)C(O)(phenyl), -C(O)C1-C 10 alkyl, -C(O)C1-C 10 alkylphenyl, -C(O)C1-C 10 haloalkyl, -OC(O)C1-C 10 alkyl, -SO2(C1-C 10 (alkyl), -SO2(phenyl), -SO2(C1-C 10 (haloalkyl), -SO2NH2, -SO2NH(C1-C 10 (alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 (alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 (haloalkyl);

[0264] represents the site at which the group is covalently linked;

[0265] M1 represents a targeting group, which has the same definition and optional scope as described above. In some embodiments, each M1 is independently selected from one of the ligands having an affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.

[0266] Those skilled in the art will understand that although for convenience L1 is defined as a linear alkylene group, it may not be a linear group or have a different name, such as an amine or alkenyl group resulting from the above substitutions and / or permutations. For the purposes of the present disclosure, the length of L1 is the number of atoms in the chain connecting the two attachment points. For this purpose, a ring (such as a heterocyclic moiety or heteroaryl) obtained by replacing the carbon atoms of the straight-chain alkylene group is counted as one atom.

[0267] When M1 is a ligand with affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 can be an integer from 1 to 3, and n3 can be an integer from 0 to 4, ensuring that the number of M1 ligands in the conjugate is at least 2. In some embodiments, n1 + n3 ≥ 2, which can result in the number of M1 ligands being at least 3, making it easier for the M1 ligand to bind to the asialoglycoprotein receptor on the liver surface, thereby promoting the conjugate to enter the cell via endocytosis. Experiments have shown that when the number of M1 ligands is greater than 3, the increased ease of binding of the M1 ligand to the asialoglycoprotein receptor on the liver surface is not significantly increased. Therefore, considering multiple factors such as ease of synthesis, structural / processing costs, and delivery efficiency, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1 + n3 = 2-3.

[0268] In some embodiments, when m1, m2 and m3 are independently selected from integers of 2-10, the spatial positions between multiple M1 ligands can be made suitable for the binding of M1 ligands to the asialoglycoprotein receptor on the liver surface. In order to make the conjugate provided by the present disclosure simpler, easier to synthesize and / or reduce costs, in some embodiments, m1, m2 and m3 are each independently an integer of 2-5. In some embodiments, m1=m2=m3.

[0269] Those skilled in the art will understand that when R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl, and C1-C 10 In some embodiments, R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each is independently selected from H, methyl and ethyl. 10 、R 11 、R 12 、R 13 、R 14 and R 15 Both are H.

[0270] According to the siRNA conjugate provided by the present disclosure, R3 is a group having the structure shown in Formula A59, wherein E1 is OH, SH or BH2. Considering the easy availability of the preparation raw materials, in some embodiments, E1 is OH or SH.

[0271] In some embodiments, R2 is selected to achieve the connection between the N atom on the nitrogen-containing backbone and A59. In the context of the present disclosure, the "nitrogen-containing backbone" refers to a chain structure in which the carbon atoms and N atoms connected to R 10 、R 11 、R 12 、R 13 、R 14 and R 15 are connected to each other. Therefore, R2 can be any linking group that can connect the A59 group to the N atom on the nitrogen-containing backbone in an appropriate manner. In some embodiments, when the siRNA conjugate of the present disclosure is prepared by a solid-phase synthesis process, the R2 group needs to contain both a linking site for connecting to the N atom on the nitrogen-containing backbone and a linking site for connecting to the P atom in R3. In some embodiments, the site in R2 for connecting to the N atom on the nitrogen-containing backbone forms an amide bond with the N atom, and the site for connecting to the P atom on R3 forms a phosphate ester bond with the P atom. In some embodiments, the length of R2 is 2-20 atoms, or 4-15 atoms. In some embodiments, R2 is B5, B6, B5' or B6':

[0272]

[0273] wherein, represents the site of covalent bond connection of the group.

[0274] The value range of q2 can be an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.

[0275] The function of L1 is to connect the M1 ligand to the N on the nitrogen-containing backbone, providing a targeting function for the siRNA conjugate of the present disclosure. In some embodiments, L1 is selected from one or more of the linking combinations of the groups of Formulae A1-A26. In some embodiments, L1 is selected from one or more of the linking combinations of A1, A4, A5, A6, A8, A10, A11 and A13; in some embodiments, L1 is selected from the linking combination of at least 2 of A1, A4, A8, A10 and A11; in some embodiments, L1 is selected from the linking combination of at least 2 of A1, A8 and A10.

[0276]

[0277]

[0278] In some embodiments, the length of L1 can be 3 to 25 atoms, 3 to 20 atoms, 4 to 15 atoms, or 5 to 12 atoms. In some embodiments, the length of L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atoms.

[0279] In some embodiments, j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R' is an alkyl group of C1-C4, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments, Ra is A27 or A28. Rb is an alkyl group of C1-C5, and in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, in formulae A1 - A26, j1, j2, R', Ra, and Rb are each selected to enable the N on the nitrogen-containing backbone to be connected to the M1 ligand and to make the spatial positions between the M1 ligands more suitable for the M1 ligand to bind to the asialoglycoprotein receptor on the liver surface.

[0280]

[0281] In some embodiments, the siRNA conjugate of the present disclosure has a structure shown in formulae (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289] Among them, Nu represents the siRNA of the present disclosure.

[0290] In some embodiments, the P atom in formula A59 can be connected to any possible position in the siRNA sequence. For example, the P atom in formula A59 can be connected to any nucleotide on the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula A59 is connected to any nucleotide on the sense strand of the siRNA. In some embodiments, the P atom in formula A59 is connected to the end of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula A59 is connected to the end of the sense strand of the siRNA. The "end" refers to the first 4 nucleotides starting from one end of the sense strand or the antisense strand. In some embodiments, the P atom in formula A59 is connected to the end of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula A59 is connected to the 3'-end of the sense strand of the siRNA. In the case of connecting to the above positions of the sense strand of the siRNA, after the conjugate provided by the present disclosure enters the cell and is unwound, the separate siRNA antisense strand can be released to inhibit the HAV viral RNA through the RNAi mechanism.

[0291] The P atom in formula A59 can be connected to any possible position on the nucleotide in the siRNA. For example, the 5'-position of the nucleotide, the 2'-position of the nucleotide, the 3'-position of the nucleotide, or the base of the nucleotide. In some embodiments, the P atom in formula A59 can be connected to the 2'-position, 3'-position, or 5'-position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments, the P atom in formula A59 is connected to the oxygen atom formed after dehydrogenation of the 3'-hydroxyl of the nucleotide at the 3'-end of the sense strand of the siRNA, or the P atom in formula A59 is connected to the nucleotide by substituting the hydrogen in the 2'-hydroxyl of a nucleotide in the sense strand of the siRNA, or the P atom in formula A59 is connected to the nucleotide by substituting the hydrogen in the 5'-hydroxyl of the nucleotide at the 5'-end of the sense strand of the siRNA.

[0292] The inventors of the present disclosure unexpectedly found that the siRNA conjugate of the present disclosure has significantly improved stability in plasma and high HAV viral RNA silencing activity. In some embodiments, the siRNA of the present disclosure can be one of the siRNAs shown in Table 1.

[0293] Table 1 siRNAs of the present disclosure

[0294]

[0295]

[0296]

[0297]

[0298]

[0299] Among them, the capital letters C, G, U, and A represent the base composition of nucleotides; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a nucleotide modified with a methoxy group; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a nucleotide modified with a fluoro group; the lowercase letter s indicates that there is a phosphorothioate subunit linkage between the two nucleotides on the left and right of the letter; P1 indicates that the nucleotide adjacent to the right of the P1 is a nucleotide modified with a 5'-phosphate nucleotide or a 5'-phosphate analog. In some embodiments, P1 is VP, Ps, or P representing specific modifications, wherein the letter combination VP indicates that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinyl phosphate (5'-(E)-vinylphosphonate, E-VP); the letter combination Ps indicates that the nucleotide adjacent to the right of the letter combination Ps is a nucleotide modified with a phosphorothioate; the capital letter P indicates that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0300] It is well known to those skilled in the art that modified nucleotide groups can be introduced into the siRNAs described in the present disclosure by using nucleoside monomers with corresponding modifications. The methods for preparing nucleoside monomers with corresponding modifications and the methods for introducing modified nucleotide groups into siRNAs are also well known to those skilled in the art. All modified nucleoside monomers can be commercially available or prepared by known methods.

[0301] The siRNA conjugates of the present disclosure can be prepared by any reasonable synthetic route. For example, for a conjugate molecule containing a targeting group and a reactive group that can react with phosphoramidite to form a covalent linkage, the reactive group in the conjugate molecule can be protected with a protecting agent first, then connected to a solid-phase support, and subsequently, by the phosphoramidite solid-phase synthesis method, according to the nucleotide types and sequences of the sense and antisense strands of the siRNA, nucleoside monomers are connected one by one in the 3' to 5' direction. The connection of each nucleoside monomer includes four steps: deprotection, coupling, capping, and oxidation or sulfurization; the sense and antisense strands of the siRNA are separated and annealed to obtain the siRNA conjugates of the present disclosure.

[0302] Furthermore, the preparation of siRNA conjugates can also be carried out with reference to the disclosure of existing literature. For example, WO2019010274A1 describes a method for sequentially connecting a linking group and a targeting ligand with a specific structure to an siRNA in Example 1. The entire content thereof is incorporated herein by reference.

[0303] Pharmaceutical composition

[0304] In one aspect, the present disclosure provides a pharmaceutical composition comprising the siRNA and / or siRNA conjugate described above as an active ingredient and a pharmaceutically acceptable carrier.

[0305] The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (such as nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), and one or more of their derivatives.

[0306] In some embodiments, in the pharmaceutical composition, there is no particular requirement for the content of the siRNA and / or siRNA conjugate and the pharmaceutically acceptable carrier. In some embodiments, the weight ratio of the siRNA and / or siRNA conjugate to the pharmaceutically acceptable carrier may be 1:(1 - 500). In some embodiments, the above weight ratio is 1:(1 - 50).

[0307] In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0308] The pH buffer solution can be a tris(hydroxymethyl)aminomethane hydrochloride buffer solution with a pH value of 7.5 - 8.5 and / or a phosphate buffer solution with a pH value of 5.5 - 8.5. For example, it can be a phosphate buffer solution with a pH value of 5.5 - 8.5.

[0309] The cryoprotectant can be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the cryoprotectant can be 0.01 - 30% by weight.

[0310] The osmotic pressure regulator can be, for example, sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200 - 700 milliosmoles per kilogram (mOsm / kg). Those skilled in the art can easily determine the content of the osmotic pressure regulator according to the required osmotic pressure. In some embodiments, the dosage of the preparation made from the pharmaceutical composition will be adjusted depending on the different administration methods during administration.

[0311] In some embodiments, the pharmaceutical composition can be a liquid preparation, such as an injection; or it can be a freeze-dried powder injection, which is mixed with a liquid excipient during administration to prepare a liquid preparation. The liquid preparation can be but is not limited to being used for subcutaneous, intramuscular, or intravenous injection, and can also be but is not limited to being delivered by nasal administration, oropharyngeal inhalation, spray administration, etc. In some embodiments, the pharmaceutical composition is administered by subcutaneous injection.

[0312] In some embodiments, the pharmaceutical composition can be in the form of a liposomal preparation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal preparation includes an amine-containing transfection compound (hereinafter also referred to as an organic amine), a co-lipid, and / or a polyethylene glycolylated lipid. Among them, the organic amine, co-lipid, and polyethylene glycolylated lipid can be respectively selected from one or more of the amine-containing transfection compounds or their pharmaceutically acceptable salts or derivatives, co-lipids, and polyethylene glycolylated lipids described in Chinese Patent Application CN103380113A (which is incorporated herein by reference in its entirety).

[0313] In some embodiments, the organic amine can be the compound shown in formula (201) described in Chinese Patent Application CN103380113A or its pharmaceutically acceptable salt:

[0314] Wherein:

[0315] X 101 and X 102 are each independently O, S, N-A, or C-A, where A is hydrogen or C1-C20 Hydrocarbon chain;

[0316] Y 101 and Z 101 each independently is C═O, C═S, S═O, CH—OH or SO2;

[0317] R 101 、R 102 、R 103 、R 104 、R 105 、R 106 and R 107 each independently is hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, a substituted or unsubstituted, branched or straight-chain heteroaryl group;

[0318] x is an integer from 1 to 10;

[0319] n is an integer from 1 to 3, m is an integer from 0 to 20, p is 0 or 1; wherein, if m = p = 0, then R 102 is hydrogen;

[0320] And, if at least one of n or m is 2, then R 103 and the nitrogen in formula (^{201}) form a structure as shown in formula (^{202}) or formula (^{203}):

[0321]

[0322] wherein, g, e and f each independently are integers from 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (^{201}).

[0323] In some embodiments, R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, R 101 and R 102 in formula (^{201}) each independently is any substituted or unsubstituted, branched or straight-chain alkyl or alkenyl group having 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.

[0324] In some embodiments, if each of n and m independently has a value of 1 or 3, then R 103 can be any one of the following formulas (^{204})-(^{213}):

[0325]

[0326]

[0327] Among them, in Formulas (204)-(213), g, e, and f are each independently an integer from 1 to 6, each "HCC" represents a hydrocarbon chain, and each * indicates R 103 Possible connection points to the nitrogen atom in Formula (201), where each H at any * position can be replaced to achieve connection to the nitrogen atom in Formula (201).

[0328] Among them, the compound shown in Formula (201) can be prepared according to the description in Chinese Patent Application CN103380113A.

[0329] In some embodiments, the organic amine is the organic amine shown in Formula (214) and / or the organic amine shown in Formula (215):

[0330]

[0331] The auxiliary lipid is cholesterol, an analogue of cholesterol, and / or a derivative of cholesterol;

[0332] The polyethylene glycolated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0333] In some embodiments, in the pharmaceutical composition, the molar ratio among the organic amine, the auxiliary lipid, and the polyethylene glycolated lipid is (19.7 - 80):(19.7 - 80):(0.3 - 50), and for example, it can be (50 - 70):(20 - 40):(3 - 20).

[0334] In some embodiments, the pharmaceutical composition particles formed by the siRNA of the present disclosure and the above amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, usually about 40 nm to about 135 nm, and more usually, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm. For example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

[0335] In some embodiments, in the pharmaceutical composition formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent, the weight ratio (weight / weight ratio) of the siRNA to all lipids (such as organic amines, co-lipids, and / or polyethylene glycolated lipids) ranges from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10. For example, the weight ratio of the siRNA of the present disclosure to all lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18.

[0336] In some embodiments, the components of the pharmaceutical composition may exist independently when sold and may exist in the form of a liquid preparation when used. In some embodiments, the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared by various known methods, only by replacing the existing siRNA with the siRNA provided by the present disclosure. In some embodiments, it can be prepared according to the following method:

[0337] Suspend the organic amine, co-lipid, and polyethylene glycolated lipid in the alcohol according to the above molar ratio and mix well to obtain a lipid solution. The amount of alcohol is such that the total mass concentration of the obtained lipid solution is 2-25 mg / mL, for example, it can be 8-18 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, and for example, it can be ethanol.

[0338] Dissolve the siRNA provided by the present disclosure in a buffer salt solution to obtain an aqueous siRNA solution. The concentration of the buffer salt solution is 0.05-0.5 M, for example, it can be 0.1-0.2 M. Adjust the pH of the buffer salt solution to 4.0-5.5, for example, it can be 5.0-5.2. The amount of the buffer salt solution is such that the concentration of the siRNA does not exceed 0.6 mg / mL, for example, it can be 0.2-0.4 mg / mL. The buffer salt is selected from one or more of soluble acetates and soluble citrates, for example, it can be sodium acetate and / or potassium acetate.

[0339] Mix the lipid solution and the aqueous siRNA solution, and incubate the obtained product at 40-60 °C for at least 2 minutes, for example, it can be 5-30 minutes, to obtain an incubated liposomal preparation. The volume ratio of the lipid solution to the aqueous siRNA solution is 1:(2-5), for example, it can be 1:4.

[0340] The incubated liposomal preparation is concentrated or diluted, impurities are removed, and sterilization is carried out to obtain the pharmaceutical composition provided by the present disclosure. Its physicochemical parameters are as follows: the pH value is 6.5 - 8, the encapsulation efficiency is not less than 80%, the particle size is 40 - 200 nm, the polydispersity index is not higher than 0.30, and the osmotic pressure is 250 - 400 mOsm / kg. For example, the physicochemical parameters can be that the pH value is 7.2 - 7.6, the encapsulation efficiency is not less than 90%, the particle size is 60 - 100 nm, the polydispersity index is not higher than 0.20, and the osmotic pressure is 300 - 400 mOsm / kg.

[0341] Among them, concentration or dilution can be carried out before, after, or simultaneously with the removal of impurities. Various existing methods can be used to remove impurities. For example, a tangential flow system or a hollow fiber column can be used, and ultrafiltration is carried out under the condition of 100 KDa, and the ultrafiltration exchange solution is phosphate buffer solution (PBS) with a pH of 7.4. Various existing methods can be used for sterilization. For example, filtration sterilization can be carried out on a 0.22 μm filter.

[0342] Applications of the siRNA and siRNA conjugate of the present disclosure and the pharmaceutical composition containing the siRNA

[0343] In some embodiments, the present disclosure provides the use of the siRNA and / or siRNA conjugate and / or pharmaceutical composition of the present disclosure in the preparation of a medicament for treating diseases or symptoms related to the regulation of HAV function.

[0344] In some embodiments, the present disclosure provides a method for treating diseases or symptoms related to the regulation of HAV function, which method comprises administering an effective amount of the siRNA / or siRNA conjugate and / or pharmaceutical composition of the present disclosure to a subject in need thereof. By administering the active ingredient of the siRNA of the present disclosure to a subject in need thereof, the purpose of treating the resulting disease can be achieved through the mechanism of RNA interference. Therefore, the siRNA and / or siRNA conjugate and / or pharmaceutical composition of the present disclosure can be used to treat diseases or symptoms related to the regulation of HAV function, or for the preparation of a medicament for treating diseases or symptoms related to the regulation of HAV function.

[0345] As used herein, the term "administer / give" refers to placing the siRNA, siRNA conjugate, and / or pharmaceutical composition of the present disclosure into a subject by a method or route that causes at least partial localization of the siRNA, siRNA conjugate, and / or pharmaceutical composition of the present disclosure to a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include topical administration and systemic administration. Generally, topical administration results in delivery of more siRNA conjugates to a specific site compared to the subject's systemic circulation; while systemic administration results in delivery of the siRNA, siRNA conjugate, and / or pharmaceutical composition of the present disclosure to the subject's general systemic circulation. Considering that the present disclosure aims to provide a means for treating hepatitis A disease, in some embodiments, a route of administration that delivers the drug to liver tissue is employed.

[0346] Administration to a subject can be by any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), nasal administration, rectal administration, and topical administration (including buccal and sublingual administration). The frequency of administration can be once or multiple times per day, per week, every two weeks, every three weeks, per month, or per year.

[0347] The dosage of the siRNA, siRNA conjugate, or pharmaceutical composition described in the present disclosure can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially the age, weight, and gender of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining LD 50 (the lethal dose that causes 50% of the population to die) and ED 50 (in a quantal response, it refers to the dose that can cause 50% of the maximum response intensity, and in a qualitative response, it refers to the dose that can cause 50% of the experimental subjects to show a positive response). The range of human dosage can be derived based on data obtained from cell culture assays and animal studies. In some embodiments, the dosage of the preparation made from the siRNA, siRNA conjugate, or pharmaceutical composition is adjusted during administration according to the different routes of administration.

[0348] When administering the siRNAs, siRNA conjugates, and / or pharmaceutical compositions described herein, for example, for male or female C57BL / 6J mice that are 6 - 12 weeks old and weigh 18 - 25 g or ob / ob mice that weigh 30 - 45 g, based on the amount of siRNA: (i) for siRNA conjugates, the dosage of siRNA can be 0.001 - 100 mg / kg body weight, in some embodiments 0.01 - 50 mg / kg body weight, in some embodiments 0.05 - 20 mg / kg body weight, in some other embodiments 0.1 - 15 mg / kg body weight, and in some other embodiments 0.1 - 10 mg / kg body weight; (ii) for pharmaceutical compositions formed by siRNA and a pharmaceutically acceptable carrier, the dosage of siRNA can be 0.001 - 50 mg / kg body weight, in some embodiments 0.01 - 10 mg / kg body weight, in some embodiments 0.05 - 5 mg / kg body weight, and in some embodiments 0.1 - 3 mg / kg body weight.

[0349] In some embodiments, the present disclosure provides a method for inhibiting HAV viral RNA in cells, the method comprising contacting an effective amount of the siRNA and / or siRNA conjugate and / or pharmaceutical composition of the present disclosure with the cells, introducing the siRNA and / or siRNA conjugate and / or pharmaceutical composition of the present disclosure into the cells, and achieving the purpose of inhibiting HAV viral RNA in the cells through the mechanism of RNA interference.

[0350] When using the method provided by the present disclosure to inhibit HAV viral RNA, the dosage of siRNA in the modified siRNA, siRNA conjugate, and / or pharmaceutical composition provided is generally an amount sufficient to reduce viral RNA and result in an extracellular concentration of 1 pM to 1 μM, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM, or 0.05 nM to about 5 nM at the surface of target cells containing viral RNA. The amount required to achieve this local concentration will vary with various factors, including the delivery method, delivery site, the number of cell layers between the delivery site and the target cells or tissues, the delivery route (local or systemic), etc. The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cells or tissues.

[0351] Kit

[0352] In one aspect, the present disclosure provides a kit that contains at least one of an effective amount of the siRNA, siRNA conjugate, and pharmaceutical composition of the present disclosure.

[0353] In some embodiments, the kits described herein may provide siRNA in one container. In some embodiments, the kits described herein may comprise a container providing a pharmaceutically acceptable excipient. In some embodiments, other components such as stabilizers or preservatives may also be included in the kit. In some embodiments, the kits described herein may comprise at least one other therapeutic agent in a container different from the container providing the siRNA described herein. In some embodiments, the kit may comprise instructions for mixing the siRNA with a pharmaceutically acceptable carrier and / or excipient or other components (if any).

[0354] In the kits of the present disclosure, the siRNA and the pharmaceutically acceptable carrier and / or excipient, and the modified siRNA, siRNA conjugate and / or pharmaceutical composition, and / or the pharmaceutically acceptable carrier and / or excipient may be provided in any form, such as in liquid form, dry form or lyophilized form. In some embodiments, the siRNA and the pharmaceutically acceptable carrier and / or excipient and the pharmaceutically acceptable carrier and / or excipient of the pharmaceutical composition and / or siRNA conjugate are substantially pure and / or sterile. In some embodiments, sterile water may be provided in the kits of the present disclosure.

[0355] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereby.

[0356] Examples

[0357] Unless otherwise specified, the reagents and culture media used in the following examples are commercially available products, and the operations such as nucleic acid electrophoresis and real-time PCR are carried out according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0358] Unless otherwise specified, the reagent ratios provided below are calculated by volume ratio (v / v). Data analysis was performed using Graphpad prism 8.0 statistical analysis software.

[0359] Unless otherwise specified, the reagents and culture media used in the following examples are commercially available products, and the operations such as nucleic acid electrophoresis and real-time PCR are carried out according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0360] Preparation Examples 1-4 Synthesis of Conjugates 1-4 Provided by the Present Disclosure

[0361] According to the preparation method described in Preparation Example 13 of CN110959011A, conjugates 1-4 in Table 2A below were prepared. The difference is that: according to the nucleotide sequences of the siRNAs of conjugates 1-conjugate 4 in Table 2A below, the sense strand and the antisense strand of the siRNA were synthesized respectively.

[0362] Conjugates 1-4 have the structure shown in formula (403)

[0363]

[0364] Wherein, Nu in formula (403) represents the siRNA of the present disclosure. Table 2A lists the siRNA nucleotide sequences of conjugates 1-4. The linking position of the conjugating group is the 3'-position of the ribose of the nucleotide at the 3'-end of the sense strand in the siRNA, and the conjugates 1-4 are in the form of sodium salts.

[0365] Table 2A siRNA Sequences in siRNA Conjugates

[0366]

[0367] Wherein, the capital letters C, G, U, A represent the base composition of the nucleotide; the lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a nucleotide modified with a methoxy group; the lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a nucleotide modified with a fluorine group; the lowercase letter s represents that there is a phosphorothioate subunit linkage between the two nucleotides on the left and right of the letter s.

[0368] After diluting each siRNA conjugate to a concentration of 0.2 mg / mL (calculated as siRNA) using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ·cm (25 °C)), the molecular weight was detected using a liquid chromatography-mass spectrometry instrument (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). Table 2B below details the results of the measured values and theoretical values of the molecular weight detection of the sense strand and antisense strand of conjugates 1-4. The measured values are consistent with the theoretical values, indicating that the synthesized conjugates 1-4 are the double-stranded nucleic acid sequences designed as the target.

[0369] Table 2B Molecular Weight Determination Results of Conjugates 1-4

[0370]

[0371] Comparison of the Synthesis of the Reference siRNA Conjugate in Preparation Example 1

[0372] The reference siRNA conjugate was prepared according to the method of Preparation Example 13 in CN110959011A, except that the nucleotide sequence used was the nucleotide sequence corresponding to NC in Table 2A, and the reference siRNA conjugate, numbered NC conjugate, was obtained. The structural formula is shown in Formula (403). The NC conjugate is an siRNA conjugate used as a negative control and has no sequence homology with the HAV viral RNA.

[0373] Experimental Example 1 This experimental example illustrates the inhibitory activity of siRNA and its conjugates in Huh7.5.1 human hepatoma cells in vitro.

[0374] The Huh7.5.1 human hepatoma cells used in this experiment were purchased from Changsha Abiway Biotechnology Co., Ltd.; HM175 / 18f hepatitis A virus (obtained from ATCC). The virus solution was diluted and cultured with DMEM medium supplemented with 2% FBS until the virus copy number was 1×10 9 GE / ml and the virus titer was 3×10 6 pfu / ml to obtain the P2 generation virus seed solution.

[0375] Take 10 μL of the above-obtained P2 generation virus seed solution of HM175 / 18f hepatitis A virus and add 90 μL of DMEM medium with 2% FBS, mix well to obtain 100 μL of DMEM medium containing HAV virus.

[0376] Select Huh7.5.1 human hepatoma cells in the logarithmic growth phase and inoculate them at 4.0x10 5 cells / well in a 96-well plate. Add 100 μL of DMEM medium to each well and culture in air containing 5% CO2 at 37 °C for 24 h. Then, aspirate the medium in the culture wells, add 100 μL of the above-obtained DMEM medium containing HAV virus to each well, continue to culture for 2 hours, aspirate the medium in the culture wells, and add 100 μL of fresh DMEM medium with 2% FBS to each well.

[0377] Each test siRNA conjugate was separately prepared into a 2 μM stock solution with opti-MEM medium (GIBCO); the test siRNA conjugates used were Conjugate 1, Conjugate 2, Conjugate 3, Conjugate 4, or Conjugate NC.

[0378] Each test siRNA conjugate was separately prepared into an siRNA conjugate working solution with 8 different concentrations of 2 μM, 200 nM, 20 nM, 2 nM, 0.2 nM, 0.02 nM, 0.002 nM, and 0.0002 nM (calculated based on the siRNA in the conjugate) with opti-MEM medium.

[0379] For each siRNA conjugate to be tested, prepare 1A1-1A8 solutions. Each 1A1-1A8 solution contains 10 μL of the conjugate working solution and 10 μL of Opti-MEM medium in sequence.

[0380] Prepare 1B solution. Each 1B solution contains 0.6 μL of Lipofectamine TM 3000 (Invitrogen) and 19.4 μL of Opti-MEM medium.

[0381] For each conjugate, mix 1 portion of 1B solution with the 1A1-1A8 solutions of each siRNA conjugate evenly, and incubate at room temperature for 10 min to obtain transfection complexes 1X1-1X8 of each siRNA conjugate.

[0382] In the culture wells containing the cell fluid infected with the above virus in a 48-well plate, add 100 μL of the above fresh 2% FBS DMEM medium respectively, and add the transfection complexes 1X1-1X8 of each siRNA conjugate respectively, mix evenly, and the addition amount is 20 μL / well to obtain transfection complexes with the final concentrations of each siRNA conjugate being approximately 50 nM, 5 nM, 0.5 nM, 0.05 nM, 0.005 nM, 0.0005 nM, 0.00005 nM, and 0.000005 nM (calculated based on the amount of siRNA in the siRNA conjugate). Transfect the transfection complexes 1X1-1X8 of each siRNA conjugate into 3 culture wells to obtain transfection mixtures containing the siRNA conjugate, denoted as the test groups. For each siRNA conjugate, denote the transfection mixtures in the 3 culture wells with the lowest transfection final concentration (0.000005 nM) of the conjugate as the control group.

[0383] After transfecting the transfection mixtures containing the siRNA conjugate in the culture wells for 6 hours, aspirate the medium in the culture wells, and add 200 μL of fresh 2% FBS DMEM medium to each well, and continue culturing for 72 hours.

[0384] According to the instruction manual provided by the supplier, use a fully automatic nucleic acid extractor (model: NP-GeneRotex 96) to extract the total RNA in the cells of each well.

[0385] For the cells in each well, take 1 μg of total RNA respectively, and use the reagents provided by the reverse transcription kit Goldenstar TM RT6 cDNA Synthesis Kit (Beijing Tsingke New Industry Biotechnology Co., Ltd.), and select Goldenstar TM Oligo(dT) 17As primers, a reverse transcription reaction system of 20 μL was prepared according to the reverse transcription operation steps in the kit instruction manual to perform reverse transcription on the total RNA of cells in each well. The conditions for reverse transcription were as follows: for each reverse transcription reaction system, the reverse transcription reaction system was incubated at 50 °C for 50 min, then incubated at 85 °C for 5 min, and finally incubated at 4 °C for 30 s. After the reaction ended, 80 μL of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA.

[0386] For each reverse transcription reaction system, 5 μL of the above-mentioned solution containing cDNA was taken as a template respectively, and the reagents provided by the SYBR qPCR SuperMix Plus kit (purchased from Novoprotein Scientific Inc., catalog number E096-01B) were used to prepare a qPCR reaction system of 20 μL. Among them, the PCR primer sequences for amplifying the target HAV reverse-transcribed DNA and the internal reference gene GAPDH are shown in Table 3, and the final concentration of each primer was 0.25 μM. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using the three-step method. The amplification program was pre-denaturation at 95 °C for 10 min, then denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s. After repeating the above denaturation, annealing, and extension processes 40 times, a product W1 containing the amplified target HAV reverse-transcribed DNA and the internal reference gene GAPDH was obtained. The product W1 was then incubated at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s in sequence. The real-time fluorescence quantitative PCR instrument collected the melting curves of the target HAV reverse-transcribed DNA and the internal reference gene GAPDH in the product W1 respectively to obtain the Ct values of the target HAV reverse-transcribed DNA and the internal reference gene GAPDH.

[0387] Table 3 Primer information for HAV reverse-transcribed DNA and internal reference human gene GAPDH

[0388]

[0389] The comparative Ct (ΔΔCt) method was used to perform relative quantitative calculation on the target HAV reverse-transcribed DNA in each test group. The calculation method was as follows:

[0390] ΔCt (test group) = Ct (target reverse-transcribed DNA in test group) – Ct (internal reference gene in test group)

[0391] ΔCt (control group) = Ct (target reverse-transcribed DNA in control group) – Ct (internal reference gene in control group)

[0392] ΔΔCt (test group) = ΔCt (test group) - ΔCt (average of control group)

[0393] ΔΔCt (control group) = ΔCt (control group) - ΔCt (average of control group)

[0394] Among them, ΔCt (average of control group) is the arithmetic mean of ΔCt (control group) of each of the 3 culture wells in the control group. Thus, each culture well in the test group and the control group corresponds to a ΔΔCt value.

[0395] Using the Ct value in the lowest concentration siRNA conjugate of each group as the control, calculate the relative amount of HAV copies under the action of siRNA conjugates at different concentrations according to the following formula.

[0396] Relative copy number of HAV virus in the test group = 2 -ΔΔCt(测试组) ;

[0397] Relative inhibition rate of HAV virus in the test group = (1 - relative copy number of HAV virus in the test group) × 100%.

[0398] Based on the relative copy number of HAV virus in the test group in Huh7.5.1 human hepatoma cells after transfection with different concentrations of the siRNA conjugate to be tested, use the non-linear regression analysis function of Graphpad 5.0 software to fit the log(inhibitor) vs. response—Variable slope (four parameters) dose-effect curve.

[0399] According to the function corresponding to the fitted dose-effect curve, calculate the IC 50 value of the target sequence targeted by the siRNA to be tested. The function is as follows

[0400]

[0401] In the formula

[0402] Y is the relative copy number of HAV virus

[0403] X is the logarithm of the siRNA transfection concentration

[0404] Bot is the Y value at the bottom of the steady state

[0405] Top is the Y value at the top of the steady state

[0406] X' is the X value corresponding to when Y is half way between the bottom and the top, and HillSlope is the slope of the curve at X'.

[0407] From this dose-effect curve and the corresponding function, determine the X 50 value corresponding to when Y = 50%, and calculate the IC 50 value of each siRNA = 10^X 50 (nM).

[0408] Figure 1A-1D is a dot plot showing the relative copy number of HAV viral RNA in Huh7.5.1 human hepatoma cells after transfection with different concentrations of siRNA conjugates. The IC 50 value calculated using the fitted curve is also shown in the figure.

[0409] Figure 1A-1D The results of 50 show that in vitro Huh7.5.1 human hepatoma cells, the IC

[0410] values of conjugate 1, conjugate 2, conjugate 3 and conjugate 4 are 1.1550 nM, 0.0110 nM, 0.0087 nM and 0.4299 nM respectively, showing very efficient HAV viral RNA inhibitory activity. Further, at a concentration of 5 nM, the relative inhibition rates of conjugate 2 and conjugate 3 are both greater than 90%, and the relative inhibition rates of conjugate 1 and conjugate 4 are also greater than 75%, all showing excellent HAV viral RNA inhibitory effects.

[0411] The primary monkey hepatocytes (product number Cat#LV-PmonH001), plating medium (product number Cat#LV-WEP007) and maintenance medium (product number Cat#LV-WEM007) used in this experiment were all purchased from Liwo Biotechnology Co., Ltd.

[0412] Take 10 μL of the HM175 / 18f hepatitis A virus P2 generation virus solution prepared in Experimental Example 1 and add it to 90 μL of maintenance medium, mix well to obtain 100 μL of maintenance medium containing HAV virus.

[0413] Seed the primary monkey hepatocytes at a plating density of 1.8x10 5 cells / cm 2 in a 96-well plate, add 100 μL of plating medium to each well, after culturing for 24 h, aspirate the medium in the culture wells, add 100 μL of the above-obtained maintenance medium containing HAV virus to each well, and continue to culture for 24 hours. Aspirate the medium in the culture wells, and add 100 μL of fresh maintenance medium supplemented with 2% FBS to each well.

[0414] Prepare a 2 μM conjugate working solution of each siRNA conjugate to be tested using maintenance medium. The siRNA conjugates to be tested used are conjugate 1, conjugate 2, conjugate 3, conjugate 4 or conjugate NC.

[0415] For each siRNA conjugate to be tested, prepare 2A solutions respectively, and each 2A solution contains 10 μL of the conjugate working solution and 10 μL of the maintenance medium.

[0416] In a 48-well plate, add 100 μL of the maintenance medium, and add the 2A solutions of each siRNA conjugate respectively, mix evenly, and the addition amount is 10 μL / well to obtain transfection mixtures 2X with the final concentration of each siRNA conjugate being approximately 50 nM (calculated based on the amount of siRNA in the siRNA conjugate). Transfect 3 culture wells with the 2A solution of each siRNA conjugate. For conjugates 1 - 4, obtain the transfection mixtures containing the siRNA conjugates, denoted as the test groups. For conjugate NC, obtain the transfection mixture containing conjugate NC, denoted as the control group.

[0417] For each transfection mixture 2X, after culturing in an incubator with 5% CO2 in air at 37 °C for 6 hours, aspirate the medium in the culture wells, and add 100 μL of fresh maintenance medium supplemented with 2% FBS to each well, and continue culturing for 48 hours.

[0418] Use a fully automatic nucleic acid extractor (model: NP - GeneRotex 96) to extract the total RNA in the cells of each well. According to the method of Experimental Example 1, test the relative copy number of HAV virus, with the difference that the test results of the conjugate NC group are used as the control group for calculation. The results are as Figure 2 shown.

[0419] Figure 2 is a bar chart showing the relative copy number of HAV virus after transfection with the siRNA conjugate or the comparative conjugate of the present disclosure at a concentration of 50 nM in primary monkey cells in vitro. As Figure 2 the results show, compared with the control group given conjugate NC, in primary monkey cells in vitro, the relative copy number of HAV virus of conjugate 3 is 0.18, that is, the relative inhibition rate is 82%; the relative copy numbers of the viruses of conjugates 1 and 2 are both 0.28, that is, the relative inhibition rate is 72%; the relative copy number of the virus of conjugate 4 is 0.43, that is, the relative inhibition rate is 57%. It can be seen that under the condition of not using a transfection reagent, the conjugates of the present disclosure can effectively enter primary monkey hepatocytes and exert an inhibitory effect on HAV virus, and conjugates 1 - 4 all show good inhibitory activity on HAV virus RNA.

[0420] Experimental Example 3 This experimental example illustrates the inhibitory activity of the siRNA and its conjugates of the present disclosure after infection with different strains in Huh7.5.1 human hepatoma cells in vitro.

[0421] (3 - 1) Inhibitory activity against 18F strain

[0422] Take 10 μL of the HM175 / 18f hepatitis A virus solution cultured in Experimental Example 1, add 90 μL of fresh DMEM medium supplemented with 2% FBS, and mix evenly to obtain 100 μL of DMEM medium containing HAV virus 18F.

[0423] Select logarithmically growing Huh7.5.1 human hepatoma cells and inoculate them into a 96-well plate at 4.0x10 5 cells / well, with 100 μL of DMEM medium in each well. After culturing for 24 h at 37 °C in an air environment containing 5% CO2, aspirate the medium in the culture wells, and then add 100 μL of DMEM medium containing HAV virus 18F to each culture well and continue culturing for 24 hours.

[0424] Use Opti-MEM medium (GIBCO) to prepare a 2 μM conjugate working solution for each of the siRNA conjugates to be tested; the siRNA conjugates to be used are Conjugate 1, Conjugate 2, Conjugate 3, Conjugate 4, or Conjugate NC.

[0425] For each siRNA conjugate to be tested, prepare 3A solutions, and each 3A solution contains 10 μL of the conjugate working solution and 10 μL of Opti-MEM medium in sequence.

[0426] Prepare a 3B solution, and each 3B solution contains 0.6 μL of Lipofectamine TM 3000 (Invitrogen) and 19.4 μL of Opti-MEM medium.

[0427] For each conjugate, mix 1 portion of the 3B solution with the 3A solution of each siRNA conjugate evenly and incubate at room temperature for 10 min to obtain the transfection complex 3Xa of each siRNA conjugate.

[0428] In the culture wells, add 100 μL of the above DMEM medium containing 2% FBS (containing Huh7.5.1 cells and hepatitis A virus) respectively, add the transfection complex 3Xa of each siRNA conjugate respectively, mix evenly, and the addition amount is 20 μL / well to obtain transfection complexes with the final concentration of each siRNA conjugate being approximately 50 nM (calculated based on the amount of siRNA in the siRNA conjugate). The transfection complex 3Xa of each siRNA conjugate is transfected into 3 culture wells. For Conjugate 1 - Conjugate 4, obtain transfection mixtures containing siRNA conjugates, denoted as test groups. For Conjugate NC, obtain a transfection mixture containing Conjugate NC, denoted as the control group.

[0429] After transfecting the above transfection mixture in the culture wells for 6 hours, aspirate the culture medium in the culture wells, and add an additional 100 μL of fresh DMEM medium supplemented with 2% FBS to each well, and continue culturing for 72 hours.

[0430] Use a fully automatic nucleic acid extractor (model: NP-GeneRotex 96) to extract the total RNA from the cells in each well. According to the method of Experimental Example 1, test the relative copy number of HAV virus. The difference is that the transfection mixture containing the conjugate NC is used as a control group for normalization, and the relative copy number is calculated.

[0431] (3-2) Inhibitory activity against H2 strain

[0432] According to the method of (3-1) above, the relative copy number of HAV virus of the live attenuated hepatitis A vaccine H2 strain in Huh7.5.1 human hepatoma cells was tested. The difference is that the 18F strain was replaced with the live attenuated hepatitis A vaccine H2 strain, which was preserved by the Academy of Military Sciences of the Chinese People's Liberation Army, passaged with 2BS cells, and the virus copy number measured by qPCR was 1×10 8 GE / ml. Take 10 μL of the P2 generation virus solution of the hepatitis A virus H2 strain and add it to 90 μL of DMEM medium containing 2% FBS, mix well to obtain 100 μL of DMEM medium containing the HAV virus H2 strain.

[0433] Figure 3 is a bar graph showing the relative copy numbers of HAV viruses of different strains after transfecting the siRNA conjugate or the control conjugate of the present disclosure at a concentration of 50 nM in Huh7.5.1 human hepatoma cells. From Figure 3 the results, it can be seen that at a concentration of 50 nM, the inhibition rates of conjugate 1, conjugate 2, conjugate 3, or conjugate 4 on the relative RNA copy number of the 18F strain in Huh7.5.1 human hepatoma cells are all greater than 84%, and the relative inhibition rates on the H2 strain are all greater than 60%. Compared with the NC group, each siRNA conjugate shows good inhibitory activity against both strains.

[0434] Experimental Example 4 This experimental example illustrates the inhibitory activity of siRNA and its conjugate at different time points in vitro in Huh7.5.1 human hepatoma cells.

[0435] Use Lipofectamine RNAiMAX (Invitrogen) to transfect Huh7.5.1 cells. Add 4.0×10 5Huh7.5.1 human hepatoma cells were seeded into 96-well plates, cultured overnight, and transfected. The final concentration of the siRNA conjugate was 5 nM, and the siRNA conjugates were Conjugate 1, Conjugate 2, Conjugate 3, Conjugate 4, or Conjugate NC, respectively.

[0436] Take 10 μL of HM175 / 18f-Nluc hepatitis A reporter virus (1×10 8 GE / ml) solution and add it to 90 μL of DMEM medium containing 2% FBS, and mix well. Among them, HM175 / 18f-Nluc (recombinant hepatitis A virus labeled with Nluc) was obtained according to the method described by Krishnamurthy Konduru et al., Hepatitis A virus (HAV) packaging size limit. Virology Journal volume 6, Article number: 204 (2009), with the only difference being that the luciferase Nluc gene ( luciferase) was used to replace the Blasticidin (Bsd) gene.

[0437] According to the method of Experimental Example 1, add 100 μL of DMEM medium containing 2% FBS (containing 10 μL of HM175 / 18f-Nluc hepatitis A reporter virus) to each well of one portion and continue culturing. Samples were taken and stored at -80 °C on the 1st, 2nd, 3rd, and 4th days after transfection (sampling every 24 hours).

[0438] For each conjugate, the co-transfected cells were washed once with PBS and operated according to the instructions of the luciferase detection kit, and detected with a 96-well microplate luminometer to obtain the luminescence values (RLU values) of each group of cells. A growth curve was made as Figure 4 shown.

[0439] Figure 4 It is a line graph showing the proliferation degree of HAV virus in Huh7.5.1 human hepatoma cells from day 0 to day 4 after transfection with the siRNA conjugate or the control conjugate of the present disclosure, respectively. Further, according to the proliferation degree of HAV virus in each group of cells, the relative inhibition rate of each conjugate from day 1 to day 4 relative to the NC group was calculated and recorded in Table 4:

[0440] Table 4 Relative inhibition rates of Conjugates 1-4

[0441] Number of days Conjugate 1 Conjugate 2 Conjugate 3 Conjugate 4 Day 1 18.70％ 39.80％ 29.40％ 32.70％ Day 2 80.90％ 77.80％ 79.90％ 70.60％ Day 3 77.50％ 73.80％ 78.30％ 72.00％ Day 4 86.40％ 88.80％ 86.10％ 89.90％

[0442] Among them, the relative inhibition rate of the conjugate = (RLU value (NC) - RLU value (test conjugate)) / RLU value (NC) × 100%.

[0443] According to the results in Table 4, compared with the negative control group NC, starting from the second day, Conjugate 1, Conjugate 2, Conjugate 3 or Conjugate 4 all showed a relative inhibition rate greater than 70%; the relative inhibition rate on the fourth day reached more than 86%, showing a good and stable inhibitory effect on HAV virus. Further, on the fourth day, the conjugate of the present disclosure was able to reduce the HAV fluorescence value in the cells to the level before transfection (i.e., on the 0th day), showing excellent therapeutic effects.

[0444] Experimental Example 5 This experimental example illustrates the in vivo activity test of siRNA and its siRNA conjugate

[0445] This experimental example investigated the activity of Conjugate 3 in mice under the condition of the same concentration and different administration methods. The Conjugate 3 prepared in Preparation Example 3 was dissolved in PBS to a solution of 1 mg / ml (calculated based on siRNA). Then the following treatments were carried out:

[0446] (1) The C57BL / 6 mice lacking the type I interferon receptor (female, about 25 g in weight, 6 - 8 weeks old, purchased from Cyagen Biosciences Inc.) were randomly divided into 3 groups, with 9 mice in each group, and numbered respectively. All mice were infected by tail vein injection with 2x10 7 The HM175 / 18f strain of GE.

[0447] (2) By the method of tail vein injection in mice, the above siRNA Conjugate 3 solution was respectively administered to the 9 mice in the first group. The weight was measured and recorded before administration, and the drug was administered according to the weight. The single - dose volume was 9 μL / g of mouse body weight. It can be calculated that the single - dose was 9 mg / kg, as Test Group 1.

[0448] By the method of subcutaneous injection in the nape of the neck in mice, the above siRNA Conjugate 3 solution was respectively administered to the 9 mice in the second group. The weight was measured and recorded before administration, and the drug was administered according to the weight. The single - dose volume was 9 μL / g of mouse body weight. It can be calculated that the single - dose was 9 mg / kg, as Test Group 2.

[0449] By the method of tail vein injection in mice, PBS was respectively administered to the 9 mice in the third group, and the single - dose volume was 9 μL / g, as the blank control group.

[0450] (3) Calculate with the administration time point as the first day. Collect mouse feces on the 7th and 14th days after administration. Stir the fecal samples and PBS evenly at a ratio of 1 g:4 ml. Centrifuge the processed fecal samples at a speed of 20,000 for 20 minutes using a centrifuge. After centrifugation, transfer 200 μL of the supernatant to a new centrifuge tube.

[0451] Use a fully automatic nucleic acid extractor (model: NP-GeneRotex 96) to extract the total RNA from the virus particles in the supernatant.

[0452] Reverse transcribe using the same method as in Experimental Example 1 to obtain the Ct values of the target HAV reverse transcribed DNA in each test group, and quantitatively calculate the copy number of HAV virus RNA in the fecal samples of each test group. The results are shown in Table 5.

[0453] Table 5 Average copy number of HAV virus RNA in mouse feces

[0454] Number of days Blank control Intravenous injection Subcutaneous injection Day 7 <![CDATA[1×10 4.77 GE / ml]]> <![CDATA[1×10 4.58 GE / ml]]> <![CDATA[1×10 4.05 GE / ml]]> Day 14 <![CDATA[1×10 5.47 GE / ml]]> <![CDATA[1×10 3.94 GE / ml]]> <![CDATA[1×10 4.26 GE / ml]]>

[0455] As can be seen from the results in Table 5, on the 7th day after administering the siRNA conjugate 3 of the present disclosure, the average copy number of HAV virus in the fecal samples of the blank control group is 1×10 4.77 GE / ml, the average copy number of HAV virus in the fecal samples of the intravenous injection group is 1×10 4.58 GE / ml, the average copy number of HAV virus in the fecal samples of the subcutaneous injection group is 1×10 4.05 GE / ml. Compared with the blank control, it shows that conjugate 3 has an inhibitory effect on the HAV virus. Further, on the 14th day after administering the siRNA conjugate 3 of the present disclosure, the average copy number of HAV virus in the fecal samples of the blank control group is 1×10 5.47 GE / ml, the average copy number of HAV virus in the fecal samples of the intravenous injection 1 is 1×10 3.94 GE / ml, the average copy number of HAV virus in the fecal samples of the subcutaneous injection group is 1×10 4.26 GE / ml.

[0456] Using the average copy number of the blank control group as a control, calculate the inhibition rate of conjugate 3 on the HAV virus by different administration methods according to the following formula.

[0457] Relative inhibition rate of HAV virus in the test group = (1 - average copy number of HAV virus in the test group / average copy number of HAV virus in the blank control group) × 100%.

[0458] On the 14th day after administration of the siRNA conjugate 3 of the present disclosure, in the intravenous injection group, the average inhibition rate of conjugate 3 against HAV virus was 95.5%, and in the subcutaneous injection group, the average inhibition rate of conjugate 3 against HAV virus was 93.5%. Compared with the blank control, whether by intravenous injection or subcutaneous injection, conjugate 3 showed significant inhibitory effects on HAV virus.

[0459] Calculating with the administration time point as the 1st day, on the 21st day after administration, the liver tissues of each mouse were taken and stored with RNAlater. Total RNA in hepatocytes was extracted using a fully automatic nucleic acid extractor (model: NP-GeneRotex 96). Using uninfected mice as controls, the Ct values of viral RNA and GADPH were measured in the same method as in Experimental Example 1, and the relative copy number of viral RNA in the livers of mice in each group was calculated using the comparative Ct (ΔΔCt) method in Experimental Example 1. Taking the relative copy number of the blank control group as the control, the inhibition rate of different administration methods against HAV virus was calculated according to the following formula.

[0460] Relative inhibition rate of HAV virus in the test group = (1 - relative copy number of HAV virus in the test group / relative copy number of HAV virus in the blank control group) × 100%.

[0461] The results are shown in Table 7.

[0462] The primer information used is shown in Table 6.

[0463] Table 6 Primer information of HAV reverse transcription DNA and internal reference mouse gene GAPDH

[0464]

[0465] Table 7 Inhibition rate of HAV virus in liver cells of mice on the 21st day with different administration methods

[0466] Number of days Intravenous injection Subcutaneous injection Day 21 61.5％ 55.1％

[0467] As can be seen from the results in Table 7, on the 21st day after administration of the siRNA conjugate 3 of the present disclosure, compared with the blank control group, the inhibition rate of HAV virus in the intravenous injection group was 61.5%, and the inhibition rate of HAV virus in the subcutaneous injection group was 55.1%. This indicates that the conjugate 3 of the present disclosure has significant and long-lasting inhibitory effects on HAV virus.

[0468] The preferred embodiments of the present disclosure have been described in detail above. However, the present disclosure is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0469] It should be further noted that, in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present disclosure will not separately describe various possible combinations. In addition, any combination can be made among the various different embodiments of the present disclosure, as long as it does not violate the idea of the present disclosure, and it should also be regarded as the content disclosed by the present disclosure.

Claims

1. An siRNA, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein, The sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. Among them, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:1 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO:2 and has no more than 3 nucleotide differences: 5'-CCAGUUUGGGAAUUGACAZ a1 -3'(SEQ ID NO:1); 5'-Z a2 UGUCAAUUCCCAAACUGG-3'(SEQ ID NO:2), Wherein, Z a1 is A, and Z a2 is U. The nucleotide sequence I contains a nucleotide Z a1 corresponding to the position of Z a3 , and the nucleotide sequence II contains a nucleotide Z a2 corresponding to the position of Z a4 . The Z a4 is the first nucleotide at the 5'-end of the antisense strand; Alternatively, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:33 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO:34 and has no more than 3 nucleotide differences: 5'-GAUAUGUCUUGUGUUGAUZ b1 -3'(SEQ ID NO:33); 5'-Z b2 AUCAACACAAGACAUAUC-3'(SEQ ID NO:34), where Z b1 is U, and Z b2 is A, and the nucleotide sequence I contains a nucleotide Z b1 at a position corresponding to Z b3 , and the nucleotide sequence II contains a nucleotide Z b2 at a position corresponding to Z b4 , and the Z b4 is the first nucleotide at the 5'-end of the antisense strand; Alternatively, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:65 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO:66 and has no more than 3 nucleotide differences: 5'-GGUGGAACUUACUAUUCAZ c1 -3'(SEQ ID NO:65); 5'-Z c2 UGAAUAGUAAGUUCCACC-3'(SEQ ID NO:66), where Z c1 is A, Z c2 is U, and the nucleotide sequence I contains a nucleotide Z at a position corresponding to Z c1 , and the nucleotide sequence II contains a nucleotide Z at a position corresponding to Z c3 , and the Z c2 is the first nucleotide at the 5'-end of the antisense strand; c4 c4 ​​ Alternatively, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:97 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO:98 and has no more than 3 nucleotide differences: 5'-GGUACAACAGUUGAUUUAZ d1 -3'(SEQ ID NO:97); 5'-Z d2 UAAAUCAACUGUUGUACC-3' (SEQ ID NO:98), where Z d1 is A, Z d2 is U, and the nucleotide sequence I contains a nucleotide Z d1 at a position corresponding to Z d3 , and the nucleotide sequence II contains a nucleotide Z d2 at a position corresponding to Z d4 , and the Z d4 is the first nucleotide at the 5'-end of the antisense strand.

2. The siRNA according to claim 1, wherein, There are no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1, and / or there are no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2; Alternatively, there are no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:33, and / or there are no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:34; Alternatively, there are no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:65, and / or there are no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:66; Alternatively, there are no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:97, and / or there are no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:

98.

3. The siRNA according to claim 1 or 2, wherein, The nucleotide differences between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 include Z a4 the difference at the position, and Z a4 is selected from A, C or G; Alternatively, the differences between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 34 include the difference at position Z b4 and Z b4 is selected from U, G or C; Alternatively, the differences between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 66 include the difference at position Z c4 and Z c4 is selected from A, G or C; Alternatively, the differences between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 98 include the differences at position Z d4 and Z d4 is selected from A, G or C.

4. The siRNA according to any one of claims 1-3, wherein, The said Z a3 is a nucleotide complementary to Z a4 ; or the said Z b3 is a nucleotide complementary to Z b4 ; or the said Z c3 is a nucleotide complementary to Z c4 ; or the said Z d3 is a nucleotide complementary to Z d4 .

5. The siRNA according to any one of claims 1-4, wherein, The nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; the essentially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; the completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

6. The siRNA according to any one of claims 1-5, wherein, The sense strand and the antisense strand have the same or different lengths. The length of the sense strand is 19 - 23 nucleotides, and the length of the antisense strand is 19 - 26 nucleotides; and the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:3, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:4: 5'-CCAGUUUGGGAAUUGACAZ a3 -3'(SEQ ID NO:3); 5'-Z a4 UGUCAAUUCCCAAACUGG-3'(SEQ ID NO:4), Among them, the Z a4 is the first nucleotide at the 5' end of the antisense strand, and Z a3 is selected from A, U, G or C, and Z a4 is a nucleotide complementary to Z a3 ; Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:35, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:36: 5'-GAUAUGUCUUGUGUUGAUZ b3 -3'(SEQ ID NO:35); 5'-Z b4 AUCAACACAAGACAUAUC-3'(SEQ ID NO:36), Among them, the said Z b4 is the first nucleotide at the 5'-end of the antisense strand, and Z b3 is selected from A, U, G or C, and Z b4 is a nucleotide complementary to Z b3 ; Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:67, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:68: 5'-GGUGGAACUUACUAUUCA Z c3 -3'(SEQ ID NO:67); 5'-Z c4 UGAAUAGUAAGUUCCACC-3'(SEQ ID NO:68), Wherein, said Z c4 is the first nucleotide at the 5'-end of the antisense strand, and Z c3 is selected from A, U, G or C, and Z c4 is a nucleotide complementary to Z c3 ; Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:99, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:100: 5'-GGUACAACAGUUGAUUUAZ d3 -3'(SEQ ID NO:99); 5'-Z d4 UAAAUCAACUGUUGUACC-3' (SEQ ID NO:100), Among them, said Z d4 is the first nucleotide at the 5'-end of the antisense strand, and Z d3 is selected from A, U, G or C, and Z d4 is a nucleotide complementary to Z d3 ​ 7. The siRNA according to claim 6, wherein Z a3 is A, and Z a4 is U; Z b3 is U, and Z b4 is A; Z c3 is A, and Z c4 is U; Z d3 is A, and Z d4 is U.

8. The siRNA according to any one of claims 1-7, wherein, The sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. The lengths of nucleotide sequence III and nucleotide sequence IV are each independently 1 - 4 nucleotides. Nucleotide sequence III is linked to the 5'-end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3'-end of nucleotide sequence II. Nucleotide sequence III and nucleotide sequence IV have equal lengths and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; the completely reverse complementary means that there is no mismatch between the two nucleotide sequences. Nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence, and the second nucleotide sequence refers to the nucleotide sequence adjacent to the 5'-end of the nucleotide sequence represented by SEQ ID NO:1 or SEQ ID NO:33 or SEQ ID NO:65 or SEQ ID NO:97 in the HAV virus RNA and having the same length as nucleotide sequence IV.

9. The siRNA according to claim 8, wherein, The nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:1 and has no more than 3 nucleotide differences. Moreover, the lengths of nucleotide sequences III and IV are both 1 nucleotide, and the base of nucleotide sequence III is C; or the lengths of nucleotide sequences III and IV are both 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is AC; or the lengths of nucleotide sequences III and IV are both 3 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is UAC; or the lengths of nucleotide sequences III and IV are both 4 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is AUAC; Alternatively, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:33, with no more than 3 nucleotide differences, and the lengths of the nucleotide sequences III and IV are both 1 nucleotide, and the base of the nucleotide sequence III is A; or the lengths of the nucleotide sequences III and IV are both 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AA; or the lengths of the nucleotide sequences III and IV are both 3 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AAA; or the lengths of the nucleotide sequences III and IV are both 4 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is CAAA; Alternatively, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:65, with no more than 3 nucleotide differences, and the lengths of the nucleotide sequences III and IV are both 1 nucleotide, and the base of the nucleotide sequence III is A; or the lengths of the nucleotide sequences III and IV are both 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GA; or the lengths of the nucleotide sequences III and IV are both 3 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AGA; or the lengths of the nucleotide sequences III and IV are both 4 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is UAGA; Alternatively, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO:97, with no more than 3 nucleotide differences, and the lengths of the nucleotide sequences III and IV are both 1 nucleotide, and the base of the nucleotide sequence III is U; or the lengths of the nucleotide sequences III and IV are both 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AU; or the lengths of the nucleotide sequences III and IV are both 3 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GAU; or the lengths of the nucleotide sequences III and IV are both 4 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is UGAU.

10. The siRNA according to any one of claims 1-9, wherein, The antisense strand further contains a nucleotide sequence V, the length of the nucleotide sequence V is 1 to 3 nucleotides, which is connected to the 3'-end of the antisense strand to form a 3'-overhang of the antisense strand; and / or the sense strand further contains a nucleotide sequence VI, the length of the nucleotide sequence VI is 1 to 3 nucleotides, which is connected to the 3'-end of the sense strand to form a 3'-overhang of the sense strand.

11. The siRNA according to claim 10, wherein, The length of the nucleotide sequence V and / or the nucleotide sequence VI is 2 nucleotides.

12. The siRNA according to claim 10 or 11, wherein, The nucleotide sequence V and / or VI is two consecutive thymidine deoxyribonucleotides or two consecutive uridine ribonucleotides; or the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the HAV virus RNA, and / or the nucleotide sequence VI is the same as the nucleotide at the corresponding position of the HAV virus RNA.

13. The siRNA according to any one of claims 1-12, wherein, The sense strand of the siRNA contains the nucleotides shown in SEQ ID NO:5, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:6: 5'-CCAGUUUGGGAAUUGACAZ a3 -3'(SEQ ID NO:5); 5'-Z a4 UGUCAAUUCCCAAACUGGGU-3'(SEQ ID NO:6); wherein, said Z a4 is the first nucleotide at the 5'-end of the antisense strand, and Z a3 is selected from A, U, G or C, and Z a4 is a nucleotide complementary to Z a3 ; Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:7, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:8: 5'-ACCCAGUUUGGGAAUUGACAZ a3 -3'(SEQ ID NO:7); 5'-Z a4 UGUCAAUUCCCAAACUGGGUAU-3' (SEQ ID NO:8); Among them, the Z a4 is the first nucleotide at the 5'-end of the antisense strand, and Z a3 is selected from A, U, G or C, and Z a4 is a nucleotide complementary to Z a3 ; Alternatively, the sense strand of the siRNA contains the nucleotides shown in SEQ ID NO:37, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:38: 5'-GAUAUGUCUUGUGUUGAUZ b3 -3'(SEQ ID NO:37); 5'-Z b4 AUCAACACAAGACAUAUCUU-3'(SEQ ID NO:38); wherein, said Z b4 is the first nucleotide at the 5'-end of the antisense strand, and Z b3 is selected from A, U, G or C, and Z b4 is a nucleotide complementary to Z b3 ; Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:39, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:40: 5'-AAGAUAUGUCUUGUGUUGAUZ b3 -3'(SEQ ID NO:39); 5'-Z b4 AUCAACACAAGACAUAUCUUUG-3' (SEQ ID NO:40); Among them, the said Z b4 is the first nucleotide at the 5'-end of the antisense strand, and Z b3 is selected from A, U, G or C, and Z b4 is the nucleotide complementary to Z b3 ; Alternatively, the sense strand of the siRNA contains the nucleotides shown in SEQ ID NO:69, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:70: 5'-GGUGGAACUUACUAUUCAZ c3 -3'(SEQ ID NO:69); 5'-Z c4 UGAAUAGUAAGUUCCACCUC-3'(SEQ ID NO:70); Among them, the Z c4 is the first nucleotide at the 5'-end of the antisense strand, and Z c3 is selected from A, U, G or C, and Z c4 is a nucleotide complementary to Z c3 ; Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:71, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:72: 5'-GAGGUGGAACUUACUAUUCAZ c3 -3'(SEQ ID NO:71); 5'-Z c4 UGAAUAGUAAGUUCCACCUCUA-3'(SEQ ID NO:72); wherein, said Z c4 is the first nucleotide at the 5'-end of the antisense strand, and Z c3 is selected from A, U, G or C, and Z c4 is a nucleotide complementary to Z c3 ; Alternatively, the sense strand of the siRNA contains the nucleotides shown in SEQ ID NO:101, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:102: 5'-GGUACAACAGUUGAUUUAZ d3 -3'(SEQ ID NO:101); 5'-Z d4 UAAAUCAACUGUUGUACCAU-3' (SEQ ID NO:102); Among them, the Z d4 is the first nucleotide at the 5'-end of the antisense strand, and Z d3 is selected from A, U, G or C, and Z c4 is a nucleotide complementary to Z d3 ; Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:103, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:104: 5'-AUGGUACAACAGUUGAUUUAZ d3 -3'(SEQ ID NO:103); 5'-Z d4 UAAAUCAACUGUUGUACCAUCA-3' (SEQ ID NO:104); Wherein, said Z d4 is the first nucleotide at the 5'-end of the antisense strand, and Z d3 is selected from A, U, G or C, and Z d4 is a nucleotide complementary to Z d3 ​ 14. The siRNA according to any one of claims 1-13, wherein, The siRNA is one of siHAVa1, siHAVa2, siHAVa3, siHAVb1, siHAVb2, siHAVb3, siHAVc1, siHAVc2, siHAVc3, siHAVd1, siHAVd2, and siHAVd3.

15. The siRNA according to any one of claims 1-14, wherein, At least one nucleotide in the sense strand and the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group with a modifying group.

16. The siRNA according to any one of claims 1-15, wherein, Each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

17. The siRNA according to claim 16, wherein, The fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and, in the direction from the 5'-end to the 3'-end, at least the 7th, 8th, and 9th nucleotides of nucleotide sequence I are fluorinated modified nucleotides; in the direction from the 5'-end to the 3'-end, at least the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence II are fluorinated modified nucleotides.

18. The siRNA according to any one of claims 1-17, wherein, The siRNA is one of siHAVa1-M1, siHAVa2-M1, siHAVa3-M1, siHAVb1-M1, siHAVb2-M1, siHAVb3-M1, siHAVc1-M1, siHAVc2-M1, siHAVc3-M1, siHAVd1-M1, siHAVd2-M1 and siHAVd3-M1.

19. The siRNA according to claim 15, wherein, The phosphoryl group with a modifying group is a phosphorothioate subunit formed by substituting at least one oxygen atom in the phosphodiester bond of the phosphoryl group with a sulfur atom.

20. The siRNA according to any one of claims 1-19, wherein The siRNA is one of siHAVa1-M1S, siHAVa1-M1X, siHAVa2-M1S, siHAVa2-M1X, siHAVa3-M1S, siHAVa3-M1X, siHAVb1-M1S, siHAVb1-M1X, siHAVb2-M1S, siHAVb2-M1X, siHAVb3-M1S, siHAVb3-M1X, siHAVc1-M1S, siHAVc1-M1X, siHAVc2-M1S, siHAVc2-M1X, siHAVc3-M1S, siHAVc3-M1X, siHAVd1-M1S, siHAVd1-M1X, siHAVd2-M1S, siHAVd2-M1X, siHAVd3-M1S and siHAVd3-M1X.

21. The siRNA according to any one of claims 1-20, wherein, The nucleotide at the 5'-end of the antisense strand is a 5'-phosphonucleotide or a nucleotide modified with a 5'-phosphate analog.

22. The siRNA according to claim 21, wherein, The siRNA is one of siHAVa1-M1P1, siHAVa2-M1P1, siHAVa3-M1P1, siHAVa1-M1SP1, siHAVa2-M1SP1, siHAVa3-M1SP1, siHAVa1-M1XP1, siHAVa2-M1XP1, siHAVa3-M1XP1, siHAVb1-M1P1, siHAVb2-M1P1, siHAVb3-M1P1, siHAVb1-M1SP1, siHAVb2-M1SP1, siHAVb3-M1SP1, siHAVb1-M1XP1, siHAVb2-M1XP1, siHAVb3-M1XP1, siHAVc1-M1P1, siHAVc2-M1P1, siHAVc3-M1P1, siHAVc1-M1SP1, siHAVc2-M1SP1, siHAVc3-M1SP1, siHAVc1-M1XP1, siHAVc2-M1XP1, siHAVc3-M1XP1, siHAVd1-M1P1, siHAVd2-M1P1, siHAVd3-M1P1, siHAVd1-M1SP1, siHAVd2-M1SP1, siHAVd3-M1SP1, siHAVd1-M1XP1, siHAVd2-M1XP1 and siHAVd3-M1XP1.

23. An siRNA conjugate, the siRNA conjugate contains the siRNA described in any one of claims 1-22 and a conjugating group conjugated to the siRNA, the conjugating group includes a linker and a pharmaceutically acceptable targeting group, and the siRNA, the linker and the targeting group are covalently or non-covalently connected in sequence, and each of the targeting groups is selected from ligands capable of binding to cell surface receptors.

24. The siRNA conjugate according to claim 23, wherein, The siRNA conjugate has the structure shown in formula (403): Where Nu represents the siRNA or any one of siRNA1, siRNA2, siRNA3 and siRNA4, the connecting position of the conjugating group is the 3'-position of the ribose of the nucleotide at the 3'-end of the sense strand in the siRNA, and the siRNA conjugate is in the form of sodium salt.

25. A pharmaceutical composition, the pharmaceutical composition contains the siRNA described in any one of claims 1-22 and / or the siRNA conjugate described in any one of claims 23-24, and a pharmaceutically acceptable carrier.

26. The pharmaceutical composition according to claim 25, wherein, The weight ratio of the siRNA and / or siRNA conjugate to the pharmaceutically acceptable carrier is 1:(1-500).

27. The pharmaceutical composition according to claim 26, wherein, The weight ratio of the siRNA and / or siRNA conjugate to the pharmaceutically acceptable carrier is 1:(1-50).

28. Use of the siRNA according to any one of claims 1 - 22 and / or the siRNA conjugate according to any one of claims 23 - 24 and / or the pharmaceutical composition according to any one of claims 25 - 27 in the preparation of a medicament for treating a pathological condition or disease caused by hepatitis A virus.

29. A method for inhibiting HAV viral RNA in a cell, the method comprising contacting the cell with an effective amount of the siRNA according to any one of claims 1 - 22 and / or the siRNA conjugate according to any one of claims 23 - 24 or the pharmaceutical composition according to any one of claims 25 - 27.

30. A kit, the kit comprising the siRNA according to any one of claims 1 - 22 and / or the siRNA conjugate according to any one of claims 23 - 24 or the pharmaceutical composition according to any one of claims 25 - 27.

Images