WO2024255748A1 - NUCLEIC ACID TARGETING HYDROXYSTEROID 17-β DEHYDROGENASE 13 AND USE THEREOF - Google Patents

Abstract

The present invention relates to the technical field of biology, and discloses a nucleic acid targeting hydroxysteroid 17-β dehydrogenase 13 and a use thereof. The provided nucleic acid can effectively inhibit the expression of hydroxysteroid 17-β dehydrogenase 13, and can thus treat and/or prevent diseases associated with hydroxysteroid 17-β dehydrogenase 13 activation.

Description

Nucleic acid targeting 17-β-hydroxysteroid dehydrogenase 13 and use thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to Chinese patent application No. 202310718422.1 filed on June 16, 2023, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates to the field of biotechnology, in particular to a nucleic acid targeting 17-β-hydroxysteroid dehydrogenase 13 and a use thereof.

Background Art

17-β-Hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) is an enzyme expressed by the HSD17B13 (also known as FLDP, HMFN0376, NIIL497, SCDR9, SDR16C3) gene. HSD17B13 belongs to the HSD17 protein family, which has 15 enzymes that mainly regulate fatty acid metabolism, cholesterol synthesis, bile synthesis and sex hormone activation. HSD17B13 is mainly expressed specifically in hepatocytes and is confined to the surface of lipid droplets (MacParland, SA et al., Nat. Commun. 2018, 9: 4383). In patients with nonalcoholic fatty liver disease (NAFLD), the expression of HSD17B13 is significantly increased (Su W et al., Proc. Natl. Acad. Sci. USA 2014, 111: 11437–42.). In GWAS studies, multiple HSD17B13 gene variants were also found to be associated with NAFLD (Anstee QM et al., J. Hepatol. 2020, 73: 505–515), and a HSD17B13 loss-of-function variant (rs72613567:TA) can reduce the risk of NAFLD (Abul-Husn NS et al., N. Engl. J. Med. 2018, 378: 1096–1106). In animal models, excessive expression of HSD17B13 is associated with NAFLD. The expression of HSD17B13 will significantly increase the number and size of lipid droplets (Su W et al., Proc. Natl. Acad. Sci. USA 2014, 111: 11437–42.). Currently, the number of NAFLD patients is increasing significantly, and the number of cases of nonalcholic steatohepatitis (NASH) is also increasing. However, there is currently no effective drug to treat it. Targeting HSD17B13 may provide a potential means of treating NAFLD.

RNA interference (RNAi) refers to the highly conserved phenomenon in the evolutionary process, which is induced by double-stranded small interfering RNA (siRNA) to efficiently and specifically degrade homologous mRNA. RNAi drugs also have the advantage of having a longer duration of efficacy than antibodies. Therefore, it is of great significance to study and develop siRNA targeting HSD17B13.

Summary of the invention

The purpose of the present invention is to overcome the problems existing in the prior art and provide a new nucleic acid targeting HSD17B13 and its use.

The first aspect of the present invention provides a nucleic acid, which includes a sense chain and an antisense chain, wherein the sense chain contains a sequence having more than 80% sequence identity with the sequence shown in any one of SEQ ID NOs: 1 to 80, and the antisense chain contains a sequence having more than 80% sequence identity with the sequence shown in any one of SEQ ID NOs: 81 to 160.

A second aspect of the present invention provides a targeted drug delivery system, which comprises a targeting group, a linking group, and the nucleic acid as described above connected to the targeting group via the linking group.

The third aspect of the present invention provides a pharmaceutical composition, which contains the nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

The fourth aspect of the present invention provides a method for inhibiting the expression of 17-β-hydroxysteroid dehydrogenase 13 gene in a cell, the method comprising: contacting the cell with the nucleic acid, targeted drug delivery system or pharmaceutical composition as described above to inhibit the 17-β-hydroxysteroid dehydrogenase in the cell. Expression of 13 genes.

The fifth aspect of the present invention provides the use of the nucleic acid, targeted drug delivery system or pharmaceutical composition as described above in any of the following aspects: 1) treating and/or preventing diseases associated with 17-β-hydroxysteroid dehydrogenase 13; 2) preparing drugs for treating and/or preventing diseases associated with 17-β-hydroxysteroid dehydrogenase 13.

The nucleic acid of the present invention can effectively reduce the level of HSD17B13, and can effectively prevent or treat related diseases, such as non-alcoholic fatty liver disease.

DETAILED DESCRIPTION

The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.

Explanation of terms

Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

The terms "and/or", "or/and", and "and/or" used herein include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and/or", "or/and", and "and/or", it should be understood that in this application, the technical solution undoubtedly includes all technical solutions connected by "logical AND". The technical scheme also undoubtedly includes the technical schemes that are all connected by "logical OR". For example, "A and/or B" includes three parallel schemes of A, B and A+B. For another example, the technical scheme of "A, and/or, B, and/or, C, and/or, D" includes any one of A, B, C, and D (that is, the technical schemes that are all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four items of A, B, C, and D (that is, the technical schemes that are all connected by "logical AND").

As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.

In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

In the present invention, "optionally", "optional", "optionally", "optionally", "optional", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel schemes of "yes" or "no". If multiple "optional" or "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.

In the present invention, the term "nucleic acid" refers to a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules, which can degrade or inhibit (e.g., degrade or inhibit under appropriate conditions) the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. The nucleic acid can act through an RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex or RISC)), or through any alternative mechanism or approach. The scope of the nucleic acids disclosed herein including sense and antisense strands includes, but is not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and dicer substrates.

As used herein, the terms "silencing", "reducing", "inhibiting", "downregulating" or "knockdown" when referring to the expression of a given gene, mean that the expression of the gene is reduced when the cell, cell population, tissue, organ or subject is treated with a nucleic acid described herein, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from mRNA in a cell, cell population, tissue, organ or subject in which the gene is transcribed, compared to a second cell, cell population, tissue, organ or subject not so treated.

In the present invention, "fully complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, all (100%) bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may include all or part of the first nucleotide sequence or the second nucleotide sequence.

In the present invention, "partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 70% but not all of the bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may include all or part of the first nucleotide sequence or the second nucleotide sequence.

In the present invention, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 85% but not all of the bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may include the first nucleotide sequence or all or part of a second nucleotide sequence.

In the present invention, "at least partially complementary" means that the first oligonucleotide and the second oligonucleotide are partially complementary, substantially complementary or completely complementary in a hybridization pair of nucleobase or nucleotide sequence molecules.

In the present invention, the term "treatment" means a method or step taken to provide relief or reduction in the number, severity and/or frequency of one or more disease symptoms in a subject. The treatment may include prevention, management, prophylactic treatment, and/or inhibition or reduction of the number, severity and/or frequency of one or more disease symptoms in a subject.

In the present invention, the term "connection" means that two compounds or molecules are joined by a covalent bond. Unless otherwise specified, as used herein, the term "connection" may refer to a connection between a first compound and a second compound with or without any intermediate atoms or atomic groups.

Nucleic Acids

The present invention provides a (modified or unmodified) nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand contains 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%) of the sequence shown in any one of SEQ ID NOs: 1 to 80. , 99%) or more sequence identity, the antisense strand contains a sequence having 80% (e.g. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) or more sequence identity with the sequence shown in any one of SEQ ID NOs: 81 to 160.

In some embodiments, the antisense strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).

In some embodiments, the sense strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).

In specific implementation, those skilled in the art can consider the complementarity of the sense strand and the antisense strand. The sequences provided in the present invention are combined to obtain a combined nucleic acid (siRNA).

In a preferred embodiment of the present invention, as shown in Table 1, the nucleic acid is selected from at least one of siRNA-1 whose sense chain sequence is SEQ ID NO: 1 and whose antisense chain sequence is SEQ ID NO: 81, siRNA-2 whose sense chain sequence is SEQ ID NO: 2 and whose antisense chain sequence is SEQ ID NO: 82, siRNA-3 whose sense chain sequence is SEQ ID NO: 3 and whose antisense chain sequence is SEQ ID NO: 83, siRNA-4 whose sense chain sequence is SEQ ID NO: 4 and whose antisense chain sequence is SEQ ID NO: 84, siRNA-5 whose sense chain sequence is SEQ ID NO: 5 and whose antisense chain sequence is SEQ ID NO: 85..., siRNA-78, siRNA-79, and siRNA-80.

In some preferred embodiments, the antisense strand contains at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from the sequence shown in any one of SEQ ID NOs: 81 to 160 by no more than 0, 1, 2 or 3 nucleotides, and the positive strand contains a nucleotide sequence that is at least partially complementary (such as partially complementary, substantially complementary or completely complementary) to the antisense strand.

In some preferred embodiments, the positive strand contains at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from the sequence shown in any one of SEQ ID NOs: 1 to 80 by no more than 0, 1, 2 or 3 nucleotides.

In the present invention, the sense strand and the antisense strand may have the same length or different lengths.

All nucleotide groups in the above nucleic acid may be chemically unmodified, or may contain at least one modified nucleotide group, and the modification may be on the nucleotide at any position.

In some embodiments, the sense and antisense strands may be partially complementary, substantially complementary, or fully complementary to each other.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from any one of SEQ ID NOs: 89, 100, 106, 109, 135, 136, 149, 150, 152, 153, 154, 156, 157, 158, 159, 160 by 0, 1 or 2 nucleotides, and the forward strand contains a nucleotide sequence that is at least partially complementary to the antisense strand. When the sequence was expressed, double-stranded RNA had a significantly better inhibitory effect on HSD17B13.

In some embodiments, the positive strand contains a nucleotide sequence that differs by 0, 1 or 2 nucleotides from the sequence shown in any one of SEQ ID NO: 9, 20, 26, 29, 55, 56, 69, 70, 72, 73, 74, 76, 77, 78, 79, 80.

In some embodiments, the antisense strand contains a nucleotide sequence that differs by 0, 1 or 2 nucleotides from the sequence shown in any one of SEQ ID NOs: 109, 135, 136, 149, 150, 152, 153, 154, 156, 157, 158, and 159, and the sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand. When the antisense strand has this sequence, the inhibitory effect of the double-stranded RNA with different modifications on HSD17B13 is further improved.

In some embodiments, the positive strand contains a nucleotide sequence that differs by 0, 1 or 2 nucleotides from the sequence shown in any one of SEQ ID NOs: 29, 55, 56, 69, 70, 72, 73, 74, 76, 77, 78, and 79.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 109 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 29 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 135 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 55 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 136 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 56 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 149 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 69 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains the sequence shown in SEQ ID NO: 150 The sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 70 by 0, 1 or 2 nucleotides. The sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 70 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 152 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 72 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 153 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 73 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 154 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 74 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 156 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 76 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 157 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 77 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 158 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 78 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 159 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 79 by 0, 1 or 2 nucleotides.

In some embodiments, when the sequence identity of the sense strand or antisense strand in the nucleic acid with the corresponding sequence mentioned in the present invention is less than 100% or differs by more than 1 nucleotide, it still has the same identity as the corresponding sequence mentioned in the present invention. The inhibitory effect of the corresponding sequence on HSD17B13 is similar or equivalent. For example, the UU connected to the 3' end of the antisense strand of the nucleic acid is replaced by AA, CC, GG or UG, etc., or a combination of any two nucleic acids. Such nucleic acid sequences also belong to the protection scope of the present invention.

The above-mentioned technical solution for naked sequence (i.e., unmodified sequence) mentioned in the present invention has an effect advantage that does not depend on the selection of modification method or targeting vector. The following is an introduction to the applicable modification scheme and further preferred modification scheme:

According to the nucleic acid of the present invention, the nucleic acid contains a nucleotide group as a basic structural unit, and the nucleotide group contains a phosphate group, a ribose group and a base. Preferably, the nucleic acid contains at least one modified nucleotide group. The inhibition efficiency of the modified nucleic acid on HSD17B13 is not less than 50% (such as 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).

According to the nucleic acid of the present invention, wherein the modified nucleotide group is a nucleotide group in which a phosphate group and/or a ribose group is modified. The modified site can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 of the 1st, 2nd, 3rd, 4th, 5th, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30th nucleotide of the sense strand and/or the antisense strand.

For example, modification of the phosphate group refers to modification of the oxygen in the phosphate group, including phosphorothioate modification and boranophosphate modification. As shown in the following formula, the oxygen in the phosphate group is replaced by sulfur, borane, amine, alkyl or alkoxy. These modifications can stabilize the structure of nucleic acids and maintain high specificity and high affinity of base pairing.

In the above structural formula, BASE represents the base A, U, C, G or T. X can be oxygen (O) or sulfur (S). R in the above structure can be the same or different, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, amino, cyanoethyl, acetyl, etc., R' and R" can each independently be hydrogen (H), methyl (CH3), ethyl (CH2CH3), propyl (CH2CH2CH3), isopropyl (CH(CH3)2), allyl, propargyl, acyloxybenzyl, acyloxyethyl.

The modification of the ribose group refers to the modification of the 2′-hydroxyl (2′-OH) in the ribose group. After introducing certain substituents such as methoxy or fluorine at the 2′-hydroxyl position of the ribose group, the nucleic acid is not easily cut by ribonucleases, thereby increasing the stability of the nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis. The modification of the 2′-hydroxyl in the pentose of nucleotides includes 2′-fluoro modification (2′-fluoromodification, such as 2′-arabino-fluoro modification), 2′-methoxy modification (2′-OME), 2′-methoxyethyl modification (2′-MOE), 2′-2,4-dinitrophenol modification (2′-DNP modification), cyclolocked ethyl modification (2′,4′-constrained ethyl modification), 2'-Amino modification, 2'-deoxy modification, BNA, acyclic nucleic acid modification, misplaced nucleic acid modification, L-type nucleic acid modification, etc. BNA (endocyclic bridged nucleotide) refers to a constrained or inaccessible nucleotide. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C 3'-endo sugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose ring to provide a 2', 4'-BNA nucleotide, such as locked ethyl modification (LNA), ring locked ethyl modification (ENA) and ethyl locked nucleic acid modification (cET BNA). Acyclic nucleic acids are nucleotides formed by opening the sugar ring of the nucleotide, such as unlocked nucleic acid (UNA) nucleotides and glycerol nucleic acid (GNA) nucleotides. Misplaced nucleic acid modification refers to the replacement of the 3', 5'-phosphate bond link by a 2', 5'-phosphate bond chain. L-type nucleic acid modification refers to the replacement of the naturally occurring D-type nucleic acid with its mirror stereo equivalent L-type nucleic acid.

Wherein, BASE represents the base A, U, C, G or T. R in the above structure can be the same or different, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, acetyl, etc.

According to the nucleic acid of the present invention, preferably, the nucleotide group in which the ribose group is modified is a nucleotide group in which the 2'-OH of the ribose group is substituted by a methoxy group or a fluorine group.

According to a particularly preferred embodiment of the present invention, the nucleotide group containing uracil base or cytosine base in the sense strand of the nucleic acid is a nucleotide group in which the ribose group is modified, that is, the nucleotide group containing uracil base or cytosine base in the sense strand of the nucleic acid The 2'-OH of the ribose group in the nucleic acid is replaced by a methoxy group or a fluorine group. More preferably, the 3' end of the sense strand and the antisense strand of the nucleic acid can be connected to dTdT; or, the 3' end of the antisense strand of the nucleic acid can be connected to AA or UU or a combination of any two nucleic acids (which can be but is not limited to CC, GG or UG), so that the sequence has a specific inducement to mRNA degradation. The nucleic acid with the above modifications shows a more excellent in vivo inhibitory effect, and the above modifications can further reduce the immunogenicity of the nucleic acid of the present invention in vivo.

The nucleic acid of the present invention may also include a modification of a monophosphate nucleoside connected to the 5' end of the antisense strand. Since the 5'-monophosphate at the terminal of the siRNA guide strand is important for RISC recognition. Among them, the phosphorylation of the 5'-hydroxyl group plays a certain role in whether the siRNA can be effectively loaded into the Ago2 inside the cell. The 5'-terminal monophosphate of the guide strand in the siRNA has an H bond interaction with Argonaute-2 (Ago2), thereby ensuring accurate positioning and precise cutting of the mRNA target. Commonly used 5'-monophosphate nucleoside derivatives are as follows. Such phosphate nucleoside derivatives have been proven to have certain stability in biological metabolic media and have a certain effect on promoting the loading of siRNA guide strands into Ago2 inside cells (Nucleic Acids Research, 2015, 43, 2993-3011). According to the nucleic acid of the present invention, wherein, preferably, trans-vinyl phosphate (VP) is the first choice, and monophosphate nucleoside derivatives other than those described above may also be included.

In the above structure, BASE represents the base A, U, C, G or T. R in the above structure can be The radicals may be the same or different, such as hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, amino, acetyl, etc.

In some embodiments, at least one nucleotide in the nucleic acid is a modified nucleotide or includes a modified linkage.

In some preferred embodiments, the modified nucleotide is selected from one or more of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-open ring nucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitols, reverse nucleotides, reverse 2'-O-methyl nucleotides, reverse 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, nucleotides containing vinyl phosphonate, nucleotides containing cyclopropyl phosphonate and 3'-O-methyl nucleotides; the modified nucleotide is further preferably selected from one or more of 2'-O-methyl nucleotides and 2'-fluoro nucleotides.

In some preferred embodiments, the modified inter-linkage is preferably selected from one or more of phosphorothioate inter-nucleotide linkages and methylphosphonate inter-nucleotide linkages; the modified inter-linkage is further preferably selected from one or more of phosphorothioate monoester inter-nucleotide linkages and phosphorothioate diester inter-nucleotide linkages.

In some embodiments, the antisense strand contains two phosphorothioate internucleotide bonds at the 5' end and the 3' end, respectively, and contains 4 to 6 2'-fluoro nucleotides, and the remaining nucleotides are 2'-O-methyl nucleotides.

In some embodiments, the 5' end of the sense strand contains two phosphorothioate internucleotide bonds and 2 to 4 2'-fluoro nucleotides, and the remaining nucleotides are all 2'-O-methyl nucleotides.

In some preferred embodiments, the antisense strand has 2'-fluoro nucleotides at the 2nd, 4th, 6th, 14th and 16th nucleotides counted from the 5' end.

In some preferred embodiments, the sense strand has 2'-fluoro nucleotides at the 7th, 9th and 11th nucleotides counted from the 5' end.

In some preferred embodiments, the antisense strand has at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from the antisense strand sequence of any one of Table 2 by no more than 0, 1, 2 or 3 nucleotides, and the sense strand has at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from the sense strand sequence of any one of Table 2 by no more than 0, 1, 2 or 3 nucleotides.

In some preferred embodiments, the sense strand and the antisense strand form a siRNA having any one of the sequences shown in Table 3.

In some preferred embodiments, the antisense strand contains a nucleotide sequence from the 5' end to the 3' end that differs from the following nucleotide sequence by 0, 1 or 2 nucleotides:

SN-5553: asUfsaGfaUfcucuucuCfuGfuugcusgsc;

SN-5564: asUfsaCfaGfgccuaaaUfcUfugugcsusu;

SN-5573:usAfsaGfuAfuuccaucUfaUfcagacsusu;

SN-5577: csAfsuAfuCfauuaucaUfgCfauacasusc;

SN-5606:usUfsaUfaCfagaagacAfgAfaucaasgsu;

SN-5607: asGfsuUfaUfacagaagAfcAfgaaucsusu;

SN-52389:asCfsuAfuUfcauaucaUfuAfucaugscsa;

SN-52391:usCfsaUfaAfuguaucuUfaUfaagacsusa;

SN-52393: asCfsaAfaGfaaaaaggUfuAfucaugsusu;

SN-52394: asUfsuAfcAfaagaaaaAfgGfuuaucsusu;

SN-52395: asUfsaUfaUfgaaagcaUfaAfauuacsusu;

SN-52397: asCfsaAfgAfuuagucuUfgAfugaugsusg;

SN-52398: asAfsaAfcAfagauuagUfcUfugaugsusa;

SN-52399: asCfsaCfaCfaaaacaaGfaUfuagucsusu;

SN-52400:usAfsuAfaUfacaugugAfaAfaacacsasc;

SN-52401:usCfsuAfuAfauacaugUfgAfaaaacsasc;

The sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand.

In some preferred embodiments, the sense strand contains a nucleotide sequence from the 5' end to the 3' end that differs by 0, 1 or 2 nucleotides from any of the following nucleotide sequences:

SN-2553: asgscaacAfgAfgAfagagaucuau;

SN-2564:gscsacaaGfaUfuAfuggccuguau;

SN-2573:gsuscugaUfaGfaUfggaauacuua;

SN-2577:usgsuaugCfaUfgAfuaaugauaug;

SN-2606:ususgauuCfuGfuCfuucuguuaa;

SN-2607: gsasuucuGfuCfuUfcuguauaacu;

SN-22389:csasugauAfaUfgAfuaugaauagu;

SN-22391: gsuscuuaUfaAfgAfuacauuauga;

SN-22393:csasugauAfaCfcUfuuuucuuugu;

SN-22394:gsasuaacCfuUfuUfucuuuguaau;

SN-22395:gsusaauuUfaUfgCfuuucauauau;

SN-22397: csusacauCfaAfgAfcuaaucuugu;

SN-22398: csasucaaGfaCfuAfaucuuguuuu;

SN-22399:gsascuaaUfcUfuGfuuuugugu;

SN-22400:gsusguuuUfuCfaCfauguauuaua;

SN-22401: gsusuuuuCfaCfaUfguauuauaga.

In each sequence of the present invention, a nucleotide represented by a lowercase letter represents that the nucleotide is 2'-O- Methyl nucleotide; f means that the adjacent nucleotide on its left is a 2'-fluoro nucleotide; s means that the two adjacent nucleotides on the left and right are connected by a phosphorothioate diester bond.

In some preferred embodiments, the antisense strand contains a nucleotide sequence from the 5' end to the 3' end that differs from the following nucleotide sequence by 0, 1 or 2 nucleotides:

SN-5577: csAfsuAfuCfauuaucaUfgCfauacasusc;

SN-5606:usUfsaUfaCfagaagacAfgAfaucaasgsu;

SN-5607: asGfsuUfaUfacagaagAfcAfgaaucsusu;

SN-52389: asCfsuAfuUfcauaucaUfuAfucaugscsa;

SN-52391:usCfsaUfaAfuguaucuUfaUfaagacsusa;

SN-52393: asCfsaAfaGfaaaaaggUfuAfucaugsusu;

SN-52394: asUfsuAfcAfaagaaaaAfgGfuuaucsusu;

SN-52395: asUfsaUfaUfgaaagcaUfaAfauuacsusu;

SN-52397: asCfsaAfgAfuuagucuUfgAfugaugsusg;

SN-52398: asAfsaAfcAfagauuagUfcUfugaugsusa;

SN-52399: asCfsaCfaCfaaaacaaGfaUfuagucsusu;

SN-52400:usAfsuAfaUfacaugugAfaAfaacacsasc;

The sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand.

In some preferred embodiments, the sense strand contains a nucleotide sequence from the 5' end to the 3' end that differs by 0, 1 or 2 nucleotides from any of the following nucleotide sequences:

SN-2577:usgsuaugCfaUfgAfuaaugauaug;

SN-2606:ususgauuCfuGfuCfuucuguuaa;

SN-2607: gsasuucuGfuCfuUfcuguauaacu;

SN-22389: csasugauAfaUfgAfuaugaauagu;

SN-22391: gsuscuuaUfaAfgAfuacauuauga;

SN-22393:csasugauAfaCfcUfuuuucuuugu;

SN-22394:gsasuaacCfuUfuUfucuuuguaau;

SN-22395:gsusaauuUfaUfgCfuuucauauau;

SN-22397: csusacauCfaAfgAfcuaaucuugu;

SN-22398: csasucaaGfaCfuAfaucuuguuuu;

SN-22399:gsascuaaUfcUfuGfuuuugugu;

SN-22400: gsusguuuUfuCfaCfauguauuaua.

In specific implementation, those skilled in the art can combine the sense and antisense strand sequences mentioned above in consideration of the complementarity of the sense and antisense strands. In a preferred embodiment, the sense and antisense strand sequences with the same last three digits are combined to obtain the corresponding nucleic acid (siRNA). For example, SN-5577 and SN-2577 are combined, and SN-52389 and SN-22389 are combined to obtain the corresponding nucleic acid (siRNA).

The nucleic acid according to the present invention can be obtained by conventional methods in the art, such as solid phase synthesis and liquid phase synthesis, and the solid phase synthesis has commercial custom services and can therefore be obtained commercially. The modified nucleotide group can be introduced by a nucleotide monomer having a corresponding modification.

Based on the nucleic acid (siRNA) synthesized as above, the present invention can further construct an shRNA expression plasmid having the same or similar function as the above siRNA. The method for constructing the expression plasmid is well known to those skilled in the art and will not be described in detail here.

The present invention also provides a targeted gene site of the nucleic acid as described above. In some embodiments, the targeted gene site is annotated as any one of the items in column 1 of Table 1.

Table 1

Note: The first column refers to the position of the first base of the targeted gene in the HSD17B13 gene sequence, and so on; the numbers in the third and fifth columns represent the sequence numbers, for example, "1" represents SEQ ID NO: 1.

Among them, the reference sequence of the targeted gene is the coding sequence of human HSD17B13 NM_178135.5

(SEQ ID NO: 161):

Targeted drug delivery system

The present invention also provides a targeted drug delivery system, characterized in that the targeted drug delivery system comprises a targeting group, a linking group and the above-mentioned of nucleic acid.

In combination with common sense in the art, the nucleic acid (siRNA) of the present invention has a better inhibitory effect when applied to different targeted drug delivery systems. In other words, the effect advantage of the naked sequence and the modified sequence in the present invention does not depend on the selection of the targeting carrier. In order to further improve the bioavailability and therapeutic effect of siRNA, the present invention also optimizes the targeted drug delivery system and obtains the following technical scheme.

In some embodiments, the linker is linked to the 3' end or the 5' end of the sense strand or the antisense strand of the nucleic acid.

In some embodiments, the linker is attached to the 3' end of the sense strand of the nucleic acid.

In some embodiments, the targeted drug delivery system comprises a ligand and the nucleic acid linked to the ligand.

In some embodiments, the ligand is a GalNAc derivative.

In some embodiments, the ligand is one or more GalNAc derivatives connected by single-stranded, double-stranded, or triple-stranded branched linkers.

In some preferred embodiments, the structure of the targeted drug delivery system is shown in Formula I below:

In formula I, Nu represents the nucleic acid (siRNA). In some embodiments, the compound part in the system can be connected to the 5' end or 3' end of the sense strand of the siRNA through a phosphodiester bond, and can also be connected to the 5' end or 3' end of the antisense strand of the siRNA through a phosphodiester bond. The targeted drug delivery system utilizes the structural characteristics on its left side to improve the cell penetration ability of the nucleic acid drug (Nu), enhance its stability in the cell, and has a simple preparation process and strong practicality.

Pharmaceutical composition

The present invention also provides a pharmaceutical composition, which contains the nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

The pharmaceutical composition can be prepared from the nucleic acid and the pharmaceutically acceptable carrier by conventional methods. For example, the pharmaceutical composition can be an injection. The injection can be used for subcutaneous, intramuscular or intravenous injection.

According to the pharmaceutical composition of the present invention, there is no special requirement for the amount of the nucleic acid or the targeted drug delivery system and the pharmaceutically acceptable carrier. Generally, relative to 1 part by weight of the nucleic acid (or 1 part by weight of the targeted drug delivery system calculated as nucleic acid), the content of the pharmaceutically acceptable carrier can be 1-100000 parts by weight (such as 1 part by weight, 5 parts by weight, 10 parts by weight, 50 parts by weight, 100 parts by weight, 500 parts by weight, 1000 parts by weight, 5000 parts by weight, 10000 parts by weight, 50000 parts by weight, 100000 parts by weight or any value between any two of the above values).

According to the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier may be various carriers conventionally used in the art, for example, it may include at least one of a pH buffer, a protective agent and an osmotic pressure regulator. The pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, preferably a phosphate buffer with a pH of 5.5-8.5. The protective agent may be at least one of inositol, sorbitol and sucrose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight (such as 0.01% by weight, 0.05% by weight, 0.1% by weight, 0.5% by weight, 1% by weight, 5% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight). The osmotic pressure regulator may be sodium chloride and/or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 mOsmole/kg. According to the desired osmotic pressure, those skilled in the art can determine the content of the osmotic pressure regulator.

According to a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is a liposome. The liposome can be any liposome capable of encapsulating nucleic acid, and its diameter can be 25-1000 nm, and can include but not limited to cholesterol and its analogs or derivatives.

The dosage of the pharmaceutical composition of the present invention can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially according to the age, weight and gender of the subject. For example, for female, 3-4 month old mice weighing 25-30 g, based on the amount of the nucleic acid in the pharmaceutical composition, the dosage of the pharmaceutical composition can be 0.01-100 mg/kg body weight, preferably 1-10 mg/kg body weight.

Methods and uses

The present invention also provides a method for inhibiting the expression of 17-β-hydroxysteroid dehydrogenase 13 gene in a cell, the method comprising: contacting the cell with the nucleic acid, targeted drug delivery system or pharmaceutical composition as described above to inhibit the expression of 17-β-hydroxysteroid dehydrogenase 13 gene in the cell.

In some embodiments, the cell is in a subject, e.g., a human subject, e.g., a subject suffering from a 17-β-hydroxysteroid dehydrogenase 13-associated disease.

In some embodiments, the cell is in vitro. The method is for research purposes or for constructing an animal model.

In some embodiments, contacting the cell with the nucleic acid inhibits expression of HSD17B13 by at least 50%, 60%, 70%, 80%, 90%, 95% (e.g., compared to the expression level of HSD17B13 before the cell was first contacted with the nucleic acid; e.g., before the first dose of the nucleic acid was administered to the subject). In certain embodiments, inhibiting expression of HSD17B13 reduces the level of HSD17B13 protein in a serum sample of the subject by at least 50%, 60%, 70%, 80%, 90%, 95%. 80%, 90% or 95%, for example, compared to the expression level of HSD17B13 before the cell is first contacted with the nucleic acid.

The present invention also provides the use of the nucleic acid as described above, the targeted drug delivery system as described above, or the pharmaceutical composition as described above in any of the following aspects: 1) treating and/or preventing diseases associated with 17-β-hydroxysteroid dehydrogenase 13; 2) preparing drugs for treating and/or preventing diseases associated with 17-β-hydroxysteroid dehydrogenase 13.

In some embodiments, the disease is: (i) a disease associated with increased or elevated levels of 17-β-hydroxysteroid dehydrogenase 13; or (ii) a disease that would benefit from decreased expression of 17-β-hydroxysteroid dehydrogenase 13.

In some embodiments, the disease is selected from one or more of the following: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, alcoholic steatohepatitis (ASH), alcoholic fatty liver disease (ALD), cirrhosis, hepatitis, hepatitis C virus (HCV)-related cirrhosis, drug-induced liver injury, and hepatocellular necrosis.

In the present invention, the subject may be a mammal, including primates (such as humans, non-human primates, such as monkeys and chimpanzees), non-primates (such as cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats or mice) or birds. In some embodiments, the subject is preferably a primate, more preferably a human. Administration can be by a variety of routes, depending on whether local or systemic treatment is required. The mode of administration can be, but is not limited to, intravenous administration, intra-arterial administration, subcutaneous administration, intraperitoneal administration, transdermal administration (such as by implantation of a device) and administration into soft tissue. The dosage can refer to the above, and will not be repeated here.

In some embodiments, the nucleic acid, the targeted drug delivery system or the pharmaceutical composition is administered to the subject by subcutaneous administration, intravenous administration and/or intramuscular administration.

Example

The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions should be referred to the instructions given in the present invention first, and can also be used according to the knowledge of the art. The experimental manual or routine conditions may also refer to other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

Example 1 In vitro screening of siRNA

After screening to obtain the sense strand and antisense strand sequences shown in Table 1, each sense strand and antisense strand was modified, and the modified sequences are shown in Table 2.

Table 2

In the table, a/c/g/u = 2'-OMe nucleotides; Af/Cf/Gf/Uf = 2'-F nucleotides; s = phosphorothioate diester bond.

The sense strand and antisense strand in Table 2 were synthesized into the modified double-stranded siRNA in Table 3 by solid phase synthesis.

Table 3

In the table, a/c/g/u = 2'-OMe nucleotides; Af/Cf/Gf/Uf = 2'-F nucleotides; s = phosphorothioate diester bond.

The small interfering nucleic acids (siRNAs) in Table 3 and SN-252704 as a comparison were applied to HEK293T cells transfected with HSD17B13/luciferase plasmids to detect the knockdown effect on HSD17B13 expression. Among them, SN-252704 as a comparison was prepared according to the public information of US11180757B1, and the sense chain sequence (5'-3') was AUGCUUUUGCAUGGACUAUCU (SEQ ID NO: 162), which was modified to asusgcuuUfuGfCfAfuggacuaucu, and the antisense chain sequence (5'-3') was AGAUAGUCCAUGCAAAAGCAUUC (SEQ ID NO: 163), which was modified to asGfsauaguccaugcAfaAfagcaususc. The letters in each modified sequence have the same meaning as described in Table 3.

The specific process of the experiment is as follows: 50 microliters of cell culture medium (DMEM, 10% calf serum) containing 25,000 HEK293T cells were added to the 96-well cell culture dish coated with gelatin, and siRNA transfection was performed after incubation overnight. 20X sample working solution was prepared in Opti-MEM, and plasmids containing human HSD17B13 gene and luciferase-renilla fragment were prepared in Opti-MEM to make the final concentration of plasmid 5ng/mL. 0.5mL Lipofectamine was added to 24.5mL Opti-MEM. 12mL of 20X sample working solution was taken and mixed with 48mL plasmid and Opti-MEM, and then 60mL Lipofectamine mixture was added and incubated at room temperature for 30 minutes. 50mL of the above mixture was added to each well of HEK293T cells incubated overnight and incubated for 5 hours. Then the culture medium was aspirated and replaced with fresh cell culture medium (DMEM, 10% calf serum) and incubated for 43 hours. 75mL Glo-Luciferase was added to each well, incubated at room temperature for 15 minutes, and the luminesence value was read at 590nm. After reading, 75mL of stop solution was added and incubated for 15 minutes, and the renilla fluorescence value was read in the full band. The ratio of renilla fluorescence value to firefly fluorescence value was used to calculate the effect of sample silencing HSD17B13 expression. The results are shown in Table 4.

Table 4. Knockdown effect of siRNA on HSD17B13 expression in HEK293T cells transfected with HSD17B13/luciferase plasmid

Example 2 siRNA effect verification

In order to further verify the activity of siRNA compounds, human and monkey homologous siRNA (Table 5) was screened in the above HEK293T cell system at 8 doses (0.001, 0.011, 0.033, 0.06, 0.11, 0.33, 1.1, 3.3 nM).

Table 5

In the table, a/c/g/u = 2'-OMe nucleotides; Af/Cf/Gf/Uf = 2'-F nucleotides; s = phosphorothioate diester bond.

The obtained doses required for 50% knockdown of HSD17B13 (IC ₅₀ ) are shown in Table 6.

Table 6 IC ₅₀ values of siRNA inhibiting HSD17B13 expression

From the results, it can be seen that SN-25577, SN-252397, SN-252398, SN-252400, SN-25606, SN-25607, SN-252389, SN-252391, SN-252393, SN-252394, SN-252395, and SN-252399 have significantly better knockdown effects on HSD17B13 than other groups. At the same time, it is also significantly better than the currently known dominant sequence SN-252704. It can be seen that the effect of the modified sequence in the present invention is significantly better than the existing dominant sequence, and the above advantages do not depend on the selection of the targeting vector.

In addition, coupling the above-mentioned dominant group of siRNAs with Tri-GalNAc (its specific structure is shown in the aforementioned formula I, and the sequences involved are shown in Table 7) can further enhance the liver targeting of siRNA drugs.

Table 7

In the table, a/c/g/u = 2'-OMe nucleotides; Af/Cf/Gf/Uf = 2'-F nucleotides; s = phosphorothioate diester bond.

When the siRNA drugs shown in Table 7 were tested for efficacy in human HSD17B13 transgenic mice, they still had significant efficacy advantages. The siRNA drugs of the present invention can achieve better efficacy at a lower dose.

In addition, by comparing the silencing effect of unmodified siRNA on HSD17B13 gene expression in cells, it can be confirmed that the siRNA of the present invention has obvious effect advantages at the naked sequence level, and its effect in silencing the HSD17B13 gene is significantly better than the existing advantageous sequences.

The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. Those skilled in the art may make several modifications and improvements without departing from the concept of the present invention, all of which fall within the protection scope of the present invention.

Claims (20)

A nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand contains a sequence having more than 80% sequence identity with a sequence shown in any one of SEQ ID NOs: 1 to 80, and the antisense strand contains a sequence having more than 80% sequence identity with a sequence shown in any one of SEQ ID NOs: 81 to 160. The nucleic acid according to claim 1, wherein the antisense strand contains at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the sequence shown in any one of SEQ ID NOs: 81 to 160, and the sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand; optionally, the sense strand contains at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the sequence shown in any one of SEQ ID NOs: 1 to 80. The nucleic acid according to claim 1, wherein the antisense strand contains a nucleotide sequence that differs by 0, 1 or 2 nucleotides from the sequence shown in any one of SEQ ID NOs: 89, 100, 106, 109, 135, 136, 149, 150, 152, 153, 154, 156, 157, 158, 159, 160, and the positive strand contains a nucleotide sequence that is at least partially complementary to the antisense strand; optionally, the positive strand contains a nucleotide sequence that differs by 0, 1 or 2 nucleotides from the sequence shown in any one of SEQ ID NOs: 9, 20, 26, 29, 55, 56, 69, 70, 72, 73, 74, 76, 77, 78, 79, 80. The nucleic acid according to claim 3, wherein the antisense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 109, 135, 136, 149, 150, 152, 153, 154, 156, 157, 158, 159 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand; optionally, the sense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 29, 55, 56, 69, 70, 72, 73, 74, 76, 77, 78, 79 by 0, 1 or 2 nucleotides. The nucleic acid of claim 4, wherein the antisense strand comprises a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 109 by 0, 1 or 2 nucleotides, and the sense strand comprises a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 29 by 0, 1 or 2 nucleotides; Or, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 135 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 55 by 0, 1 or 2 nucleotides; Or, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 136 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 56 by 0, 1 or 2 nucleotides; Or, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 149 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 69 by 0, 1 or 2 nucleotides; Or, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 150 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 70 by 0, 1 or 2 nucleotides; Or, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 152 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 72 by 0, 1 or 2 nucleotides; Or, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 153 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 73 by 0, 1 or 2 nucleotides; Or, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 154 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 74 by 0, 1 or 2 nucleotides; Or, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 156 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 76 by 0, 1 or 2 nucleotides; Or, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 157 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 77 by 1, 2 or 3 nucleotides. Nucleotide sequences differing by 0, 1, or 2 nucleotides; Or, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 158 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 78 by 0, 1 or 2 nucleotides; Alternatively, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 159 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 79 by 0, 1 or 2 nucleotides. The nucleic acid according to claim 1, wherein at least one nucleotide in the nucleic acid is a modified nucleotide or comprises a modified internucleotide; The modified nucleotide is preferably selected from one or more of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-open ring nucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitols, reverse nucleotides, reverse 2'-O-methyl nucleotides, reverse 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, vinyl phosphonate containing nucleotides, cyclopropyl phosphonate containing nucleotides and 3'-O-methyl nucleotides; the modified nucleotide is further preferably selected from one or more of 2'-O-methyl nucleotides and 2'-fluoro nucleotides; The modified internucleotide bond is preferably selected from one or more of phosphorothioate internucleotide bonds and methylphosphonate internucleotide bonds; the modified internucleotide bond is further preferably selected from one or more of phosphorothioate monoester internucleotide bonds and phosphorothioate diester internucleotide bonds. The nucleic acid according to claim 6, wherein the antisense strand contains 2 phosphorothioate internucleotide bonds at the 5' end and the 3' end, respectively, and contains 4 to 6 2'-fluoro nucleotides, and the remaining nucleotides are 2'-O-methyl nucleotides; the 5' end of the sense strand contains 2 phosphorothioate internucleotide bonds and contains 2 to 4 2'-fluoro nucleotides, and the remaining nucleotides are 2'-O-methyl nucleotides; preferably, the 2nd, 4th, 6th, 14th and 16th nucleotides of the antisense strand counting from the 5' end are 2'-fluoro nucleotides, and the 7th, 9th and 11th nucleotides of the sense strand counting from the 5' end are 2'-fluoro nucleotides. The nucleic acid according to claim 1, wherein the antisense strand has any of the sequences in Table 2. The antisense strand sequence shown in one embodiment differs by no more than 3 nucleotides for at least 15 consecutive nucleotides, and the sense strand has a sense strand sequence as shown in any one of Table 2 that differs by no more than 3 nucleotides for at least 15 consecutive nucleotides; preferably, the sense strand and the antisense strand form an siRNA as shown in any one of Table 3. The nucleic acid according to claim 1, wherein the antisense strand contains a nucleotide sequence from the 5' end to the 3' end that differs from the following nucleotide sequence by 0, 1 or 2 nucleotides:
SN-5553: asUfsaGfaUfcucuucuCfuGfuugcusgsc;
SN-5564: asUfsaCfaGfgccuaaaUfcUfugugcsusu;
SN-5573:usAfsaGfuAfuuccaucUfaUfcagacsusu;
SN-5577: csAfsuAfuCfauuaucaUfgCfauacasusc;
SN-5606:usUfsaUfaCfagaagacAfgAfaucaasgsu;
SN-5607: asGfsuUfaUfacagaagAfcAfgaaucsusu;
SN-52389:asCfsuAfuUfcauaucaUfuAfucaugscsa;
SN-52391:usCfsaUfaAfuguaucuUfaUfaagacsusa;
SN-52393: asCfsaAfaGfaaaaaggUfuAfucaugsusu;
SN-52394: asUfsuAfcAfaagaaaaAfgGfuuaucsusu;
SN-52395: asUfsaUfaUfgaaagcaUfaAfauuacsusu;
SN-52397: asCfsaAfgAfuuagucuUfgAfugaugsusg;
SN-52398: asAfsaAfcAfagauuagUfcUfugaugsusa;
SN-52399: asCfsaCfaCfaaaacaaGfaUfuagucsusu;
SN-52400:usAfsuAfaUfacaugugAfaAfaacacsasc;
SN-52401:usCfsuAfuAfauacaugUfgAfaaaacsasc; The sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand; Preferably, the sense strand contains a nucleotide sequence from the 5' end to the 3' end that differs by 0, 1 or 2 nucleotides from any of the following nucleotide sequences:
SN-2553: asgscaacAfgAfgAfagagaucuau;
SN-2564:gscsacaaGfaUfuAfuggccuguau;
SN-2573:gsuscugaUfaGfaUfggaauacuua;
SN-2577:usgsuaugCfaUfgAfuaaugauaug;
SN-2606:ususgauuCfuGfuCfuucuguuaa;
SN-2607: gsasuucuGfuCfuUfcuguauaacu;
SN-22389: csasugauAfaUfgAfuaugaauagu;
SN-22391: gsuscuuaUfaAfgAfuacauuauga;
SN-22393: csasugauAfaCfcUfuuuucuuugu;
SN-22394:gsasuaacCfuUfuUfucuuuguaau;
SN-22395:gsusaauuUfaUfgCfuuucauauau;
SN-22397:csusacauCfaAfgAfcuaaucuugu;
SN-22398: csasucaaGfaCfuAfaucuuguuuu;
SN-22399:gsascuaaUfcUfuGfuuuugugu;
SN-22400:gsusguuuUfuCfaCfauguauuaua;
SN-22401: gsusuuuuCfaCfaUfguauuauaga; In each sequence, a nucleotide represented by a lowercase letter indicates that the nucleotide is a 2'-O-methyl nucleotide; f indicates that the nucleotide adjacent to its left is a 2'-fluoro nucleotide; and s indicates that the two adjacent nucleotides on the left and right are connected by a phosphorothioate diester bond. The nucleic acid according to claim 1, wherein the antisense strand contains a nucleotide sequence from the 5' end to the 3' end that differs from the following nucleotide sequence by 0, 1 or 2 nucleotides:
SN-5577: csAfsuAfuCfauuaucaUfgCfauacasusc;
SN-5606:usUfsaUfaCfagaagacAfgAfaucaasgsu;
SN-5607: asGfsuUfaUfacagaagAfcAfgaaucsusu;
SN-52389:asCfsuAfuUfcauaucaUfuAfucaugscsa;
SN-52391:usCfsaUfaAfuguaucuUfaUfaagacsusa;
SN-52393: asCfsaAfaGfaaaaaggUfuAfucaugsusu;
SN-52394: asUfsuAfcAfaagaaaaAfgGfuuaucsusu;
SN-52395: asUfsaUfaUfgaaagcaUfaAfauuacsusu;
SN-52397: asCfsaAfgAfuuagucuUfgAfugaugsusg;
SN-52398: asAfsaAfcAfagauuagUfcUfugaugsusa;
SN-52399: asCfsaCfaCfaaaacaaGfaUfuagucsusu;
SN-52400:usAfsuAfaUfacaugugAfaAfaacacsasc; The sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand; Preferably, the sense strand contains a nucleotide sequence from the 5' end to the 3' end that differs by 0, 1 or 2 nucleotides from any of the following nucleotide sequences:
SN-2577:usgsuaugCfaUfgAfuaaugauaug;
SN-2606:ususgauuCfuGfuCfuucuguuaa;
SN-2607: gsasuucuGfuCfuUfcuguauaacu;
SN-22389:csasugauAfaUfgAfuaugaauagu;
SN-22391: gsuscuuaUfaAfgAfuacauuauga;
SN-22393:csasugauAfaCfcUfuuuucuuugu;
SN-22394:gsasuaacCfuUfuUfucuuuguaau;
SN-22395:gsusaauuUfaUfgCfuuucauauau;
SN-22397: csusacauCfaAfgAfcuaaucuugu;
SN-22398: csasucaaGfaCfuAfaucuuguuuu;
SN-22399:gsascuaaUfcUfuGfuuuugugu;
SN-22400:gsusguuuUfuCfaCfauguauuaua; In each sequence, a nucleotide represented by a lowercase letter indicates that the nucleotide is a 2'-O-methyl nucleotide; f indicates that the nucleotide adjacent to its left is a 2'-fluoro nucleotide; and s indicates that the two adjacent nucleotides on the left and right are connected by a phosphorothioate diester bond. A targeted drug delivery system, characterized in that the targeted drug delivery system comprises a targeting group, a linking group and the nucleic acid according to any one of claims 1 to 10 connected to the targeting group via the linking group. The targeted drug delivery system according to claim 11, wherein the linking group is connected to the 3' end or the 5' end of the sense strand or the antisense strand of the nucleic acid. The targeted drug delivery system according to claim 11 or 12, wherein the targeted drug delivery system comprises a ligand and the nucleic acid connected to the ligand; preferably the ligand is a GalNAc derivative; more preferably the ligand is one or more GalNAc derivatives connected by single-stranded, double-stranded or triple-stranded branched linkers. The targeted drug delivery system according to claim 11, wherein the structure of the targeted drug delivery system is shown as follows:
In the formula, Nu represents the nucleic acid. A pharmaceutical composition, characterized in that the pharmaceutical composition contains the nucleic acid according to any one of claims 1 to 10 or the targeted drug delivery system according to any one of claims 11 to 14 and a pharmaceutically acceptable carrier. A method for inhibiting the expression of 17-β-hydroxysteroid dehydrogenase 13 gene in a cell, the method comprising: contacting the cell with the nucleic acid described in any one of claims 1 to 10, the targeted drug delivery system described in any one of claims 11 to 14, or the pharmaceutical composition described in claim 15, so as to inhibit the expression of 17-β-hydroxysteroid dehydrogenase 13 gene in the cell. Use of the nucleic acid according to any one of claims 1 to 10, the targeted drug delivery system according to any one of claims 11 to 14, or the pharmaceutical composition according to claim 15 in any of the following aspects: 1) Treating and/or preventing diseases related to 17-β-hydroxysteroid dehydrogenase 13; 2) Preparation of drugs for treating and/or preventing diseases associated with 17-β-hydroxysteroid dehydrogenase 13. The use according to claim 17, wherein the disease is: (i) a disease associated with increased or elevated levels of 17-β-hydroxysteroid dehydrogenase 13; or (ii) Diseases that would benefit from reduced expression of 17-β-hydroxysteroid dehydrogenase 13. The use according to claim 17, wherein the disease is selected from the following: Several: nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, liver fibrosis, alcoholic steatohepatitis, alcoholic fatty liver disease, cirrhosis, hepatitis, hepatitis C virus-related cirrhosis, drug-induced liver injury, and hepatocellular necrosis. The use according to claim 17, wherein the nucleic acid, the targeted drug delivery system or the pharmaceutical composition is administered to the subject by subcutaneous administration, intravenous administration and/or intramuscular administration.