CN116042627A

CN116042627A - siRNA and conjugate thereof

Info

Publication number: CN116042627A
Application number: CN202310169084.0A
Authority: CN
Inventors: 唐春雷; 薛凡; 范为正; 姜虹羽; 袁昕
Original assignee: Sanorri Biomedical Technology Wuxi Co ltd
Current assignee: Sanorri Biomedical Technology Wuxi Co ltd
Priority date: 2023-02-27
Filing date: 2023-02-27
Publication date: 2023-05-02

Abstract

The present invention provides an siRNA and conjugates thereof. The siRNA and the conjugate thereof and the pharmaceutical composition containing the siRNA and the conjugate can specifically target the liver, so that the expression of the dipeptidyl peptidase 4 gene can be inhibited, and the treatment of diseases and/or symptoms (particularly metabolic diseases) related to the expression of the dipeptidyl peptidase 4 can be realized.

Description

siRNA and conjugate thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to siRNA for inhibiting dipeptidyl peptidase 4 (DPP 4) gene expression and a conjugate thereof, a pharmaceutical composition containing the siRNA or the conjugate thereof, and siRNA conjugates, as well as a preparation method and application of the siRNA and the conjugate thereof and the pharmaceutical composition.

Background

Dipeptidyl peptidase 4 (DPP 4), also known as T cell surface antigen 26 (CD 26), was first found in 1966 as a cell surface serine protease that cleaves amino-terminal dipeptides from the N-terminal L-proline or L-alanine of polypeptides.

DPP4 exists in two forms in vivo, can be used as a cell membrane binding receptor, is widely expressed on the surfaces of various tissues and cells of a human body, such as liver, lung, lymph node, vascular endothelial cells, T lymphocytes and the like, and can be used as a soluble enzyme protein (solubledipeptidyl peptidase-4, sDPP 4) to fall off and exist in body fluid so as to maintain the enzyme activity. Although the source of sDPP4 is not completely understood, studies have shown that lymphocytes may be the main source of sDPP4, DPP4 also shows its biological effects through two different mechanisms of action, on the one hand DPP4 can function as a dipeptidyl peptidase 4 hydrolysing polypeptide and thus modulate the activity of peptide hormones, neuropeptides, cytokines and growth factors. For example, the peptide hormones insulin-like polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) promote insulin synthesis and secretion by binding to specific receptors on islet beta cells, inhibit glucagon secretion, and increase the sensitivity of the body to insulin. And DPP4 can degrade GIP and GLP-1, so DPP4 inhibitors such as sitagliptin and the like are clinically applied to inhibit DPP4 activity, thereby achieving the purpose of controlling blood sugar. In addition to GLP-1 and GIP, DPP4 has various substrates including chemokine ligand 5 (CCL 5), interferon inducible protein 10 (CXCL 10), neuropeptide NPY and the like, so DPP4 is involved in various pathophysiological processes such as organism metabolism, cellular immunity, tumor suppression and the like.

DPP4 has also been shown to have non-glycemic effects by controlling the progression of inflammation, which is probably mediated more by direct protein-protein interactions than catalytic activity in the case of non-alcoholic fatty liver disease (NAFLD), obesity and type 2 diabetes (T2D). Failure to address inflammation that leads to chronic subclinical activation of the immune system may affect the development of metabolic disorders. In addition, elevated circulating DPP4 activity and elevated soluble plasma DPP4 levels are found in many metabolic diseases including diabetes, obesity, cardiovascular diseases and non-alcoholic fatty liver diseases. Thus, DPP4 exhibits a variety of effects on the progression of metabolic diseases through its cleavage and regulation of the biological activity of peptide hormones and its effect on inflammation.

Heretofore, various DPP-4 inhibitors have been marketed worldwide including sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, gemagliptin, tigliptin, and the like. However, DPP-4 inhibitors suffer from problems such as selectivity, long-lasting, safety and tolerability. With the continued intensive research on the DPP family, an enzyme family having DPP-4-like activity was discovered, and these enzymes mainly include Fibroblast Active Protein (FAP), DPP-2, DPP-8 and DPP-9, and the physiological functions and actions of these enzymes are not yet clear. If DPP-4 inhibitors affect the function of these enzymes, new problems and risks may arise. Despite the efforts in this area of research, current treatments do not fully meet patient needs and additional treatments suitable for most affected patient populations are highly desirable.

One study reported in Nature shows that a high fat diet induces obesity in mice, which stimulates hepatocyte synthesis and secretion of dipeptidyl peptidase 4 (DPP 4); DPP4 works together with plasma factor Xa, resulting in Adipose Tissue Macrophage (ATM) inflammation; blocking DPP4 expression in hepatocytes inhibits Visceral Adipose Tissue (VAT) inflammation and insulin resistance, whereas the oral DPP4 inhibitor sitagliptin does not show similar effects; blocking the expression of either cellular protein-1 or PAR2 in ATM can also inhibit inflammation and insulin resistance, both of which mediate the effects of DPP4 and Xa, respectively; the target blocking of the secretion of DPP4 by liver cells can inhibit VAT inflammation and insulin resistance, and has the effect which cannot be achieved by oral DPP4 inhibitors.

Therefore, the RNAi mechanism is developed to selectively and effectively default the liver DPP4 gene, so that the RNAi mechanism has application value and clinical significance.

Disclosure of Invention

The siRNA and the modified sequence thereof provided by the invention can specifically inhibit the gene expression of the dipeptidyl peptidase 4 (DPP 4), and the pharmaceutical composition or the siRNA conjugate containing the siRNA can specifically target the liver, so that the gene expression of the dipeptidyl peptidase can be inhibited, and the treatment of diseases and/or symptoms (especially metabolic diseases) related to the gene expression of the dipeptidyl peptidase 4 is realized.

In one aspect, the invention provides an 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 comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, which are at least partially complementary in opposite phase to form a double-stranded region, the nucleotide sequence I and the nucleotide sequence II being selected from the group of sequences set forth in (I) - (vi):

(i) The nucleotide sequence I and SEQ ID NO:1, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:2, and optionally having no more than 3 nucleotide differences:

5’-CUUAUAGACUGAAGUUAUAUU-3’(SEQ ID NO：1)

5’-UAUAACUUCAGUCUAUAAGUA-3’(SEQ ID NO：2)

(ii) The nucleotide sequence I and SEQ ID NO:3, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:4, and optionally having no more than 3 nucleotide differences:

5’-GCAACUUCCAUACAAAUCAUU-3’(SEQ ID NO：3)

5’-UGAUUUGUAUGGAAGUUGCAU-3’(SEQ ID NO：4)

(iii) The nucleotide sequence I and SEQ ID NO:5, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:6, and optionally having no more than 3 nucleotide differences:

5’-GUUACAGACACAUUUGUUAUU-3’(SEQ ID NO：5)

5’-UAGCAAAUGUGUCUGUAACCU-3’(SEQ ID NO：6)

(iv) The nucleotide sequence I and SEQ ID NO:7, and optionally with NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:8, and optionally having no more than 3 nucleotide differences:

5’-CGGAAAGGUGUCAGUACUAUU-3’(SEQ ID NO：7)

5’-UAGUACUGACACCUUUCCGGA-3’(SEQ ID NO：8)

(v) The nucleotide sequence I and SEQ ID NO:9, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:10, and optionally having no more than 3 nucleotide differences:

5’-CAGUAAAGAGGCGAAGUAUUU-3’(SEQ ID NO：9)

5’-AUACUUCGCCUCUUUACUGAA-3’(SEQ ID NO：10)

(vi) The nucleotide sequence I and SEQ ID NO:11, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:12, and optionally having no more than 3 nucleotide differences:

5’-CGUGUUCAAGUGUGGAAUAUU-3’(SEQ ID NO：11)

5’-UAUUCCACACUUGAACACGCC-3’(SEQ ID NO：12)。

in another aspect, the present invention provides an siRNA conjugate comprising the siRNA described above and a conjugate group conjugated to the siRNA.

In yet another aspect, the invention provides a pharmaceutical composition comprising an siRNA of the invention and/or an siRNA conjugate as described above, and optionally a pharmaceutically acceptable carrier.

In a further aspect, the invention provides the use of said siRNA and/or siRNA conjugate and/or pharmaceutical composition for the manufacture of a medicament for the treatment of a disease and/or condition caused by dipeptidyl peptidase 4 gene expression.

In a further aspect, the invention provides the use of said siRNA and/or said siRNA conjugate in the manufacture of a medicament for inhibiting the expression of a dipeptidyl peptidase 4 gene in a cell, particularly a hepatocyte.

In yet another aspect, the present invention provides a method of treating a disease and/or disorder caused by dipeptidyl peptidase 4 gene expression, comprising administering to a subject in need thereof an effective amount of the siRNA and/or siRNA conjugate and/or pharmaceutical composition of the invention.

In another aspect, the invention provides a method of inhibiting dipeptidyl peptidase 4 gene expression comprising contacting an effective amount of the siRNA and/or siRNA conjugate and/or pharmaceutical composition of the invention with a cell, particularly a hepatocyte.

Advantageous effects

The siRNA, the siRNA conjugate and the pharmaceutical composition provided by the invention have good stability, higher inhibition activity of the mRNA of the dipeptidyl peptidase 4, lower off-target effect and/or can remarkably treat diseases and/or symptoms related to the expression of the dipeptidyl peptidase 4.

In some embodiments, the siRNA, siRNA conjugates, and pharmaceutical compositions provided herein exhibit excellent target gene inhibition activity in vitro experiments. In some embodiments, the siRNA, siRNA conjugates, and pharmaceutical compositions provided herein exhibit a target gene expression inhibition rate of at least 50%, 60%, 70%, 80%, 90%, or 95% in hepatocytes.

The siRNA, siRNA conjugates and pharmaceutical compositions provided by the invention do not show significant off-target effects. The off-target effect may be, for example, inhibition of normal gene expression of non-target genes. It is believed that the off-target effect is not significant if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20% or 10% compared to the effect at the target gene.

Therefore, the siRNA conjugate and the pharmaceutical composition provided by the invention can inhibit the expression of the dipeptidyl peptidase 4 gene, effectively treat diseases and/or symptoms related to the expression of the dipeptidyl peptidase 4, and have good application prospects.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Detailed Description

The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

In the present invention, DPP4 mRNA means mRNA having a sequence shown in Genbank accession No. NM-001935.4. Further, unless otherwise indicated, the term "target gene" as used herein refers to a gene expressing DPP4 mRNA, and the term "target mRNA" refers to the DPP4 mRNA described above.

Definition of the definition

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

The term "linked" as used herein, when referring to a link between two molecules, refers to the linking of the two molecules by covalent bonds or the association of the two molecules via non-covalent bonds (e.g., hydrogen or ionic bonds).

An "oligonucleotide" as used herein is a nucleotide sequence containing 10-50 nucleotides or nucleotide base pairs. In some embodiments of the invention, the oligonucleotide has a nucleobase sequence that is at least partially complementary to a coding sequence in a target gene expressed in a cell. The nucleotide may optionally be modified. In some embodiments of the invention, the oligonucleotide is capable of inhibiting or blocking expression of a gene in vitro or in vivo after delivery of the oligonucleotide to a cell expressing the gene.

According to the present invention, the term "dipeptidyl peptidase 4", which is used interchangeably with the term "DPP4", refers to an adenosine deaminase binding protein or cluster of differentiation 26 (CD 26), which is a serine exopeptidase capable of inactivating various oligopeptides and smaller peptides by removing the dipeptide from the N-terminus of the oligopeptides and smaller peptides, peptides having proline or alanine in the penultimate position.

Other examples of DPP4 mRNA sequences are readily available using public databases such as GenBank, uniProt and OMIM. As used herein, the term "DPP4" also refers to naturally occurring DNA sequence variants of the DPP4 genome.

The term "dipeptidyl peptidase 4 expression associated disease and/or disorder" or "DPP4 associated disease" as used herein is a disease or disorder caused by or associated with DPP4 expression. The term "DPP 4-related disease" includes diseases, disorders or conditions that benefit from reduced DPP4 gene expression or replication. Non-limiting examples of DPP 4-related diseases include, for example, diabetes.

The term "inhibit", when referring to the expression of a given gene, means that the expression of the gene is reduced when the cell, cell population or tissue is treated with the siRNA, siRNA conjugate and pharmaceutical composition of the invention, as compared to a cell, cell population or tissue that has not been so treated.

The term "inhibit" is used interchangeably with "reduce," "silence," "down-regulate," "inhibit," and other similar terms, and includes any level of inhibition. Preferably, inhibition comprises a statistically significant inhibition or a clinically significant inhibition.

As used herein, the phrase "inhibiting expression of DPP 4" or "inhibiting expression of DPP4 gene" includes inhibiting expression of any DPP4 gene (e.g., DPP4 gene expressed by DPP4 in DPP4, DPP4 gene expressed by an expression construct in a cell) as well as a variant or mutant of DPP4 gene encoding DPP4 protein. The term includes the knockdown of any DPP4 transcript encoding one or more DPP4 proteins, a variant or mutant of the DPP4 gene.

Each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide. In the context of the present invention, unless otherwise indicated, "conjugated" means that two or more chemical moieties each having a specific function are linked to each other by covalent linkage; accordingly, "conjugate" refers to a compound formed by covalent linkage between the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently attaching one or more chemical moieties having specific functions to an siRNA. Hereinafter, the siRNA conjugate of the present invention is also sometimes simply referred to as "conjugate". siRNA conjugates are to be understood as the generic term of siRNA conjugates, either the first or the second siRNA conjugate, or the siRNA sense strand conjugate or the siRNA antisense strand conjugate, depending on the context.

In some embodiments, the conjugate group may be attached to the phosphate group, the 2 '-hydroxyl group, the 5' -hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugate group may also be attached to the 3' -hydroxyl group, in which case the nucleotides are linked using a 2' -5' phosphodiester linkage. When a conjugate group is attached to the end of the siRNA strand, the conjugate group is typically attached to the phosphate group of the nucleotide; when a conjugate group is attached to the internal sequence of the siRNA, the conjugate group is typically attached to a ribose sugar ring or base. Various connection means can be referred to as: 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.

In some embodiments, the siRNA and the conjugate group may be linked by acid labile, or reducible, chemical bonds that degrade in the acidic environment of the intracellular inclusion bodies, thereby allowing the siRNA to be in a free state. For non-degradable conjugation, the conjugation group can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.

In the above and below, unless otherwise specified, "G", "C", "a", "T" and "U" each represent a nucleotide containing guanine, cytosine, adenine, thymine and uracil as bases. However, it is understood that the term "ribonucleotide" or "nucleotide" may also refer to modified nucleotides, nucleotide analogs (surrogate replacement moiety), as described in further detail below.

Wherein a, c, g and u are 2 '-O-methyladenosine-3' -phosphate, 2 '-O-methylcytidine-3' -phosphate, 2 '-O-methylguanosine-3' -phosphate and 2 '-O-methyluridine-3' -phosphate, respectively;

af. Cf, gf and Uf are 2 '-fluoroadenosine-3' -phosphate, 2 '-fluorocytidine-3' -phosphate, 2 '-fluoroguanosine-3' -phosphate and 2 '-fluorouridine-3' -phosphate, respectively;

dA. dC, dG and dT are 2 '-deoxyadenosine-3' -phosphate, 2 '-deoxycytidine-3' -phosphate, 2 '-deoxyguanosine-3' -phosphate and 2 '-deoxythymidine-3' -phosphate, respectively;

(Agn) is an adenosine-diol nucleic acid (GNA); (Cgn) is cytidine-diol nucleic acid (GNA); (Ggn) is guanosine-diol nucleic acid (GNA); (Tgn) is thymidine-diol nucleic acid (GNA); and s is a phosphorothioate chain.

In the context of the present invention, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meaning known to the person skilled in the art, i.e. in a double stranded nucleic acid molecule the bases of one strand are each paired with a base on the other strand in a complementary manner. In DNA, the purine base adenine (a) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary to each other, and the sequence of the strand can be deduced from the sequence of its complementary strand. Accordingly, "mismatch" means in the art that bases at corresponding positions do not exist in complementary pairs in a double-stranded nucleic acid.

In the above and in the following, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than 1 base mismatch between two nucleotide sequences; "complete reverse complement" means that there is no base mismatch between the two nucleotide sequences. In the above and below, the "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position is changed as compared with the former, for example, when one nucleotide base is A in the latter, when the corresponding nucleotide base 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 the position. In some embodiments, a nucleotide difference is also considered to occur at an original position when the nucleotide is replaced with an abasic nucleotide or its equivalent.

In the above and in the following, particularly in describing the methods of preparation of the siRNA, siRNA conjugates, siRNA and/or siRNA conjugate-containing pharmaceutical compositions of the invention, the nucleoside monomer (nucleoside monomer) refers to a modified or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites also referred to as Nucleoside phosphoramidites) used in phosphoramidite solid phase synthesis depending on the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared, unless otherwise specified. Phosphoramidite solid phase synthesis is a method well known to those skilled in the art for use in RNA synthesis. Nucleoside monomers useful in the present invention are commercially available.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted" alkyl "includes" alkyl "and" substituted alkyl "as defined below, as will be understood by those of skill in the art, for any group comprising one or more substituents, such groups are not intended to introduce any substitution or pattern that is sterically impractical, synthetically infeasible, and/or inherently unstable.

The term "subject" as used herein refers to any animal, such as a mammal or a pouched animal. Subjects of the invention include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, cows, rats, or any kind of poultry.

As used herein, "treatment" refers to a method of achieving a beneficial or desired result, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is obtained by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

In one aspect, the invention provides an 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 comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, which are at least partially complementary in antiphase to form a double-stranded region, the nucleotide sequence I and the nucleotide sequence II being selected from the group of sequences set forth in (I) - (vi):

5’-CUUAUAGACUGAAGUUAUAUU-3’(SEQ ID NO：1)

5’-UAUAACUUCAGUCUAUAAGUA-3’(SEQ ID NO：2)

5’-GCAACUUCCAUACAAAUCAUU-3’(SEQ ID NO：3)

5’-UGAUUUGUAUGGAAGUUGCAU-3’(SEQ ID NO：4)

5’-GUUACAGACACAUUUGUUAUU-3’(SEQ ID NO：5)

5’-UAGCAAAUGUGUCUGUAACCU-3’(SEQ ID NO：6)

5’-CGGAAAGGUGUCAGUACUAUU-3’(SEQ ID NO：7)

5’-UAGUACUGACACCUUUCCGGA-3’(SEQ ID NO：8)

5’-CAGUAAAGAGGCGAAGUAUUU-3’(SEQ ID NO：9)

5’-AUACUUCGCCUCUUUACUGAA-3’(SEQ ID NO：10)

5’-CGUGUUCAAGUGUGGAAUAUU-3’(SEQ ID NO：11)

5’-UAUUCCACACUUGAACACGCC-3’(SEQ ID NO：12)。

accordingly, the present invention provides the first to sixth siRNAs capable of inhibiting the expression of DPP4 gene. This will be described in detail in turn.

The siRNA of the present invention contains a nucleotide group as a basic structural unit, which is well known to those skilled in the art, and the nucleotide group contains a phosphate group, a ribose group and a base, and is not described herein.

First siRNA

According to the present invention, the siRNA may be a first siRNA.

The first siRNA comprises a sense strand and an antisense strand, each nucleotide in the first siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, which are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is complementary to SEQ ID NO:1, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:2, and optionally having no more than 3 nucleotide differences:

5’-CUUAUAGACUGAAGUUAUAUU-3’(SEQ ID NO:1)

5’-UAUAACUUCAGUCUAUAAGUA-3’(SEQ ID NO：2)

In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II. In some embodiments, the nucleotide sequence I hybridizes with SEQ ID NO:1, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:2 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in figure 2.

In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary; by substantially reverse complement is meant that there are no more than 3 base mismatches between the two nucleotide sequences; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity refers to the absence of a base mismatch between two nucleotide sequences.

Second siRNA

According to the present invention, the siRNA may be a second siRNA.

The second siRNA comprises a sense strand and an antisense strand, each nucleotide in the second siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, which are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is complementary to SEQ ID NO:3, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:4, and optionally having no more than 3 nucleotide differences:

5’-GCAACUUCCAUACAAAUCAUU-3’(SEQ ID NO：3)

5’-UGAUUUGUAUGGAAGUUGCAU-3’(SEQ ID NO：4)

In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II. In some embodiments, the nucleotide sequence I hybridizes with SEQ ID NO:3, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:4 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in figure 4.

In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary.

Third siRNA

According to the present invention, the siRNA may be a third siRNA.

The third siRNA comprises a sense strand and an antisense strand, each nucleotide in the third siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, which are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is complementary to SEQ ID NO:5, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:6, and optionally having no more than 3 nucleotide differences:

5’-GUUACAGACACAUUUGUUAUU-3’(SEQ ID NO：5)

5’-UAGCAAAUGUGUCUGUAACCU-3’(SEQ ID NO：6)

In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II. In some embodiments, the nucleotide sequence I hybridizes with SEQ ID NO:5, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:6 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in fig.

Fourth siRNA

According to the present invention, the siRNA may be a fourth siRNA.

The fourth siRNA comprises a sense strand and an antisense strand, each nucleotide in the fourth siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, which are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is complementary to SEQ ID NO:7, and optionally with NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:8, and optionally having no more than 3 nucleotide differences:

5’-CGGAAAGGUGUCAGUACUAUU-3’(SEQ ID NO：7)

5’-UAGUACUGACACCUUUCCGGA-3’(SEQ ID NO：8)

In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II. In some embodiments, the nucleotide sequence I hybridizes with SEQ ID NO:7 and/or said nucleotide sequence II and the nucleotide sequence set forth in SEQ ID No. 8 optionally have NO more than 1 nucleotide difference therebetween.

Fifth siRNA

According to the present invention, the siRNA may be a fifth siRNA.

The fifth siRNA comprises a sense strand and an antisense strand, each nucleotide in the fifth siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, which are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is complementary to SEQ ID NO:9, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:10, and optionally having no more than 3 nucleotide differences:

5’-CAGUAAAGAGGCGAAGUAUUU-3’(SEQ ID NO：9)

5’-AUACUUCGCCUCUUUACUGAA-3’(SEQ ID NO：10)

In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II. In some embodiments, the nucleotide sequence I hybridizes with SEQ ID NO:9, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:10 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown.

Sixth siRNA

According to the present invention, the siRNA may be a sixth siRNA.

The sixth siRNA comprises a sense strand and an antisense strand, each nucleotide in the sixth siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, which are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is complementary to SEQ ID NO:11, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:12, and optionally having no more than 3 nucleotide differences:

5’-CGUGUUCAAGUGUGGAAUAUU-3’(SEQ ID NO：11)

5’-UAUUCCACACUUGAACACGCC-3’(SEQ ID NO：12)

In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II. In some embodiments, the nucleotide sequence I hybridizes with SEQ ID NO:11, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:12 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown.

As previously mentioned, the nucleotides in the sirnas disclosed herein are each independently modified or unmodified nucleotides. In some embodiments, each nucleotide in the siRNA of the invention is an unmodified nucleotide. In some embodiments, some or all of the nucleotides in the siRNA of the invention are modified nucleotides, and such modifications on the nucleotide groups do not result in a significant impairment or loss of the function of the siRNA conjugates of the invention to inhibit DPP4 gene expression.

In some embodiments, the siRNA of the invention may be any of the unmodified sirnas listed in table 1.

TABLE 1 unmodified sequences of the disclosure

In some embodiments, the presently disclosed siRNA contains at least 1 modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide analogue formed by substitution of the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with other groups, or a nucleotide having a modified base. The modified nucleotide does not result in a significant impairment or loss of function of the siRNA to inhibit gene expression. For example, modified nucleotides disclosed in J.K.Watts, G.F.Deleavey, and M.J.damha, chemically modified siRNA: tools and applications. Drug discovery Today,2008,13 (19-20): 842-55 may be selected.

In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided herein is a modified nucleotide, and/or at least one phosphate group is a phosphate group having a modifying group. In other words, at least a portion of the phosphate groups and/or ribose groups in at least one single-stranded phosphate-sugar backbone in the sense strand and the antisense strand are phosphate groups and/or ribose groups having a modifying group.

In some embodiments, all of the 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 fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

In the context of the present disclosure, a "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with fluorine, which has a structure represented by the following formula (1). "non-fluoro modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' -position of the ribosyl of the nucleotide is replaced with a non-fluoro group. In some embodiments, each non-fluoro modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by substitution of the hydroxyl group at the 2' position of the ribosyl of the nucleotide with a non-fluoro group.

Nucleotides in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group are well known to those skilled in the art, and may be, for example, one or more selected from the group consisting 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. In other words, the non-fluorine group may be selected from H, alkoxy (e.g., C1-C3 alkoxy, such as methoxy), substituted alkoxy (e.g., C1-C3 alkoxy substituted with C1-C3 alkoxy, such as methoxyethoxy), alkyl (e.g., C1-C3 alkyl, such as methyl), substituted alkyl (e.g., C1-C3 alkoxy substituted with C1-C3 alkyl, such as methoxymethyl, methoxyethyl), amino (-NH) ₂ ) Substituted amino groups (e.g., mono-or di-substituted amino groups such as methylamino, ethylamino groups with C1-C3 alkyl groups), but are not limited thereto.

In some embodiments, the 2' -alkoxy-modified nucleotide is a 2' -methoxy (2 ' -OMe) -modified nucleotide, as shown in formula (2) below. In some embodiments, the 2' -substituted alkoxy modified nucleotide may be a 2' -methoxyethyl (2 ' - Μ o e) modified nucleotide as shown in formula (3) below. In some embodiments, 2 '-amino (2' -n-h) ₂ ) The modified nucleotide is shown as a formula (4). In some embodiments, 2' -Deoxynucleotides (DNA) are represented by formula (5) below.

Wherein Base represents a nucleobase, e.g., A, U, G, C or T.

Nucleotide analogs refer to groups that are capable of replacing nucleotides in a nucleic acid, but that differ in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide analog may be an iso nucleotide, a bridged nucleotide, or an acyclic nucleotide.

The bridged nucleotides (bridged nucleic acid, abbreviated BNA) may contain a five-ring-, six-or seven-membered ring bridging structure with "fixed" C3' -endo-sugar tucking. The bridge is typically incorporated at the 2'-, 4' -position of the ribose to provide a 2',4' -BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cETBNA, etc., wherein LNA is shown as formula (6), ENA is shown as formula (7), ctbna is shown as formula (8).

Acyclic nucleotides are a class of nucleotides in which the sugar ring of a nucleotide is opened. In some embodiments, the acyclic nucleotide can be an Unlocking Nucleic Acid (UNA) or a Glycerol Nucleic Acid (GNA), wherein UNA is represented by formula (9), and GNA is represented by formula (10):

wherein Base represents a nucleobase, e.g. A, U, G, C or T, ra is selected from H, OH or C ₁ -C ₁₀ Alkoxy (O-alkyl).

An isopucleotide refers to a compound in which the position of a base on the ribose ring is changed in a nucleotide. In some embodiments, the isonucleotide may be a compound formed by a base moving from the 1' -position to the 2' -position or the 3' -position of the ribose ring, as shown in formula (11) or (12):

in the compounds represented by the above formula (11) and formula (12), base represents a nucleobase, for example A, U, G, C or T; r is R _b Selected from H, OH, F or a non-fluoro group as described above.

In some embodiments, the nucleotide analog is selected from one of an iso-nucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluoro modified nucleotide is a methoxy modified nucleotide, which in the foregoing and below refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

In the above and in the following, the meaning of "fluoro modified nucleotide", "2 '-fluoro modified nucleotide", "nucleotide with 2' -hydroxyl of ribose group substituted by fluoro" and "nucleotide with 2 '-fluoro ribose group" are the same, and all refer to a compound having a structure as shown in formula (1) formed by substituting 2' -hydroxyl of nucleotide by fluoro; "methoxy-modified nucleotide", "2 '-methoxy-modified nucleotide", "nucleotide in which the 2' -hydroxyl group of the ribose group is replaced by methoxy" and "nucleotide having a 2 '-methoxyribosyl" are the same in meaning, and refer to a compound having a structure shown in formula (2) in which the 2' -hydroxyl group of the ribosyl group of the nucleotide is replaced by methoxy.

In some embodiments, the sense strand or the antisense strand of the siRNA provided herein may comprise a base modification or substitution.

The siRNA with the modification can ensure that ribonuclease in blood is not easy to cut nucleic acid, thereby increasing the stability of the nucleic acid and ensuring that the nucleic acid has stronger performance of resisting nuclease hydrolysis. Meanwhile, the modified siRNA has higher activity of inhibiting target mRNA.

In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA provided herein are phosphate groups having a modifying group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of the phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (13):

This modification stabilizes the double-stranded structure of the siRNA, maintaining high specificity and high affinity for base pairing.

In some embodiments, the invention provides siRNA wherein the phosphorothioate linkage is present at least one of 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 above. In some embodiments, phosphorothioate linkages are present at all of the above positions except the 5' end of the sense strand. In some embodiments, phosphorothioate linkages are present at all of the above positions except the 3' end of the sense strand.

In some embodiments, the 5' -terminal nucleotide of the siRNA antisense strand is a 5' -phosphonucleotide or a 5' -phosphoanalog modified nucleotide.

Commonly used nucleotides modified by such 5' -phosphonucleotides or 5' -phosphoanalogs are well known to those skilled in the art, e.g., the 5' -phosphonucleotides may have the structure of formula (14):

for another example, anastasia Khvorova and Jonathan K.Watts, the chemical evolution of oligonucleotide therapies of clinical U.S. Nature Biotechnology,2017,35 (3): 238-48 discloses the following 5' -phosphate analog modified nucleotides:

Wherein R is selected from the group consisting of H, OH, methoxy and fluorine; base represents a nucleobase selected from A, U, C, G or T.

In some embodiments, the 5' -phosphate nucleotide is a nucleotide comprising a 5' -phosphate modification represented by formula (14), the 5' -phosphate analogue modified nucleotide is a nucleotide comprising a vinyl phosphate modification represented by formula (15), or is a phosphorothioate modified nucleotide represented by formula (17).

Abbreviations for the modified nucleotide monomers disclosed herein are shown in table 2.

TABLE 2 abbreviations for nucleotide monomers disclosed in the present invention

Abbreviations (abbreviations)	Nucleotide(s)	Abbreviations (abbreviations)	Nucleotide(s)
				A	Adenosine-3' -phosphate	Cs	2 '-O-methylcytidine-3' -thiophosphoric acid
Af	2 '-fluoroadenosine-3' -phosphate	G	2 '-O-methylguanosine-3' -monophosphate
				Afs	2 '-fluoroadenosine-3' -thiophosphoric acid	Gs	2 '-O-methylguanosine-3' -thiophosphoric acid
As	Adenosine-3' -thiophosphoric acid	T	2 '-O-methyl-5' -methylUridine-3' -monophosphate
				C	Cytidine-3' -phosphate	Ts	2' -O-methyl-5 ' -methyluridine-3 ' -thiophosphoric acid
Cf	2 '-fluorocytidine-3' -phosphate	U	2 '-O-methyluridine-3' -monophosphate
				Cfs	2 '-fluorocytidine-3' -thiophosphoric acid	us	2 '-O-methyluridine-3' -thiophosphoric acid
Cs	Cytidine-3' -thiophosphoric acid	S	Phosphorothioate linkages
				G	Guanosine-3' -monophosphate	P1	5' phosphorylation modification
Gf	2 '-fluoroguanosine-3' -monophosphate	(Agn)	Adenosine-diol nucleic acids
				Gfs	2’-fluoroguanosine-3' -thiophosphoric acid	(Cgn)	Cytidine-diol nucleic acids
Gs	Guanosine 3' -thiophosphoric acid	(Ggn)	Guanosine-diol nucleic acids
				T	5 '-methyluridine-3' -monophosphate	(Tgn)	Thymidine-diol nucleic acid
Tf	2' -fluoro-5 ' -methyluridine-3 ' -monophosphate	dA	2 '-deoxyadenosine-3' -phosphate
				Tfs	2' -fluoro-5 ' -methyluridine-3 ' -thiophosphoric acid	dAs	2 '-deoxyadenosine-3' -thiophosphoric acid
Ts	5 '-methyluridine-3' -thiophosphoric acid	dC	2 '-deoxycytidine-3' -phosphate
				U	Uridine-3' -monophosphate	dCs	2' -deoxycytidine-3' -thiophosphoric acid
Uf	2 '-fluorouridine-3' -monophosphate	dG	2 '-deoxyguanosine-3' -monophosphate
				Ufs	2 '-fluorouridine-3' -thiophosphoric acid	dGs	2 '-deoxyguanosine-3' -thiophosphoric acid
Us	Uridine-3' -thiophosphoric acid	dT	2 '-deoxythymidine-3' -phosphate
				A	2 '-O-methyladenosine-3' -phosphate	dTs	2 '-deoxythymidine-3' -thiophosphoric acid
As	2 '-O-methyladenosine-3' -thiophosphoric acid	dU	2' -deoxyuridine
				C	2 '-O-methylcytidine-3' -phosphate	dUs	2 '-deoxyuridine-3' -thiophosphoric acid

In some embodiments, the siRNA provided by the invention is any one of the sirnas listed in table 3.

TABLE 3 modification sequences disclosed in the present invention

The siRNA provided by the invention not only has obviously enhanced stability of plasma and lysosomes, but also has very high target mRNA inhibition activity.

The siRNA provided by the present invention may be obtained by methods of siRNA preparation conventional in the art (e.g., solid phase synthesis). Among them, solid phase synthesis is already commercially available as a custom service. Methods of preparing nucleoside monomers having corresponding modifications and methods of introducing modified nucleotide groups into siRNA can also be known to those of skill in the art by introducing modified nucleotide groups into siRNA described herein using nucleoside monomers having corresponding modifications.

siRNA conjugates

The present invention provides an siRNA conjugate comprising the above siRNA and a conjugate group conjugated to the siRNA.

In general, the conjugate group comprises at least one pharmaceutically acceptable targeting group and optionally a linker (linker), and the siRNA, the linker and the targeting group are sequentially linked. In some embodiments, the targeting group is 2-4. The siRNA molecule may be non-covalently or covalently conjugated to the conjugate group, e.g., may be covalently conjugated to the conjugate group. The conjugation site of the siRNA to the conjugation group may be at the 3 'or 5' end of the sense strand of the siRNA, or may be in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3 'end or the 5' end of the sense strand of the siRNA.

In some embodiments, when the conjugation site of the siRNA to the conjugation group is at the 3 'end of the siRNA sense strand, the TT at the 3' end in the siRNA sequence is replaced with UU to facilitate cleavage of the conjugate.

In some embodiments, the conjugate group may be attached to the phosphate group, the 2' -hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugate group may also be attached to the 3' -hydroxyl group, in which case the nucleotides are linked using a 2' -5' phosphodiester linkage. When a conjugate group is attached to the end of the siRNA strand, the conjugate group is typically attached to the phosphate group of the nucleotide; when a conjugate group is attached to the internal sequence of the siRNA, the conjugate group is typically attached to a ribose sugar ring or base. Various connection means can be referred to as: muthiah Manoharan et al, siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytocytocytosis, ACS Chemical biology,2015,10 (5): 1181-7.

In some embodiments, the siRNA and the conjugate group may be linked by acid labile or reducible chemical bonds that degrade in the acidic environment of the intracellular inclusion bodies, thereby allowing the siRNA to be in a free state. For non-degradable conjugation, the conjugation group can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.

In some embodiments, the pharmaceutically acceptable targeting group can be a ligand conventionally used in the art of siRNA administration, such as the various ligands described in W02009082607A2, the entire disclosure of which is incorporated herein by reference.

In some embodiments, the targeting group comprises an asialoglycoprotein receptor ligand. In some embodiments, the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives. As used herein, the term "galactose derivative" includes galactose and lactose derivatives having an affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. Galactose derivatives include, but are not limited to: galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-N-butyryl-galactosamine and N-isobutyryl galactosamine (see, e.g., iobst, S.T. and Drickamer, K.J.B.C.1996, vol 271, page 6686). Galactose derivatives and galactose derivative clusters that can be used for targeting oligonucleotides and other molecules to the liver in vivo are known in the art (see, e.g., baenziger and Fiete,1980, cell,22,611-620;Connolly et al, 1982, j. Biol. Chem.,257, 939-945). Galactose derivatives have been used to target molecules to hepatocytes in vivo through their binding to asialoglycoprotein receptors (ASGPr) expressed on the surface of hepatocytes. Binding of ASGPr ligands to ASGPr(s) facilitates cell-specific targeting of hepatocytes and entry of endocytic molecules into hepatocytes. The ASGPr ligand may be a monomer (e.g., having a single galactose derivative) or a multimer (e.g., having multiple galactose derivatives). Galactose derivatives or galactose derivative clusters can be attached to the 3 'or 5' end of the siRNA using methods known in the art.

In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent, tetravalent. It should be understood that the monovalent, divalent, trivalent, tetravalent means that after the siRNA molecule forms an siRNA conjugate with a conjugate group containing galactose or N-acetylgalactosamine molecules as a targeting group, the molar ratio of siRNA molecule to galactose or N-acetylgalactosamine molecules in the siRNA conjugate is 1:1, 1:2, 1:3, or 1:4, respectively. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA of the invention is conjugated to a conjugate group comprising N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, the N-acetylgalactosamine molecule is trivalent when the siRNA of the invention is conjugated to a conjugate group comprising N-acetylgalactosamine.

The targeting group can be attached to the siRNA molecule via a suitable linker, which can be selected by one skilled in the art depending on the particular type of targeting group. The types of these linkers, targeting groups, and the manner of attachment to the siRNA can be found in the disclosure of W02015006740A2, which is incorporated herein by reference in its entirety.

In some embodiments, a suitable linker may be of the structure shown in formula (19):

wherein m is an integer of 1 to 3;

L ^A is a chain-like moiety comprising an amide bond having a structure represented by formula (20), each of the L ^A At both ends thereof with one of said targeting group and said L ^C Part is connected by ether linkage:

L ^B is a N-acyl pyrrolidine-containing chain moiety having a structure represented by the formula (21), the chain moiety having a carbonyl group at one end thereof and being bonded to the L ^C Part is linked by an amide bond, has an oxygen atom at the other end and is linked to the siRNA by a phosphate bond:

L ^C is a 2-4 valent linking group based on hydroxymethyl aminomethane, dimethylol aminomethane or trimethylol aminomethane, said L ^C Via an oxygen atom with each of said L ^A Part is linked by an ether linkage and is bound to the L via a nitrogen atom ^B The moieties are linked by amide linkages.

In some embodiments, the linker is- (L) ^A ) ₃ Trimethylolaminomethane-L ^B -linking N-acetylgalactosamineAn siRNA conjugate formed from a molecule and an siRNA molecule, the structure of which is represented by formula (22):

in the formula, the double helix structure represents the siRNA of the present invention.

Also, the conjugation site of the siRNA to the conjugation group may be at the 3 'end or 5' end of the sense strand of the siRNA, or may be in the internal sequence of the siRNA.

In some embodiments, the 3 '-end of the sense strand of the siRNA of the present invention is linked to the 3' -end of the sense strand via a linker- (L) ^A ) ₃ Trimethylolaminomethane-L ^B Covalent conjugation with three N-acetylgalactosamine (GalNAc) molecules, resulting in siRNA conjugates with a molar ratio of siRNA molecules to GalNAc molecules of 1:3, which may also be referred to as (GalNAc) hereinafter ₃ -siRNA having the structure shown in formula (23):

wherein the double helix structure represents the siRNA of the invention and the linker is attached to the 3' end of the sense strand of the siRNA of the invention.

In some embodiments, the linker is attached to the 5' end of the sense strand of the siRNA of the invention.

In some embodiments, the siRNA conjugates have a structure as shown in formula (24), (25) or (26)

Wherein,,

R ₂ a group having a structure represented by the formula (S1):

wherein E is ₁ OH or SH, nu is the siRNA of the invention;

R ₁ is a linear or cyclic alkylene group of 1 to 20 carbon atoms in length, 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 、C ₂ -C ₁₀ Alkenylene, C ₂ -C ₁₀ Alkynylene, C ₆ -C ₁₀ Arylene group, C ₃ -C ₁₈ Heterocyclylene and C ₅ -C ₁₀ Heteroarylene; and wherein R is ₁ Optionally having substituents of any one or more of the group consisting of: c (C) ₁ -C ₁₀ Alkyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkyl, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituent, -OH, -SH, -NH ₂ 、-C ₁ -C ₁₀ alkyl-NH ₂ 、-N(C ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -NH (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkylphenyl), -NH (C) ₁ -C ₁₀ Alkylphenyl), cyano, -CO ₂ H、C(O)O(C ₁ -C ₁₀ Alkyl), -CON (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -CONH (C) ₁ -C ₁₀ Alkyl), -CONH ₂ 、-NHC(O)(C ₁ -C ₁₀ Alkyl), -NHC (O) (phenyl), -N (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (C ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ Alkyl, -C (O) C ₁ -C ₁₀ Alkylphenyl, -C (O) C ₁ -C ₁₀ Haloalkyl, -OC (O) C ₁ -C ₁₀ Alkyl, -SO ₂ (C ₁ -C ₁₀ Alkyl), -SO ₂ (phenyl) -SO ₂ (C ₁ -C ₁₀ Haloalkyl) -SO ₂ NH ₂ 、-SO ₂ NH(C ₁ -C ₁₀ Alkyl), -SO ₂ NH (phenyl) -NHSO ₂ (C ₁ -C ₁₀ Alkyl), -NHSO ₂ (phenyl) and-NHSO ₂ (C ₁ -C ₁₀ A haloalkyl group);

each L ₁ Independently is a linear alkylene group of 1 to 40 carbon atoms in length, 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 、C ₂ -C ₁₀ Alkenylene, C ₂ -C ₁₀ Alkynylene, C ₆ -C ₁₀ Arylene group, C ₃ -C ₁₈ Heterocyclylene and C ₅ -C ₁₀ Heteroarylene; and wherein R is ₁ Optionally having substituents of any one or more of the group consisting of: c (C) ₁ -C ₁₀ Alkyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkyl, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituent, -OH, -SH, -NH ₂ 、-C ₁ -C ₁₀ alkyl-NH ₂ 、-N(C ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -NH (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkylphenyl), -NH (C) ₁ -C ₁₀ Alkylphenyl), cyano, -CO ₂ H、C(O)O(C ₁ -C ₁₀ Alkyl), -CON (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -CONH (C) ₁ -C ₁₀ Alkyl), -CONH ₂ 、-NHC(O)(C ₁ -C ₁₀ Alkyl), -NHC (O) (phenyl), -N (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (C ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ Alkyl, -C (O) C ₁ -C ₁₀ Alkylphenyl, -C (O) C ₁ -C ₁₀ Haloalkyl, -OC (O) C ₁ -C ₁₀ Alkyl, -SO ₂ (C ₁ -C ₁₀ Alkyl), -SO ₂ (phenyl) -SO ₂ (C ₁ -C ₁₀ Haloalkyl) -SO ₂ NH ₂ 、-SO ₂ NH(C ₁ -C ₁₀ Alkyl), -SO ₂ NH (phenyl) -NHSO ₂ (C ₁ -C ₁₀ Alkyl), -NHSO ₂ (phenyl) and-NHSO ₂ (C ₁ -C ₁₀ A haloalkyl group);

in some embodiments, L ₁ May be selected from the group consisting of A1-a14 groups or any linked combination thereof, wherein the structures and definitions of A1-a14 are as follows:

wherein each k1 is independently an integer from 1 to 20;

each k2 is independently an integer from 1 to 20;

each R _c Independently C ₁ -C ₁₀ An alkyl group;

each R _d Selected from the group consisting of a15-a19 and any combination thereof:

each R _e Independently C ₁ -C ₁₀ An alkyl group;

indicating the site of covalent attachment of the group.

In some embodiments, L ₁ Is a linked combination of at least 2 of the groups A1, A4, A8, A10, A11.

In some embodiments, L ₁ Is 3 to 20 atoms in length.

In some embodiments, k1 is an integer from 3 to 5, k2 is an integer from 3 to 5, R _c Is one selected from methyl, ethyl and isopropyl, R _d Is A15 or A16, R _e Is one selected from methyl, ethyl, isopropyl and butyl.

In some embodiments, each of the targeting groups is independently one selected from the group consisting of D-galactose, L-galactose, a-D-glucopyranose, β -D-glucopyranose, a-D-glucofuranose, β -D-glucofuranose, a-D-fructofuranose, a-D-fructopyranose, a-D-galactopyranose, β -D-galactopyranose, a-D-galactofuranose, β -D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine.

In some embodiments, at least one or each of the targeting groups is galactose or N-acetylgalactosamine.

In some embodiments, R ₁ Containing both a linking site attached to an N atom on a nitrogen-containing backbone and R ₂ A linking site to which the P atom is linked.

In some embodiments, R ₁ The above-mentioned site bonded to N atom on the nitrogen-containing skeleton forms an amide bond with N, the above-mentioned site bonded to R ₂ The P atom on the substrate is linked to P to form a phosphate bond or a phosphorothioate bond.

The skilled artisan will appreciate that L, although for convenience ₁ Is defined as a linear alkylene group, but it may not be a linear group or be named differently, such as an amine or alkenyl group resulting from the substitution and/or substitution described above. For the purposes of the present disclosure, L ₁ Length of (2)Degree is the number of atoms in the chain connecting the two points of attachment. For this purpose, the ring (e.g., heterocyclylene or heteroarylene) resulting from substitution of the carbon atom of the linear alkylene group is counted as one atom.

M ₁ Represents a targeting group, the definition and optional scope of which are the same as the targeting groups described above. In some embodiments, each M ₁ Independently selected from one of the ligands having an affinity for asialoglycoprotein receptors on the surface of mammalian liver cells.

R ₂ A group of the structure represented by the formula (S1), wherein E ₁ OH or SH.

R ₁ Is selected to achieve a linkage to S1 from the N atom on the nitrogen-containing backbone. R is R ₁ Any linking group capable of linking the S1 group to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, where the siRNA conjugate of formula (24), (25) or (26) is prepared by a process of solid phase synthesis, R ₁ The group needs to contain both a linking site to the N atom on the nitrogen-containing skeleton and R ₂ A junction site to which the P atom of (C) is attached. In some embodiments, R ₁ Wherein the site bonded to the N atom on the nitrogen-containing skeleton forms an amide bond with the N atom, the site bonded to R ₂ The P atom-attached site forms a phosphate bond with the P atom.

In some embodiments, the siRNA conjugates have a structure as shown by the formula (Z1-Nu), (Z2-Nu), (Z3-Nu), (Z4-Nu), (Z5-Nu), (Z6-Nu), (Z7-Nu), (Z8-Nu), (Z9-Nu), (Z10-Nu), (Z11-Nu), (Z12-Nu), (Z13-Nu), (Z14-Nu), (Z15-Nu), (Z16-Nu), (Z17-Nu), (Z18-Nu), (Z19-Nu), (Z20-Nu), (Z21-Nu), (Z22-Nu), (Z23-Nu), (Z24-Nu), (Z25-Nu), (Z26-Nu), (Z27-Nu), (Z28-Nu), (Z29-Nu), (Z30-Nu), (Z31-Nu), (Z32-Nu), wherein Z1-Z32 is a conjugated group.

In some embodiments, the P atom in formula S1 can be attached to any possible position in the siRNA sequence, e.g., the P atom in formula S1 can be attached to any one of the nucleotides of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula S1 is attached to any one nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in formula S1 is attached to the end of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula S1 is attached to the 3' end of the sense strand of the siRNA. In the case of the above-described position of the sense strand linked to the siRNA, the siRNA conjugate shown in (24), (25) or (26) can release the separate siRNA antisense strand upon unwinding to block the process of translation of the protein by DPP4 mRNA and inhibit the expression of DPP4 gene after entering the cell.

In some embodiments, the P atom in formula S1 can be attached 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 S1 can be linked to the 2', 3', or 5' position of a nucleotide in the siRNA by formation of a phosphodiester linkage. In some embodiments, the P atom in formula S1 is attached to an oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the siRNA sense strand (in this case, the P atom in S1 can also be considered as a P atom in a phosphate group contained in the siRNA), or the P atom in formula S1 is attached to the nucleotide by replacing hydrogen in the 2' -hydroxyl group of one nucleotide in the siRNA sense strand, or the P atom in formula S1 is attached to the nucleotide by replacing hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide of the siRNA sense strand.

The siRNA conjugate of the invention has remarkably improved stability in blood plasma, low off-target effect and higher DPP4 mRNA silencing activity. In some embodiments, the siRNA of the invention may be one of the sirnas shown in table 1 or table 3. siRNA conjugates containing these siRNAs exhibit higher DPP4 mRNA silencing activity.

In the siRNA or siRNA conjugate, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate bond, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate bond carries negative charge, the siRNA or siRNA conjugate can exist in a hydroxyl or sulfhydryl form, and hydrogen ions in the hydroxyl or sulfhydryl can be partially or completely replaced by cations. The cation may be any cation, such as a metal cation, ammonium ion NH4 ⁺ One of organic ammonium cations. In one embodiment, the cation is selected from one or more of an alkali metal ion, a tertiary amine-forming ammonium cation, and a quaternary ammonium cation for improved solubility. The alkali metal ion may be and/or Na+, and the cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N, N-diisopropylethylamine. Thus, the siRNA or siRNA conjugate of the invention may be at least partially present in salt form.In one mode, the non-bridging oxygen or sulfur atoms in the phosphodiester or phosphorothioate linkages are at least partially bound to sodium ions and the siRNA or siRNA conjugates of the invention are in the form of sodium salts or partial sodium salts.

It is known to those skilled in the art that modified nucleotide groups can be introduced into the siRNA of the present invention by using nucleoside monomers having corresponding modifications. Methods of preparing nucleoside monomers with corresponding modifications and methods of introducing modified nucleotide groups into siRNA are also well known to those of skill in the art. All modified nucleoside monomers are commercially available or can be prepared using known methods.

Preparation of siRNA conjugates represented by the formulas (24), (25) or (26)

The siRNA conjugates of formulas (24), (25) or (26) may be prepared using any reasonable synthetic route.

In some embodiments, the siRNA conjugate of formula (24), (25) or (26) can be prepared by a method comprising sequentially ligating nucleoside monomers in a 3 'to 5' direction under conditions of phosphoramidite solid phase synthesis according to the nucleotide species and sequence of the sense strand and the antisense strand of the siRNA, respectively, the ligating of each nucleoside monomer comprising a deprotection, coupling, capping, oxidation or sulfidation four-step reaction; separating a sense strand and an antisense strand of the siRNA, and annealing, wherein the siRNA is the siRNA of the invention; and, the method further comprises contacting the compound represented by formula (27), (28) or (29) with a nucleoside monomer or a nucleotide sequence attached to a solid support in the presence of a coupling reagent under coupling reaction conditions, so that the compound represented by formula (27), (28) or (29) is attached to the nucleotide sequence by coupling reaction.

Hereinafter, the compound represented by the formula (27), (28) or (29) is also referred to as a conjugate molecule.

Wherein:

R ₃ is a group capable of binding to siRNA represented by Nu in the compound represented by formula (24), (25) or (26). In some embodiments, R ₃ Is a group capable of binding to siRNA represented by Nu through a covalent bond. In some embodiments, R ₃ A group that is any functional group capable of being conjugated to siRNA represented by Nu through a phosphodiester bond by reaction;

each T ₁ Independently is a group formed by substitution of all active hydroxyl groups in M1 with YCOO-groups, wherein each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and alkylphenyl; in some embodiments, Y is methyl. L (L) ₁ The definition and optional scope of (a) is as described above.

R ₃ Is selected to achieve attachment to the N atom on the nitrogen-containing backbone and to provide a suitable reaction site for synthesizing siRNA conjugates represented by formulas (24), (25) or (26). In some embodiments, R ₃ Includes R ₁ Linking group or protected R ₁ A linking group, and a functional group that can react with the siRNA to form a structure shown as S1.

In some embodiments, R ₃ Comprising the 1 st functional group which can form a phosphite with a group on a siRNA or nucleoside monomer represented by Nu, the 2 nd functional group which can react with a hydroxyl group or an amino group to form a covalent bond, or a solid support linked by the covalent bond. In some embodiments, the 1 st functional group is a phosphoramidite, a hydroxyl group, or a protected hydroxyl group. In some embodiments, the 2 nd functional group is a phosphoramidite, a carboxyl group, or a carboxylate. In some embodiments, the 2 nd functional group is a solid support attached to the rest of the molecule via a covalent bond formed by a hydroxyl or amino group. In some embodiments, the solid support is linked via a phosphate bond, a carboxylate bond, or an amide bond. In some embodiments, the solid support is a resin.

In some embodiments, the 1 st functional group contains a hydroxyl group, -ORm (R _m Is hydroxyA protecting group) or a group represented by the formula (C3); the 2 nd functional group contains a structure represented by formula (C1), (C2), (C3), (C1 ') or (C3'):

wherein q1 is an integer of 1 to 4, X is O or NH, M ⁺ Is a cation, R _m Is a hydroxyl protecting group, SPS represents a solid support,

Indicating the site at which the group attaches to the covalent moiety.

In some embodiments, the 1 st functional group contains a phosphoramidite group, as shown in formula (C3), which can undergo a coupling reaction with a hydroxyl group at any position on a nucleotide, such as a 2' -hydroxyl group, a 3' -hydroxyl group, or a 5' -hydroxyl group, to form a phosphite, and oxidized or sulfided to form a phosphodiacetyl bond or phosphorothioate bond shown in formula S1, to conjugate the conjugated molecule to the siRNA. At this time, even if the 2 nd functional group is not present, the compound represented by formula (27), (28) or (29) can be conjugated to a nucleotide without affecting the obtaining of the siRNA conjugate represented by formula (24), (25) or (26). In this case, after obtaining the sense strand or antisense strand of the siRNA via a phosphoramidite solid phase synthesis or the like, the compound represented by formula (27), (28) or (29) is reacted with a hydroxyl group on a terminal nucleotide in the nucleotide sequence, and a phosphodiester linkage or phosphorothioate linkage is formed in a subsequent oxidation or vulcanization process, and the compound represented by formula (27), (28) or (29) is conjugated to the siRNA.

In some embodiments, the 1 st functional group contains a protected hydroxyl group. In some embodiments, the 2 nd functional group comprises a group that is reactive with the solid support, the reaction providing a conjugated molecule comprising the solid support. In some embodiments, the 2 nd functional group contains a carboxyl group, carboxylate, or phosphoramidite, as shown in formula (C1), (C2), or (C3), and when the 2 nd functional group contains a carboxyl group or carboxylate, the compound of formula (27), (28), or (29) undergoes an esterification reaction or amidation reaction with a solid support, such as a hydroxyl group or an amino group on a resin, to form a conjugate molecule comprising a solid support linked via a carboxylic acid ester linkage. When the 2 nd functional group comprises a phosphoramidite functional group, the compound of formula (27), (28) or (29) is coupled to a general solid support, such as a hydroxyl group on a resin, and oxidized to form a conjugated molecule comprising a solid support linked via a phosphodiester linkage. Subsequently, the above-mentioned product after the solid phase carrier is attached is used as an initial, and nucleoside monomers are sequentially attached according to a phosphoramidite solid phase synthesis method, so as to obtain the sense strand or antisense strand of the siRNA with the attached conjugate group. During the solid phase synthesis of phosphoramidite, the 1 st functional group is deprotected and then coupled to the phosphoramidite group on the nucleoside monomer under coupling reaction conditions.

In some embodiments, the 1 st functional group contains a hydroxyl group or a protected hydroxyl group; the 2 nd functional group contains a solid phase carrier connected by a carboxylic ester bond or a solid phase carrier connected by an amide bond or a solid phase carrier connected by a phosphoric ester bond, as shown in formula (C1 ') or (C3'). At this time, nucleoside monomers were sequentially linked by phosphoramidite solid phase synthesis starting from the compounds represented by the formulas (27), (28) and (29) instead of the solid phase carrier, to obtain the sense strand or antisense strand of the siRNA to which the conjugate group was linked.

In some embodiments, each T ₁ Independently M ₁ . In some embodiments, each S ₁ M is independently ₁ At least one active hydroxyl group of the polymer is protected by a hydroxyl protecting group. In some embodiments, the protected hydroxyl group may be represented by the formula YCOO-, wherein each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; in some embodiments, Y is methyl.

In some embodiments, R _m Is one or more of MMTr (4-methoxytrityl), DMTr (4, 4 '-dimethoxytrityl) and TMTr (4, 4' -trimethoxytrityl). In some embodiments ，R _m May be DMTr, 4'-dimethoxytrityl (4, 4' -dimethoxytrityl).

Accordingly, unless otherwise indicated, in the following description relating to the preparation of conjugates and/or conjugate molecules, when reference is made to "deprotection," "coupling," "capping," "oxidation," "sulfidation," etc. reactions, it is to be understood that the reaction conditions and reagents involved in solid phase synthesis of phosphoramidite nucleic acids, which are well known in the art, are equally applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail later.

As described above, the preparation method of the siRNA conjugate represented by formula (24), (25) or (26) further comprises the steps of: the other strand of the siRNA is synthesized (e.g., when the steps described above synthesize the sense strand of the siRNA to which the conjugate molecule is attached, also include synthesizing the antisense strand of the siRNA according to a solid phase synthesis method, and vice versa), separating the sense strand and the antisense strand, and annealing. Specifically, in the separation step, the solid phase carrier linked to the nucleotide sequence and/or the conjugate molecule is cleaved, while the necessary protecting groups are removed (at this time, each S1 group in the compound represented by formula (27), (28) or (29) is converted into a corresponding M1 targeting group), and the siRNA sense strand (or antisense strand) and the corresponding antisense strand (or sense strand) linked to the conjugate molecule are obtained, and the sense strand and the antisense strand are annealed to form a double-stranded RNA structure, thereby obtaining the siRNA conjugate represented by formula (24), (25) or (26).

In some embodiments, the method of preparing the siRNA conjugate of formula (24), (25) or (26) comprises the steps of: contacting a compound shown in a formula (27), (28) or (29) with a first nucleoside monomer at the 3' end of a sense strand or an antisense strand under coupling reaction conditions and in the presence of a coupling reagent, connecting the first nucleotide in the sequence to the compound shown in the formula (27), (28) or (29), and sequentially connecting the nucleoside monomers in the 3' to 5' direction under the condition of phosphoramidite solid phase synthesis according to the expected sense strand or antisense strand nucleotide species and sequence to synthesize the sense strand or antisense strand of the siRNA; wherein the compound shown in the formula (27), (28) or (29) is a compound in which R2 contains a 1 st functional group and a 2 nd functional group, the 1 st functional group contains a protected hydroxyl group, and the 2 nd functional group has a structure shown as a formula (C1 ') or (C3'), and the compound shown in the formula (27), (28) or (29) is deprotected before being connected with the first nucleoside monomer; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or vulcanization reaction; obtaining a sense strand or an antisense strand of the nucleic acid to which the conjugate group is attached; under the condition of the solid-phase synthesis of the bony amide, sequentially connecting nucleoside monomers according to the nucleotide types and sequences of the antisense strand or the sense strand and the direction from 3 'to 5', and synthesizing the antisense strand or the sense strand of the nucleic acid; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or vulcanization reaction; removing protecting group, cutting with solid phase carrier, separating and purifying to obtain sense strand and antisense strand, and annealing.

In some embodiments, the method of preparing the siRNA conjugate of formula (24), (25) or (26) comprises the steps of: sequentially connecting nucleoside monomers according to the nucleotide types and sequences of a sense strand or an antisense strand in the double-stranded siRNA and the direction from 3 'to 5' to synthesize the sense strand and the antisense strand, wherein the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration reaction to obtain the sense strand connected to a solid carrier and the antisense strand connected to the solid carrier; contacting a compound represented by the formula (27), (28) or (29) with a sense strand attached to a solid support or an antisense strand attached to a solid support under coupling reaction conditions and in the presence of a coupling reagent to attach the compound represented by the formula (27), (28) or (29) to the sense strand or the antisense strand, wherein the compound represented by the formula (27), (28) or (29) is R ₃ The compound contains a 1 st functional group, wherein the 1 st functional group is a phosphoramidite group and is shown in the formula (27), (28) or (29); removing protecting groups, cutting with a solid phase carrier, separating and purifying to obtain a sense strand or an antisense strand of the siRNA, and annealing, wherein the sense strand or the antisense strand of the siRNA is connected with a conjugation group.

In some embodiments, the P atom in formula S1 is attached to the 3' end of the sense strand in the siRNA, and the method of preparing the siRNA conjugate of formula (24), (25) or (26) comprises:

(1) Removing the compound of formula (27), (28) or (29) (wherein, formula (27), (28) or ]29 A compound shown as R ₃ Contains a 1 st functional group and a 2 nd functional group, the 1 st functional group contains a protected hydroxyl group OR _m A hydroxyl protecting group R in a compound having a structure as shown in formula (C1 ') or (C3') as the 2 nd functional group _m The method comprises the steps of carrying out a first treatment on the surface of the Contacting the deprotected product with a nucleoside monomer under coupling reaction conditions and in the presence of a coupling reagent to obtain a nucleoside monomer attached to a solid support via a conjugate molecule;

(2) Synthesizing the sense strand of the siRNA by a phosphoramidite solid phase synthesis method according to the 3'-5' direction starting from the nucleoside monomer attached to the solid phase carrier through the conjugate molecule;

(3) Synthesizing antisense strand of siRNA through phosphoramidite solid phase synthesis method;

(4) The sense strand and the antisense strand of the siRNA are separated and annealed to obtain the siRNA conjugate represented by formula (24), (25) or (26).

After obtaining the conjugate, in some embodiments, the synthesized siRNA conjugate represented by formula (24), (25) or (26) may be further characterized by means of molecular weight detection, etc., using a method such as liquid chromatography, etc., to determine that the synthesized siRNA conjugate is the target-designed siRNA conjugate represented by formula (24), (25) or (26), and that the sequence of the synthesized siRNA is the sequence of the desired siRNA.

In some embodiments, the solid support is a solid support known in the art to be useful in solid phase synthesis of nucleic acids.

Pharmaceutical composition

In yet another aspect, the invention provides a pharmaceutical composition comprising the above siRNA, and/or the above siRNA conjugate, and optionally a pharmaceutically acceptable carrier.

The invention also includes pharmaceutical compositions and formulations comprising the siRNA and/or siRNA conjugates of the invention. In some embodiments, provided herein are pharmaceutical compositions comprising an siRNA and/or an siRNA conjugate as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising the siRNA conjugates are useful for treating diseases or disorders associated with expression or activity of the DPP4 gene. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration by parenteral delivery, such as by Subcutaneous (SC), intramuscular (IM), or Intravenous (IV) delivery. In certain embodiments, the invention provides compositions formulated for organ-specific (e.g., liver) intra-arterial, intratumoral, intradermal, intravitreal injection, topical ocular, ophthalmic (eye drops), nebulization, topical or other topical ocular route, suppository or oral administration. In a preferred embodiment, the composition is administered subcutaneously.

The pharmaceutical compositions of the invention may be administered in a dose sufficient to inhibit DPP4 gene expression. In some embodiments, the siRNA conjugate is administered at the following doses: about 0.5mg/kg to 50mg/kg per dose, or 0.3mg/kg to 20mg/kg, or 3mg/kg to 10mg/kg, or preferably 3mg/kg to 10mg/kg per dose. For example, the siRNA conjugates may be administered at a dose of about 0.5mg/kg, 1mg/kg, 1.5mg/kg, 2mg/kg, 3mg/kg, 10mg/kg, 20mg/kg, 30mg/kg, 40mg/kg, or 50mg/kg per single dose.

The composition may also be prepared and packaged in a fixed dose for the subject independent of body weight. Exemplary dosage levels may be calculated by multiplying each kilogram of body weight by the average subject's body weight. For example, average adult weight is generally considered to be about 70 kg.

Repeated dosage regimen may include periodic administration of therapeutic amounts of the siRNA conjugate, e.g., once a month, once every other month, or once every third month. In a preferred embodiment, the siRNA conjugate is administered at a frequency of no more than once a month. Following the initial treatment regimen, the treatment may be administered less frequently.

The pharmaceutical composition may be administered indefinitely, e.g. in a subject with one or more signs or symptoms of DPP4 expression. In some embodiments, treatment with the siRNA conjugate is performed for a discrete or defined period of time and provides a functional cure.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present. Furthermore, treating a subject with a therapeutically effective amount of a composition may include monotherapy or a series of therapies. As described elsewhere herein, the effective dose and in vivo half-life of the individual siRNA conjugates encompassed by the invention can be estimated using conventional methods or based on in vivo testing using an appropriate animal model.

In the present invention, the pharmaceutically acceptable carrier may include, but is not limited to, excipients, and/or other components.

A. Excipient

A "pharmaceutical excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. Such agents are well known in the art.

B. Other components

The compositions of the present invention may additionally comprise other auxiliary components conventionally present in pharmaceutical compositions at levels of use established in the art. Thus, for example, the composition may comprise additional, compatible pharmaceutically active substances, such as antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may comprise additional substances such as preservatives, antioxidants and stabilizers useful in physically formulating the compositions of the present invention in various dosage forms. However, such materials should not unduly interfere with the biological activity of the components of the compositions of the present invention when added. The formulation may be sterilized and, if desired, mixed with adjuvants which do not adversely interact with the nucleic acids of the formulation, such as preservatives, stabilizers, wetting agents, emulsifiers, salts or buffers which affect osmotic pressure, and the like.

In some embodiments, the pharmaceutical compositions characterized in the present invention comprise (a) one or more siRNA and/or siRNA conjugate compounds and (b) one or more agents that function by a non-RNAi machinery and are useful in the treatment of DPP 4-related disorders. Examples of such agents include, but are not limited to, DPP4 inhibitors.

In a further aspect, the present invention provides the use of the above siRNA, siRNA conjugate, pharmaceutical composition for the manufacture of a medicament for the treatment of a disease and/or condition caused by dipeptidyl peptidase 4 gene expression.

In yet another aspect, the present invention provides a method of treating a disease and/or disorder associated with dipeptidyl peptidase 4 gene expression, wherein the method comprises administering a therapeutically effective amount of the above siRNA, siRNA conjugate, and/or pharmaceutical composition to a subject in need thereof.

In some embodiments, the dipeptidyl peptidase 4 gene expression associated disease and/or disorder is diabetes or a lipid metabolism disorder.

In yet another aspect, the invention provides a method of inhibiting the expression of a dipeptidyl peptidase 4 gene in a cell, the method comprising contacting an effective amount of the above-described siRNA, siRNA conjugate, and/or pharmaceutical composition with the cell, thereby inhibiting the expression of the dipeptidyl peptidase 4 gene in the cell.

As noted above, in addition to their administration, the siRNA and/or siRNA conjugates characterized herein may be administered in combination with other known agents effective in inhibiting DPP4 expression. Regardless, the administering physician can adjust the amount and timing of siRNA and/or siRNA conjugate administration based on the results observed using standard efficacy measurements known in the art or described herein.

Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.

Examples

Unless otherwise specified, reagents and media used in the following examples are commercially available, and the procedures for nucleic acid electrophoresis, real-time PCR, and the like used are carried out by the method described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). The conjugate molecules (L-Z3, L-Z24, L-Z21, L-Z32) of the general formula (27), (28) or (29) are obtained from Nanjing Lei Zheng medical science and technology Co.

Example 1 design of siRNA

Since siRNA therapy has sequence specificity, it must be targeted to its target when used as a drug. DPP4 is currently an ideal target for siRNA therapy, but both treatment for DPP4 expression and treatment regimens are very challenging due to the high transcription rate. In order to inhibit expression of the target genome, the designed siRNA must be selected to be within a region of high conservation of the genome. Meanwhile, in order to avoid toxicity caused by any sequence, sequences similar to those of human genes need to be excluded.

The structure of the siRNA of the present invention is shown in Table 1.

EXAMPLE 2 preparation of siRNA or siRNA conjugates

And (3) synthesis: sense and antisense strand sequences were synthesized according to phosphoramidite solid phase synthesis techniques, on a 1. Mu. Mol scale using solid phase carrier mediated phosphoramidite chemistry on a Mermade 192 synthesizer (BioAutomation). The solid support is a controlled pore glass loaded with custom GalNAc ligand molecules (CPG,

) Or a universal solid support. Auxiliary synthesis reagents, such as 2'-F and 2' -O-methyl RNA phosphoramidite, are commercially available reagents. The corresponding phosphoramidites were used to introduce 2' -F, 2' -O-methyl, GNA (diol nucleic acid), 5' -phosphate and abasic modifications. Synthesis of 3' GalNAc conjugated single strands was performed on GalNAc modified CPG supports. CPG universal solid phase carriers are used for synthesis of antisense single strands, or synthesis of 5' GalNAc conjugated single strands. The coupling time for all phosphoramidites (dissolved in anhydrous acetonitrile, 100 mM) was 5 minutes using 5-ethylthio-1H-tetrazole (ETT) as activator (0.6M in acetonitrile). Phosphorothioate linkages were generated using a solution of 50mm 3- ((dimethylamino-methylene) amino) -3H-1,2, 4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile/pyridine (vv=1/1) for a reaction time of 3 minutes. All sequences were synthesized after the final removal of DMT groups.

Cleavage and deprotection of bound oligomers on CPG: after termination of the solid phase synthesis, the protecting group was removed by treatment with an acetonitrile solution containing 20% diethylamine for 30 minutes without cleavage of the oligonucleotide from the CPG. Subsequently, the dried CPG was treated with concentrated ammonia at 40℃for 18 hours. After centrifugation, the supernatant was transferred to a new tube and CPG was washed with ammonia. The combined solutions were concentrated to give a solid mixture.

Purifying: purification was by anion exchange HPLC using NanoQ. Buffer a was 10mM sodium perchlorate solution, 20mM Tris,1mM EDTA,pH7.4 and contained 20% acetonitrile, buffer B,500mM sodium perchlorate, 20mM Tris,1mM EDTA,pH7.4 and contained 20% acetonitrile. The target product was isolated and desalted using a reverse phase C18 column.

Annealing of the oligoribonucleotides results in siRNA conjugates: the RNA oligomer to be annealed is treated with sterile RNaseF reeH ₂ O (no RNA hydrolase) was formulated as a 200. Mu.m solution. The annealing reaction system was set up as follows, the above solution (duplex concentration 10 nmol) with total volume of 100. Mu.L was placed in a 95℃water bath for 10 minutes (. Gtoreq.100 nmol demand requires high temperature 20 minutes). Fwdarw.rapidly placed in a 60℃water bath for natural cooling. Fwdarw.the annealed solution was stored at 4 ℃. Complementary strands are mixed by combining equimolar RNA solutions.

Table 4 shows DPP4 siRNA conjugates synthesized using the above method.

TABLE 4 modified DPP4 siRNA conjugate nucleotide sequences

Example 3 in vitro Activity assay of siRNA

Quantitative determination of DPP4 mRNA content in HepG2 cells by qPCR as EC of compound ₅₀ The values are used as indicators to evaluate the inhibitory activity of the siRNA or siRNA conjugate on DPP 4.

Experimental materials and reagents:

cell line: hepG2.2.15 cells (provided by the stem cell bank of the national academy of sciences of China)

HepG2.2.15 cell culture medium (DMEM, invitrogen-11330032;10% serum, invitrogen-10099141;100units/mL penicillin and 100. Mu.g/mL streptomycin, hyclone-SV30010;1% non-essential amino acids, invitrogen-11140050;2mM L-glutamine, invitrogen-25030081;1mM sodium pyruvate, gibco-11360-070; 500. Mu.g/mL Geneticin, invitrogen-10131027).

Reagent: pancreatin (Invitrogen-25300062); DMSO (Sigma-D2650-100 ML); transfection reagent Lipofectamine RNAiMAX (Invitrogen-13778-150); MEM Medium (HyClone-SH 30024.01); ULtraPure Distilled Water (DNAse, RNAse, free) (Invitrogen-10977-015); opti-MEM I (1X) (Gibco-31985-070); phosphate Buffered Saline (PBS) (Gibco); primeScript ^TM RT reagent Kit with gDNAEraser(takara-RR047A)；ChamQ Universal SYBR qPCR Master Mix(vyzme-Q711-02)。

Consumable and instrument: 96-well cell culture plates (timing-3599); CO ₂ Incubator (HERA-CELL-240);

Microplate(Axygen-PCR-96-FLT-C)；qPCR equipment(QIANGE)。

the experimental steps are as follows:

the siRNA or siRNA conjugate was transfected into hepg2.2.15 cells by the following procedure: hepG2.2.15 cells were taken, washed with PBS, digested with trypsin, adjusted to the appropriate density, 24h later, siRNA was transferred into HepG2.2.15 cells using Lipofectamine RNAiMax, and inoculated into 48 well plates at a density of 100,000 cells per well, 500. Mu.L per well of culture medium. Cells were exposed to 5% CO ₂ Culturing in incubator at 37 deg.c for 48 hr.

The siRNA tested was tested at 2 concentration points, 2 duplicate wells. In control, 4 concentration points, 2 duplicate wells, were tested.

24 hours after transfection, cells were collected, RNA was extracted, and total intracellular DPP4-RNA was detected by RT-PCR.

The procedure for the detection of DPP4 RNA is briefly described as follows: total RNA in cells was extracted by the trizol method, and was reverse transcribed into cDNA by adding random primers, referring to the reverse transcription kit (takara) instructions, and then the DPP4 cDNA in the sample was detected by qPCR. Meanwhile, GAPDH primers and probes specifically detect GAPDH cDNA.

The PCR reaction procedure was: 95℃for 2 minutes, then enter a cyclic mode, 95℃for 10 seconds, followed by 60℃for 30 seconds for a total of 40 cycles. The DPP4 RNA content in the samples was calculated from the Ct values of the respective samples.

The expression level of the target gene DPP4 mRNA of each sample was calculated by a DeltaCt relative quantification method. The relative expression level of the target gene is expressed by using 2-delta CT, and the calculation method is as follows:

a) The Ct value is automatically calculated according to the default settings of the Quant Studio 7 software. The Ct value is exported as an Excel file.

b) The relative expression amount of the gene was calculated using the following formula:

delta ct=ct (gene of interest) -Ct (gapdh)

ΔΔΔΔ ct=Δ Ct (detection) sample) -delta Ct (Mock)

mRNA expression relative to Mock = 2 ^-ΔΔCt

Wherein Mock represents a negative control to which equal concentrations of Lipofectamine RNAiMax were added but no siRNA.

The inhibition rate was calculated as follows: (1- (2 ^-ΔΔCt ))*100％

Table 5 shows the inhibitory activity of the unmodified siRNA of the present invention against DPP4

TABLE 5 inhibition of DPP4 RNA by unmodified siRNA of the invention

Duplex name	Average inhibition ratio (20 nM)	Standard deviation of inhibition ratio (20 nM)
			JN9601	80.62	0.04
JN9602	79.56	0.06
			JN9603	75.45	0.05
JN9604	87.39	0.05
			JN9605	78.67	0.03
JN9606	86.75	0.07
			AD-1420422.1	69.82	0.04
JN9600	6.15	0.05

Table 6 shows the inhibitory activity of the modified siRNA of the present invention against DPP4

TABLE 6 inhibition of DPP4 RNA by modified siRNA of the invention

Duplex name	Average inhibition ratio (20 nM)	Standard deviation of inhibition ratio (20 nM)
			JNM96001	66.7	2.39
JNM96002	75.53	1.38
			JNM96003	73.04	3.21
JNM96004	70.23	1.29
			JNM96005	74.1	2.7
JNM96006	69.48	1.02
			JNM96007	65.89	2.24
JNM96008	69.02	3.49
			JNM96009	67.09	4.45
JNM96010	77.93	2.21
			JNM96011	82.19	1.58
JNM96012	80.34	1.23
			JNM96013	70.97	1.54
JNM96014	73.46	2.46
			JNM96015	73.02	1.33
JNM96016	78.09	1.92
			JNM96017	81.83	1.99
JNM96018	81.34	1.39
			AD_1420422.1	65.12	2.44

The siRNA conjugates of the invention have DPP4 inhibitory activity (IC ₅₀ ) Data fitting using GraphPad to derive IC ₅₀ Numerical values.

IC ₅₀ ＝log(inhibitor)vs.normalized response-Variable slope

Table 7 shows the inhibitory activity of the siRNA conjugates of the invention on DPP4 (IC ₅₀ )。

TABLE 7 inhibitory Activity of siRNA conjugates of the invention on DPP4 (IC ₅₀ )

Duplex name	DPP4 RNA inhibitory Activity IC50 (nM)
		JN L1	1.9
JN L2	2.0
		JN L3	1.8
JN L4	18
		JN L5	4.3
JN L6	4.5
		JN L7	5.8
JN L8	6.0
		AD_1420422.1 control	7.6

In this example, AD-1420422.1 (the sequence with the best silencing effect in WO 2022/066847 A1) was used as a control.

While the invention has been illustrated by the foregoing specific examples, it should not be construed as being limited thereto; but rather the invention encompasses the generic aspects previously disclosed. Various modifications and derivatives and embodiments may be made without departing from the spirit and scope of the invention.

Claims

1. An 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 comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, which are at least partially complementary in opposite phase to form a double-stranded region, the nucleotide sequence I and the nucleotide sequence II being selected from the group of sequences set forth in (I) - (vi):

5’-CUUAUAGACUGAAGUUAUAUU-3’(SEQ ID NO：1)

5’-UAUAACUUCAGUCUAUAAGUA-3’(SEQ ID NO：2)

5’-GCAACUUCCAUACAAAUCAUU-3’(SEQ ID NO：3)

5’-UGAUUUGUAUGGAAGUUGCAU-3’(SEQ ID NO：4)

5’-GUUACAGACACAUUUGUUAUU-3’(SEQ ID NO：5)

5’-UAGCAAAUGUGUCUGUAACCU-3’(SEQ ID NO：6)

5’-CGGAAAGGUGUCAGUACUAUU-3’(SEQ ID NO：7)

5’-UAGUACUGACACCUUUCCGGA-3’(SEQ ID NO：8)

5’-CAGUAAAGAGGCGAAGUAUUU-3’(SEQ ID NO：9)

5’-AUACUUCGCCUCUUUACUGAA-3’(SEQ ID NO：10)

5’-CGUGUUCAAGUGUGGAAUAUU-3’(SEQ ID NO：11)

5’-UAUUCCACACUUGAACACGCC-3’(SEQ ID NO：12)。

2. The siRNA of claim 1, wherein said nucleotide sequence I is identical to SEQ ID NO:1, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:2 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in 2;

alternatively, the nucleotide sequence I is identical to SEQ ID NO:3, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:4 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in fig;

alternatively, the nucleotide sequence I is identical to SEQ ID NO:5, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:6 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in fig. 6;

alternatively, the nucleotide sequence I is identical to SEQ ID NO:7, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:8 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in figure 8;

alternatively, the nucleotide sequence I is identical to SEQ ID NO:9, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:10 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in fig;

Alternatively, the nucleotide sequence I is identical to SEQ ID NO:11, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:12 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown.

3. siRNA according to claim 1 or 2, wherein at least one nucleotide of said sense strand or said antisense strand is a modified nucleotide, preferably all nucleotides of said sense strand and/or said antisense strand are modified nucleotides, and/or at least one phosphate group is a phosphate group with a modification group.

4. The siRNA of any of claims 1-3, wherein each nucleotide in the sense strand and the antisense strand is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

5. The siRNA according to claim 4 wherein each non-fluoro modified nucleotide is independently selected from one of nucleotides or nucleotide analogs formed by substitution of the hydroxyl group at the 2' position of the ribosyl of the nucleotide with a non-fluoro group.

6. The siRNA according to claim 5, wherein,

the nucleotide formed by substituting the hydroxyl at the 2 '-position of the ribosyl of the nucleotide with a non-fluorine group is selected from one of a 2' -alkoxy modified nucleotide, a 2 '-substituted alkoxy modified nucleotide, a 2' -alkyl modified nucleotide, a 2 '-substituted alkyl modified nucleotide, a 2' -amino modified nucleotide, a 2 '-substituted amino modified nucleotide and a 2' -deoxynucleotide;

The nucleotide analogue is selected from an iso nucleotide, a bridged nucleotide or an acyclic nucleotide;

in particular, an isonucleotide is a compound in which a base is displaced from the 1' -position to the 2' -position or the 3' -position of the ribose ring;

the bridged nucleotide is one selected from LNA shown in formula (6), ENA shown in formula (7) and cET shown in formula (8),

the acyclic nucleotide is one selected from the group consisting of UNA represented by formula (9) and GNA represented by formula (10),

wherein in the above formulae (6) to (10), base represents a nucleobase, R _a Selected from H, OH or C ₁ -C ₁₀ Alkoxy (O-alkyl).

7. The siRNA of any of claims 4-6, wherein each non-fluoro modified nucleotide is a methoxy modified nucleotide, preferably said methoxy modified nucleotide is a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with methoxy.

8. The siRNA according to claim 3, wherein the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond of a phosphate group with a sulfur atom.

9. The siRNA of claim 3 or 8, wherein the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (13):

10. The siRNA according to claim 8 or 9, wherein,

the phosphorothioate linkage is present at least one of 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 above;

alternatively, phosphorothioate linkages may be present at all of the above positions except at the 5' end of the sense strand,

alternatively, phosphorothioate linkages are present at all of the above positions except the 3' end of the sense strand.

11. The siRNA of claim 1, wherein the 5' terminal nucleotide of the antisense strand is a 5' -phosphonucleotide or a 5' -phosphoanalog modified nucleotide;

in particular, the nucleotide 5 '-phosphate is a nucleotide containing a modification of 5' -phosphate represented by the formula (14),

the 5' -phosphate analogue modified nucleotide is a nucleotide containing a vinyl phosphate modification as shown in formula (15) or a phosphorothioate modification as shown in formula (17),

12. The siRNA of any one of claims 1-11, selected from the following table:

13. An siRNA conjugate comprising the siRNA of any one of claims 1-12 and a conjugate group conjugated to the siRNA.

14. The siRNA conjugate of claim 13, wherein the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked.

15. The siRNA conjugate of claim 14, wherein the linker has a structure as shown in formula (19):

wherein m is an integer of 1 to 3;

16. The siRNA conjugate of any of claims 13 to 15, wherein said connector is attached to the 3 'end of the sense strand or the 5' end of the sense strand of said siRNA.

17. The siRNA conjugate of claim 13, wherein the conjugate has a structure represented by formula (24), (25) or (26):

wherein R is ₂ A group having a structure represented by the formula (S1):

wherein E is ₁ OH or SH;

nu is the siRNA of any one of claims 1-12;

each L ₁ Independently is a linear alkylene group of 1 to 40 carbon atoms in length, 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 、C ₂ -C ₁₀ Alkenylene, C ₂ -C ₁₀ Alkynylene, C ₆ -C ₁₀ Arylene group, C ₃ -C ₁₈ Heterocyclylene and C ₅ -C ₁₀ Heteroarylene; and wherein R is ₁ Optionally having substituents of any one or more of the group consisting of: c (C) ₁ -C ₁₀ Alkyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkyl, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ Alkyl group-SH、-SC ₁ -C ₁₀ Haloalkyl, halogen substituent, -OH, -SH, -NH ₂ 、-C ₁ -C ₁₀ alkyl-NH ₂ 、-N(C ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -NH (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkylphenyl), -NH (C) ₁ -C ₁₀ Alkylphenyl), cyano, -CO ₂ H、C(O)O(C ₁ -C ₁₀ Alkyl), -CON (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -CONH (C) ₁ -C ₁₀ Alkyl), -CONH ₂ 、-NHC(O)(C ₁ -C ₁₀ Alkyl), -NHC (O) (phenyl), -N (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (C ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ Alkyl, -C (O) C ₁ -C ₁₀ Alkylphenyl, -C (O) C ₁ -C ₁₀ Haloalkyl, -OC (O) C ₁ -C ₁₀ Alkyl, -SO ₂ (C ₁ -C ₁₀ Alkyl), -SO ₂ (phenyl) -SO ₂ (C ₁ -C ₁₀ Haloalkyl) -SO ₂ NH ₂ 、-SO ₂ NH(C ₁ -C ₁₀ Alkyl), -SO ₂ NH (phenyl) -NHSO ₂ (C ₁ -C ₁₀ Alkyl), -NHSO ₂ (phenyl) and-NHSO ₂ (C ₁ -C ₁₀ A haloalkyl group);

M ₁ represents a targeting group;

indicating the site of covalent attachment of the group.

18. The siRNA conjugate of claim 17, wherein each L ₁ Independently selected from the group consisting of ₁ -A ₁₄ And any combination of:

wherein each k1 is independently an integer from 1 to 20;

each k2 is independently an integer from 1 to 20;

each R _c Independently C ₁ -C ₁₀ An alkyl group;

each R _e Independently C ₁ -C ₁₀ An alkyl group;

indicating the site of covalent attachment of the group.

19. The siRNA conjugate of claim 18, wherein L ₁ Is a linked combination of at least 2 of the groups A1, A4, A8, A10, A11.

20. The siRNA conjugate of any of claims 17 to 19, wherein L ₁ Is 3 to 20 atoms in length.

21. The siRNA conjugate of any of claims 18 to 20, wherein k1 is an integer from 3 to 5, k2 is an integer from 3 to 5, R _c Is one of methyl, ethyl and isopropyl, R _d Is A15 or A16, R _e Is one of methyl, ethyl, isopropyl and butyl.

22. The siRNA conjugate of any of claims 14 to 21, wherein each of said targeting groups is independently one selected from the group consisting of D-galactose, L-galactose, a-D-glucopyranose, β -D-glucopyranose, a-D-glucofuranose, β -D-glucofuranose, a-D-fructofuranose, a-D-galactopyranose, β -D-galactopyranose, a-D-galactofuranose, β -D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyryl galactosamine, N-isobutyryl galactosamine.

23. The siRNA conjugate of claim 22, wherein at least one or each of said targeting groups is galactose or N-acetylgalactosamine.

24. The siRNA conjugate of any of claims 17 to 23, wherein R ₁ Containing both a linking site attached to an N atom on a nitrogen-containing backbone and R ₂ A linking site to which the P atom is linked.

25. The siRNA conjugate of any of claims 17 to 24, wherein R ₁ The above-mentioned site bonded to N atom on the nitrogen-containing skeleton forms an amide bond with N, the above-mentioned site bonded to R ₂ The P atom on the substrate is linked to P to form a phosphate bond or a phosphorothioate bond.

26. The siRNA conjugate of claims 17-25, wherein the P atom in formula (S1) is attached to the end of the sense strand or the antisense strand of the siRNA.

27. The siRNA conjugate of any of claims 17-26, wherein the P atom in formula (S1) is linked to the 2' position, the 3' position or the 5' position of a nucleotide in the siRNA via a phosphodiester linkage.

28. The siRNA conjugate according to claim 13, wherein the siRNA conjugate has a structure represented by the formula (Z1-Nu), (Z2-Nu), (Z3-Nu), (Z4-Nu), (Z5-Nu), (Z6-Nu), (Z7-Nu), (Z8-Nu), (Z9-Nu), (Z10-Nu), (Z11-Nu), (Z12-Nu), (Z13-Nu), (Z14-Nu), (Z15-Nu), (Z16-Nu), (Z17-Nu), (Z18-Nu), (Z19-Nu), (Z20-Nu), (Z21-Nu), (Z22-Nu), (Z23-Nu), (Z24-Nu), (Z25-Nu), (Z26-Nu), (Z27-Nu), (Z28-Nu), (Z29-Nu), (Z30-Nu), (Z31-Nu) or (Z32-Nu),

Wherein Nu is the siRNA of any one of claims 1-12.

29. The siRNA conjugate of claim 13, wherein the siRNA conjugate has a structure as shown in formula (23):

wherein the double helix structure represents the siRNA of any one of claims 1-12.

30. Use of the siRNA of any one of claims 1-12 and/or the siRNA conjugate of any one of claims 13-29 in the manufacture of a medicament for inhibiting dipeptidyl peptidase 4 gene expression in a cell.

31. A pharmaceutical composition comprising the siRNA of any one of claims 1-12 and/or the siRNA conjugate of any one of claims 13-29 and optionally a pharmaceutically acceptable carrier.

32. Use of the siRNA of any one of claims 1-12, the siRNA conjugate of any one of claims 13-29 and/or the pharmaceutical composition of claim 31 in the manufacture of a medicament for treating a disease and/or disorder associated with dipeptidyl peptidase 4 gene expression.

33. The use according to claim 32, wherein the disease and/or condition associated with dipeptidyl peptidase 4 gene expression is diabetes or a lipid metabolism disease.

34. A method of inhibiting the expression of a dipeptidyl peptidase 4 gene in a cell, the method comprising contacting an effective amount of the siRNA of any one of claims 1-12, the siRNA conjugate of any one of claims 13-29, and/or the pharmaceutical composition of claim 31 with the cell, thereby inhibiting the expression of the dipeptidyl peptidase 4 gene in the cell.