SiRNA, conjugate and pharmaceutical composition for reducing CIDEB expression

Abstract

The invention discloses siRNA for reducing CIDEB expression, a conjugate and a pharmaceutical composition, and belongs to the field of biological medicine. The siRNA, the conjugate of the siRNA and the pharmaceutical composition containing the siRNA and the conjugate can specifically target the liver, so that cell death-induced DFFA-like effector B gene expression can be inhibited, the siRNA and the conjugate have relatively low off-target effect and good stability, and diseases and / or symptoms related to cell death-induced DFFA-like effector B expression can be treated.

Description

Technical Field

[0001] The present invention discloses siRNAs, conjugates and pharmaceutical compositions for reducing CIDEB expression, belonging to the field of biomedicine. Background Art

[0002] Cell death-inducing DFFA-like effector B (CIDEB) is a member of the CIDE protein family and is mainly expressed in the liver and small intestine. CIDEB is an endoplasmic reticulum (ER) and lipid droplet (LD)-associated protein. Overexpression of the CIDEB protein can induce cell death. The physiological function of CIDEB is closely related to multiple lipid metabolic pathways, especially the very low density lipoprotein pathway. Apolipoprotein B (apoB) is an important apolipoprotein molecule and is a key molecule of chylomicron (CM) and very low density lipoprotein (VLDL). Abnormal synthesis and secretion of VLDL are closely related to metabolic diseases such as hyperlipidemia, fatty liver, diabetes, and atherosclerosis. Existing results show that CIDEB can regulate the secretion of triglyceride-rich very low density lipoprotein in the liver and the secretion of triglyceride-rich chylomicrons in the small intestine by interacting with apoB. CIDEB knockout reduces the levels of cholesterol and low density lipoprotein in the blood and heart, while the cholesterol level increases in the liver due to increased expression of low density lipoprotein receptors. In summary, CIDEB plays an important role in maintaining the homeostasis of triglyceride and cholesterol in the body.

[0003] Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease in all regions of the modern industrialized economies, including Korea and many other Asian countries. The incidence of non-alcoholic fatty liver in the global population exceeds one-third, paralleling the sharp rise in the incidence of obesity in the global population. The harms of non-alcoholic fatty liver are multifaceted. It not only damages the normal functions of liver cells, affects the glycolipid metabolism process in the liver and even the whole-body metabolic homeostasis. Non-alcoholic fatty liver will develop into non-alcoholic steatohepatitis (NASH). The inflammatory response will exacerbate the damage of liver cells. Further, liver cells begin to die, and the collagen in the cell stroma begins to increase, replacing the dead liver cells, and the liver begins to develop liver fibrosis. Over time, the dead liver cells are degraded, forming aggregated scar tissue. These scars gradually harden the liver and inhibit the normal functions of the liver, leading to the occurrence of liver cirrhosis. Liver cirrhosis causes the liver to lose its normal metabolic function, leading to liver failure and may induce liver cancer. More and more evidence shows that NAFLD has systemic effects and is related to type 2 diabetes, cardiovascular diseases, chronic kidney diseases, and certain types of extrahepatic malignancies, seriously threatening human health.

[0004] For the treatment of NAFLD, etiological treatment is widely adopted, including stopping the use of drugs or poisons, weight loss, and treating lipid disorders or hyperglycemia, etc. For example, for NAFLD patients with an increased risk of cardiovascular diseases, statin treatment is advocated to reduce the risk; for NAFLD patients with type 2 diabetes and chronic kidney diseases, it is recommended to consider metformin and GLP-1 analogs, or pioglitazone and SGLT2 inhibitors; lifestyle interventions such as increasing physical activity and weight loss can effectively improve the early liver diseases of NAFLD patients and improve blood glucose control, cardiovascular diseases, and risk factors for chronic kidney diseases. At present, there are also many emerging therapies for NASH targeting several different molecular pathways, including peroxisome proliferator-activated receptor alpha (PPAR-alpha), glucagon-like peptide 1 (GLP-1) modulators, and farnesoid X receptor (FXR) ligands, etc. The development of the vast majority of drugs is targeted at the targets corresponding to the driving factors of NAFLD, but so far there is no exact drug that is absolutely effective for this disease, and many drugs are still in the clinical research stage.

[0005] Therefore, there is a need to improve the methods for treating chronic liver inflammatory diseases. Summary of the Invention

[0006] The following siRNA and its modified sequences provided by the present invention can specifically inhibit the expression of the cell death-inducing DFFA-like effector B (CIDEB) gene. The pharmaceutical composition or siRNA conjugate containing the siRNA can specifically target the liver, thereby inhibiting the regulation of the cell death-inducing DFFA-like effector B (CIDEB) gene expression and achieving the treatment of diseases and / or disorders associated with non-alcoholic fatty liver disease.

[0007] In some embodiments, the present invention provides a first siRNA capable of inhibiting the expression of the cell death-inducing DFFA-like effector B (CIDEB) gene. The siRNA contains a sense strand and an antisense strand. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide. Among them, the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. Among them, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO.1 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO.2 and has no more than 3 nucleotide differences:

[0008] 5’-ACUGCUGAAUGGAGUGCUAUU-3’ (SEQ ID NO.1);

[0009] 5’-UAGCACUCCAUUCAGCAGUUU-3’ (SEQ ID NO.2).

[0010] In some embodiments, the present invention provides a second siRNA capable of inhibiting the expression of the cell death-inducing DFFA-like effector B (CIDEB) gene. The siRNA contains a sense strand and an antisense strand. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide. Among them, the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. Among them, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO.3 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO.4 and has no more than 3 nucleotide differences:

[0011] 5’-CCUUUGACGUGUACAAGCAUU-3’ (SEQ ID NO.3);

[0012] 5’-UGCUUGUACACGUCAAAGGUU-3’ (SEQ ID NO.4).

[0013] In some embodiments, the present invention provides a third siRNA capable of inhibiting the expression of cell death-inducing DFFA-like effector B (CIDEB) gene. The siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide. Among them, the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. Among them, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO.5 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO.6 and has no more than 3 nucleotide differences:

[0014] 5’-CUCUAUGAGUUGUGACUUUUU-3’ (SEQ ID NO.5);

[0015] 5’-AAAGUCACAACUCAUAGAGUU-3’ (SEQ ID NO.6).

[0016] In some embodiments, the present invention provides an siRNA conjugate, which contains the above-mentioned siRNA and a conjugate group conjugated to the siRNA.

[0017] In some embodiments, the present invention provides a pharmaceutical composition, which contains the siRNA of the present invention, and / or the siRNA conjugate, and a pharmaceutically acceptable carrier.

[0018] In some embodiments, the present invention provides the use of the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA conjugate in the preparation of a drug for treating diseases caused by the expression of cell death-inducing DFFA-like effector B (CIDEB) gene.

[0019] In some embodiments, the present invention provides a method for inhibiting the expression of cell death-inducing DFFA-like effector B (CIDEB) gene, which includes contacting an effective amount of the siRNA and / or pharmaceutical composition and / or siRNA conjugate of the present invention with the hepatocytes.

[0020] Beneficial effects

[0021] The siRNA, pharmaceutical composition, and siRNA conjugate provided by the present invention have significantly enhanced plasma and lysosomal stability, and also have high cell death-inducing DFFA-like effector B mRNA inhibitory activity, low off-target effects, and / or can significantly treat diseases and / or disorders associated with non-alcoholic fatty liver disease.

[0022] In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate provided by the present invention exhibits excellent target gene inhibitory activity in in vitro experiments. In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate provided by the present invention exhibits a target gene expression inhibition rate of at least 50%, 60%, 70%, 80%, 90%, or 95% in hepatocytes.

[0023] The siRNA, pharmaceutical composition, or siRNA conjugate provided by the present invention does not show obvious off-target effects. The off-target effect can be, for example, inhibiting the normal expression of non-target genes. It is considered 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 on-target gene effect.

[0024] This shows that the siRNA, pharmaceutical composition, and siRNA conjugate provided by the present invention can inhibit the expression of cell death-inducing DFFA-like effector B (CIDEB) gene, effectively treat diseases and / or disorders associated with non-alcoholic fatty liver disease, and have good application prospects.

[0025] Other features and advantages of the present invention will be described in detail in the following detailed description section. Detailed Description

[0026] The following provides a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not used to limit the present invention.

[0027] In the invention, CIDEB mRNA refers to the mRNA having the sequence shown in GenBank accession number NM_001393338.1. Further, unless otherwise specified, the term "target gene" used in the present invention refers to the gene that transcribes and expresses CIDEB mRNA, and the term "target mRNA" refers to the above CIDEB mRNA.

[0028] Definitions

[0029] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without a specific definition, but should be understood in its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding product or its active ingredient.

[0030] As used herein, the term "link" or "linked", when referring to the connection between two molecules, means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (e.g., hydrogen bond or ionic bond).

[0031] As used herein, an "oligonucleotide" is a nucleotide sequence containing 10 - 50 nucleotides or nucleotide base pairs. In some embodiments of the present invention, the oligonucleotide has a nucleobase sequence that is at least partially complementary to the coding sequence in the target gene expressed intracellularly. The nucleotides may be optionally modified. In some embodiments of the present invention, after the oligonucleotide is delivered to a cell expressing a gene, the oligonucleotide is capable of inhibiting or blocking gene expression in vitro or in vivo.

[0032] As used herein, "Cell Death Inducing DFFA - like Effector B" (CIDEB), which is interchangeable with the term "CIDEB", refers to a class of membrane proteins located on the endoplasmic reticulum and lipid droplets, which can regulate the secretion of very - low - density lipoproteins in the liver and participate in the regulation of cholesterol metabolism. Other examples of CIDEB mRNA sequences can be easily obtained using public databases such as GenBank, UniProt, and OMIM.

[0033] As used herein, the term "non - alcoholic fatty liver - related diseases and / or disorders" or "CIDEB - related diseases" refers to diseases or disorders caused by or associated with over - replication of CIDEB. The term "CIDEB - related diseases" includes diseases, disorders, or conditions that benefit from reduced CIDEB gene expression or replication. Non - limiting examples of CIDEB - related diseases include, for example, non - alcoholic hepatic steatosis, non - alcoholic steatohepatitis, non - alcoholic cirrhosis, liver cancer, obesity, type 2 diabetes, cardiovascular diseases, chronic kidney diseases, dyslipidemia, and certain types of extra - hepatic malignancies.

[0034] As used herein, the term "inhibit", when expressing a given gene, means that gene expression is reduced when a cell, cell population, or tissue is treated with the siRNA, pharmaceutical composition, and siRNA conjugate described herein compared to a cell, cell population, or tissue that has not been treated.

[0035] As used herein, the term "inhibit" is interchangeable with "reduce", "silence", "down - regulate", "suppress", and other similar terms, and includes any level of inhibition. Preferably, inhibition includes statistically significant inhibition or clinically significant inhibition.

[0036] As used in the present invention, the phrase "inhibiting the expression of CIDEB" or "inhibiting the expression of the CIDEB gene" includes inhibiting any CIDEB gene (e.g., the CIDEB gene expressed by CIDEB in CIDEB, the CIDEB gene expressed by an expression construct in a cell), as well as inhibiting the expression of variants or mutants of the CIDEB gene encoding the CIDEB protein. This term includes any CIDEB transcript encoding one or more CIDEB proteins, as well as knockdown of variants and mutants of the CIDEB gene.

[0037] 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 specified, "conjugation" refers to the connection of two or more chemical moieties each having a specific function to each other in a covalent linkage manner; accordingly, a "conjugate" refers to a compound formed by the covalent linkage between these individual chemical moieties. Further, an "siRNA conjugate" refers to a compound formed by covalently linking one or more chemical moieties having specific functions to an siRNA. Hereinafter, the siRNA conjugate of the present invention is sometimes also simply referred to as a "conjugate". The siRNA conjugate should be understood, according to the context, as the general term for siRNA conjugates, the first siRNA conjugate or the second siRNA conjugate, or an siRNA sense strand conjugate or an siRNA antisense strand conjugate.

[0038] In some embodiments, the conjugating group can be linked to the phosphate group, 2'-hydroxyl group, 5'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugating group can also be linked to the 3'-hydroxyl group, in which case the nucleotides are linked by a 2'-5' phosphodiester bond. When the conjugating group is linked to the end of the siRNA strand, the conjugating group is usually linked to the phosphate group of the nucleotide; when the conjugating group is linked to the internal sequence of the siRNA, the conjugating group is usually linked to the ribose sugar ring or the base. Various linking methods can be referred to in the literature: Muthiah Manoharan et.al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5):1181-7.

[0039] In some embodiments, the siRNA and the conjugating group can be linked by acid-labile or reducible chemical bonds, which can be degraded in the acidic environment of the endosome, enabling the siRNA to become free. For non-degradable conjugation methods, the conjugating group can be attached to the sense strand of the siRNA to minimize the impact of conjugation on the siRNA activity.

[0040] In the foregoing and the following, unless otherwise specified, generally, "G", "C", "A", "T", and "U" each represent a nucleotide containing guanine, cytosine, adenine, thymine, and uracil as bases. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides and nucleotide surrogates as further detailed below.

[0041] 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;

[0042] Af, Cf, Gf, and Uf are 2'-fluoroadenosine 3'-phosphate, 2'-fluorocytidine 3'-phosphate, 2'-fluoroguanosine 3'-phosphate, and 2'-fluorouridine 3'-phosphate, respectively;

[0043] dA, dC, dG, and dT are 2'-deoxyadenosine 3'-phosphate, 2'-deoxycytidine 3'-phosphate, 2'-deoxyguanosine 3'-phosphate, and 2'-deoxythymidine 3'-phosphate, respectively;

[0044] (Agn) is glycol nucleic acid (GNA); and s is a phosphorothioate linkage.

[0045] In the context of the present invention, the expressions "complementary" or "reverse complementary" can be used interchangeably and have the meanings well-known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of 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 to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand. Correspondingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at corresponding positions do not pair in a complementary form.

[0046] As used hereinabove and hereinafter, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between two nucleotide sequences involved; "essentially reverse complementary" means that there is no more than 1 base mismatch between two nucleotide sequences; "fully reverse complementary" means that there is no base mismatch between two nucleotide sequences. As used hereinabove and hereinafter, when a nucleotide sequence has a "nucleotide difference" from another nucleotide sequence, it means that compared with the latter, the base type of the nucleotide at the same position has changed. For example, when a nucleobase in the latter is A, if the corresponding nucleobase at the same position in the former is U, C, G or T, it is determined that there is a nucleotide difference between the two nucleotide sequences at this position. In some embodiments, when a nucleotide at the original position is replaced by a nucleoside without a base or its equivalent, a nucleotide difference is also considered to have occurred at this position.

[0047] As used hereinabove and hereinafter, especially when describing the preparation methods of the siRNAs, pharmaceutical compositions containing siRNAs or siRNA conjugates of the present invention, unless otherwise specified, the "nucleoside monomer" refers to the modified or unmodified RNA phosphoramidite monomers (sometimes RNA phosphoramidites are also referred to as Nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis according to the types and sequences of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid-phase synthesis is a method used in RNA synthesis well-known to those skilled in the art. The nucleoside monomers used in the present invention can all be commercially purchased.

[0048] As used herein, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and this description includes the cases where the event or condition occurs and the cases where it does not occur. For example, "optionally substituted" "alkyl" includes "alkyl" and "substituted alkyl" as defined hereinafter. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitutions or substitution patterns that are spatially impracticable, synthetically infeasible and / or inherently unstable.

[0049] The term "subject", as used herein, refers to any animal, such as a mammal or a marsupial. The subjects of the present 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.

[0050] As used herein, "treatment" refers to a method of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradicating or ameliorating the underlying disorder being treated. In addition, a therapeutic benefit is obtained by observing an improvement in a subject through eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, even though the subject may still be afflicted with the underlying disorder.

[0051] In one aspect, the present invention provides first to third siRNAs capable of inhibiting CIDEB gene expression. They are described in detail below in turn.

[0052] The siRNA of the present invention contains nucleotide groups as basic structural units. As is well known to those skilled in the art, the nucleotide groups contain phosphate groups, ribose groups, and bases, which will not be elaborated herein.

[0053] First siRNA

[0054] According to the present invention, the siRNA can be the first siRNA.

[0055] The first siRNA contains a sense strand and an antisense strand. Each nucleotide in the first siRNA is independently a modified or unmodified nucleotide. Among them, the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. Among them, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO.1 and no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO.2 and no more than 3 nucleotide differences:

[0056] 5’-ACUGCUGAAUGGAGUGCUAUU-3’(SEQ ID NO.1);

[0057] 5’-UAGCACUCCAUUCAGCAGUUU-3’(SEQ ID NO.2).

[0058] In some embodiments, the sense strand only contains the nucleotide sequence I, and the antisense strand only contains the nucleotide sequence II. In some embodiments, there are no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO.1, and / or no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO.2.

[0059] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or fully reverse complementary; the substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; the essentially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; and the fully reverse complementary means that there are no base mismatches between the two nucleotide sequences.

[0060] The second siRNA

[0061] According to the present invention, the siRNA can be the second siRNA.

[0062] The second siRNA contains a sense strand and an antisense strand. Each nucleotide in the second siRNA is independently a modified or unmodified nucleotide. Among them, the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. Among them, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO.3 and no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO.4 and no more than 3 nucleotide differences:

[0063] 5’-CCUUUGACGUGUACAAGCAUU-3’ (SEQ ID NO.3);

[0064] 5’-UGCUUGUACACGUCAAAGGUU-3’ (SEQ ID NO.4).

[0065] In some embodiments, the sense strand only contains the nucleotide sequence I, and the antisense strand only contains the nucleotide sequence II. In some embodiments, there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO.3, and / or there is no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO.4.

[0066] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or fully reverse complementary.

[0067] The third siRNA

[0068] According to the present invention, the siRNA can be the third siRNA.

[0069] The third type of siRNA contains a sense strand and an antisense strand, and each nucleotide in the third type of siRNA is independently a modified or unmodified nucleotide. Among them, the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. Among them, the nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO.5 and has no more than 3 nucleotide differences, and the nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO.6 and has no more than 3 nucleotide differences:

[0070] 5’-CUCUAUGAGUUGUGACUUUUU-3’(SEQ ID NO.5);

[0071] 5’-AAAGUCACAACUCAUAGAGUU-3’(SEQ ID NO.6).

[0072] In some embodiments, the sense strand only contains the nucleotide sequence I, and the antisense strand only contains the nucleotide sequence II. In some embodiments, there are no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO.5, and / or there are no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO.6.

[0073] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary or completely reverse complementary.

[0074] As mentioned above, the nucleotides in the siRNA disclosed in the present invention are independently modified or unmodified nucleotides. In some embodiments, each nucleotide in the siRNA of the present invention is an unmodified nucleotide. In some embodiments, some or all of the nucleotides in the siRNA of the present invention are modified nucleotides, and these modifications on the nucleotide groups do not cause the function of the siRNA conjugate of the present invention to significantly weaken or lose its ability to inhibit CIDEB gene expression.

[0075] In some embodiments, the siRNA of the present invention can be any one of the unmodified siRNAs listed in Table 1.

[0076]

[0077] In some embodiments, the siRNA disclosed in the present invention contains at least one modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" refers to a nucleotide or nucleotide analogue formed by substituting the hydroxyl group at the 2'-position of the ribose of the nucleotide with another group, or a nucleotide having a modified base. The modified nucleotide does not cause a significant weakening or loss of the function of the siRNA to inhibit gene expression. For example, the modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55 can be selected.

[0078] In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present invention 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 part of the phosphate group and / or ribose group in the phospho-sugar backbone of at least one single strand among the sense strand and the antisense strand is a phosphate group having a modifying group and / or a ribose group having a modifying group.

[0079] In some embodiments, all nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA disclosed in the present invention is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

[0080] In the context of the present disclosure, "fluorinated modified nucleotide" refers to a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose of the nucleotide with fluorine, and having the structure shown in the following formula (1). "Non-fluorinated modified nucleotide" refers to a nucleotide or nucleotide analogue formed by substituting the hydroxyl group at the 2'-position of the ribose of the nucleotide with a non-fluorine group. In some embodiments, each non-fluorinated modified nucleotide is independently selected from a nucleotide or nucleotide analogue formed by substituting the hydroxyl group at the 2'-position of the ribose of the nucleotide with a non-fluorine group.

[0081] These nucleotides formed by substituting the hydroxyl group at the 2'-position of the ribose with a non-fluorine group are well-known to those skilled in the art, and these nucleotides can be selected from one of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0082] In some embodiments, the 2'-alkoxy-modified nucleotide is a 2'-methoxy (2'-OMe)-modified nucleotide, as shown in formula (2). In some embodiments, the 2'-substituted alkoxy-modified nucleotide can be a 2'-methoxyethyl (2'-MOE)-modified nucleotide, as shown in formula (3). In some embodiments, the 2'-amino (2'-NH2)-modified nucleotide is as shown in formula (4). In some embodiments, the 2'-deoxynucleotide (DNA) is as shown in formula (5):

[0083]

[0084] A nucleotide analogue refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from that of adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide, or thymidine deoxyribonucleotide. In some embodiments, the nucleotide analogue can be a non-natural nucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0085] A bridged nucleotide (bridged nucleic acid, abbreviated as BNA) can contain a five-membered, six-membered, or seven-membered bridged structure with a "fixed" C3'-endo sugar puckering. This bridge is typically incorporated at the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, BNA can be LNA, ENA, cETBNA, etc., where LNA is as shown in formula (6), ENA is as shown in formula (7), and cETBNA is as shown in formula (8).

[0086] An acyclic nucleotide is a type of nucleotide formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide can be unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), where UNA is as shown in formula (9) and GNA is as shown in formula (10):

[0087]

[0088] In the above formulas (9) and (10), R a is selected from H, OH, or alkoxy (O-alkyl).

[0089] A non-natural nucleotide refers to a compound formed by changing the position of the base on the ribose ring in a nucleotide. In some embodiments, the non-natural nucleotide can be a compound formed by moving the base from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formulas (11) or (12):

[0090]

[0091] In the compounds shown in the above formulas (11) and (12), Base represents a nucleic acid base, such as A, U, G, C, or T; R b is selected from H, OH, F, or the non-fluorinated group as described above.

[0092] In some embodiments, the nucleotide analog is selected from one of locked nucleic acid (LNA), 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), cEt, unlocked nucleic acid (UNA), and glycol nucleic acid (GNA). In some embodiments, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide. As used herein and hereinafter, the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0093] As used herein and hereinafter, "fluorinated modified nucleotide", "2'-fluorinated modified nucleotide", "nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by fluorine", and "nucleotide having a 2'-fluorinated ribose group" have the same meaning, and all refer to a compound having the structure shown in formula (1) formed by replacing the 2'-hydroxyl group of the nucleotide with fluorine; "methoxy-modified nucleotide", "2'-methoxy-modified nucleotide", "nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group", and "nucleotide having a 2'-methoxy ribose group" have the same meaning, and all refer to a compound having the structure shown in formula (2) formed by replacing the 2'-hydroxyl group of the ribose group of the nucleotide with a methoxy group.

[0094] In some embodiments, the sense strand or the antisense strand of the siRNA provided by the present invention may further include base modification or substitution.

[0095] The siRNA with the above modifications can make ribonuclease in the blood less likely to cleave nucleic acids, thereby increasing the stability of nucleic acids and making nucleic acids have stronger resistance to nuclease hydrolysis. At the same time, the above-modified siRNA has a high activity of inhibiting target mRNA.

[0096] In some embodiments, at least a part of the phosphate ester groups in the phospho-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA provided by the present invention is a phosphate ester group having a modifying group. In some embodiments, the phosphate ester group having a modifying group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom; in some embodiments, the phosphate ester group having a modifying group is a phosphorothioate group having the structure shown in formula (13):

[0097]

[0098] This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.

[0099] In some embodiments, in the siRNA provided by the present invention, phosphorothioate linkages are present at least at one position in 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 at the 5' end of the sense strand. In some embodiments, phosphorothioate linkages are present at all of the above positions except at the 3' end of the sense strand.

[0100] In some embodiments, the 5'-terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analogue.

[0101] The commonly used 5'-phosphate nucleotides or nucleotides modified with 5'-phosphate analogues are well known to those skilled in the art. For example, the 5'-phosphate nucleotide may have a structure as shown in formula (14):

[0102]

[0103] Furthermore, as disclosed in Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48, the following 5 types of nucleotides modified with 5'-phosphate analogues are disclosed:

[0104]

[0105] Among them, R is selected from Η, OH, methoxy, fluorine; Base represents a nucleic acid base and is selected from A, U, C, G or T.

[0106] In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (14), and the nucleotide modified with a 5'-phosphate analogue is a nucleotide containing a vinyl phosphate modification, as shown in formula (15), or a nucleotide containing a phosphorothioate modification, as shown in formula (17).

[0107] Table 2 shows the abbreviations of the modified nucleotide monomers disclosed herein.

[0108] Table 2. Abbreviations of the nucleotide monomers disclosed in the present invention

[0109]

[0110]

[0111] In some embodiments, the siRNA provided by the present invention is any one of the siRNAs listed in Table 3.

[0112]

[0113] The above-mentioned siRNA provided by the present invention not only has significantly enhanced plasma and lysosomal stability, but also has a very high target mRNA inhibitory activity.

[0114] The siRNA provided by the present invention can be obtained by conventional siRNA preparation methods in the art (such as solid-phase synthesis). Among them, solid-phase synthesis has commercial customization services. Modified nucleotide groups can be introduced into the siRNA of the present invention by using nucleoside monomers with corresponding modifications, and the methods for preparing nucleoside monomers with corresponding modifications and the methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0115] siRNA conjugate

[0116] The present invention provides an siRNA conjugate, which contains the above-mentioned siRNA and a conjugate group conjugated to the siRNA.

[0117] Generally speaking, the conjugate group contains at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker and the targeting group are connected in sequence. In some embodiments, the number of the targeting groups is 2-4. The siRNA molecule can be conjugated to the conjugate group non-covalently or covalently, for example, it can be conjugated to the conjugate group covalently. The conjugation site of the siRNA and the conjugate group can be at the 3'-end or 5'-end of the sense strand of the siRNA, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and the conjugate group is at the 3'-end or 5'-end of the sense strand of the siRNA.

[0118] In some embodiments, the conjugating group can be linked to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugating group can also be linked to the 3'-hydroxyl group, in which case the nucleotides are linked by 2'-5' phosphodiester bonds. When the conjugating group is linked to the end of the siRNA strand, the conjugating group is usually linked to the phosphate group of the nucleotide; when the conjugating group is linked to the internal sequence of the siRNA, the conjugating group is usually linked to the ribose sugar ring or the base. For various linking methods, reference can be made to the literature: Muthiah Manoharan et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.

[0119] In some embodiments, the siRNA and the conjugating group can be connected by an acid-labile or reducible chemical bond, which can be degraded in the acidic environment of the endosome, so that the siRNA becomes free. For non-degradable conjugation methods, the conjugating group can be linked to the sense strand of the siRNA to minimize the impact of conjugation on the activity of the siRNA.

[0120] In some embodiments, the pharmaceutically acceptable targeting group can be a ligand commonly used in the field of siRNA administration, such as various ligands described in W02009082607A2, the entire disclosure of which is incorporated herein by reference.

[0121] 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-propionylgalactosamine, N-n-butyrylgalactosamine, and N-isobutyrylgalactosamine (see, for example, Iobst, S.T. and Drickamer, K. J. B. C. 1996, Vol. 271, p. 6686). Galactose derivatives and clusters of galactose derivatives useful for targeting the liver in vivo by oligonucleotides and other molecules are known in the art (see, for example, 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 the asialoglycoprotein receptor (ASGPr) expressed on the surface of hepatocytes. Binding of the ASGPr ligand to the ASGPr(s) facilitates cell-specific targeting of hepatocytes and endocytosis of the molecule into hepatocytes. The ASGPr ligand can be monomeric (e.g., having a single galactose derivative) or polymeric (e.g., having multiple galactose derivatives). Methods known in the art can be used to link galactose derivatives or clusters of galactose derivatives to the 3'- or 5'-end of the siRNA.

[0122] In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate after the siRNA molecule forms an siRNA conjugate with the conjugating group containing the galactose or N-acetylgalactosamine molecule as the targeting group being 1:1, 1:2, 1:3, or 1:4. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA of the present invention is conjugated to a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA of the present invention is conjugated to a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0123] The targeting group can be linked to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group. For the types of these linkers, targeting groups and the connection mode with siRNA, reference can be made to the disclosure of W02015006740A2, and the whole content thereof is incorporated into the present invention by reference.

[0124] In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker can have the structure shown in formula (19):

[0125]

[0126] Wherein, m is an integer from 1 to 3;

[0127] L A is a chain-like part containing an amide bond having the structure shown in formula (20), and each of the Ls A is respectively connected to one of the targeting groups and the L C part by an ether bond at both ends:

[0128]

[0129] L B is a chain-like part containing N-acylpyrrolidine having the structure shown in formula (21), the chain-like part has a carbonyl group at one end and is connected to the L C part by an amide bond, and has an oxygen atom at the other end and is connected to the siRNA by a phosphate bond:

[0130]

[0131] L C is a 2-4 valent linking group based on hydroxymethylaminomethane, bis(hydroxymethyl)aminomethane or tris(hydroxymethyl)aminomethane, and the L C is connected to each of the L A parts by an ether bond via an oxygen atom, and is connected to the L B part by an amide bond via a nitrogen atom.

[0132] In some embodiments, as the linker, the siRNA conjugate formed by -(L A )3 tris(hydroxymethyl)aminomethane-L B - connecting the N-acetylgalactosamine molecule and the siRNA molecule has the structure shown in the following formula (22):

[0133]

[0134] In the formula, the double helix structure represents siRNA.

[0135] Similarly, the conjugation site of the siRNA and the conjugation group can be at the 3'-end or 5'-end of the sense strand of the siRNA, or can also be in the internal sequence of the siRNA.

[0136] In some embodiments, the 3'-end of the sense strand of the siRNA of the present invention is conjugated to three N-acetylgalactosamine (GalNAc) molecules through a linker-(L A )3 tris(hydroxymethyl)aminomethane-L B - to obtain an siRNA conjugate with a molar ratio of siRNA molecule to GalNAc molecule of 1:3, which may also be referred to as (GalNAc)3-siRNA hereinafter, and its structure is shown in the following formula (23):

[0137]

[0138] Wherein, the double helix structure represents the siRNA, and the linker is connected to the 3'-end of the sense strand of the siRNA.

[0139] In some embodiments, the linker is connected to the 5'-end of the sense strand of the siRNA.

[0140] In some embodiments, the siRNA conjugate has a structure shown in formula (24), (25) or (26)

[0141]

[0142] Wherein,

[0143] R2 is a group having a structure shown in formula (S1):

[0144]

[0145] Wherein, E1 is OH or SH, and Nu is the siRNA of the present invention;

[0146] R1 is a straight-chain alkylene or cyclic alkylene having 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more of the groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18 heterocycloalkylene and C5-C 10 heteroarylene; and wherein R1 may optionally have substituents of any one or more of the groups consisting of: C1-C 10 alkyl, C6-C 10 aryl, C5-C10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Haloalkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 )Alkyl(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 )Alkylphenyl), cyano, -CO2H, C(O)O(C1-C 10 )Alkyl), -CON(C1-C 10 )Alkyl(C1-C 10 Alkyl), -CONH(C1-C 10 )Alkyl, -CONH2, -NH C(O)(C1-C 10 )Alkyl), -NHC(O)(phenyl), -N(C1-C 10 )Alkyl, -N(C1-C 10 Alkyl)C(O)(C1-C 10 )Alkyl), -N(C1-C 10 )Alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 )Alkyl, -SO2(phenyl), -SO2(C1-C 10 )Haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 )Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C) haloalkyl);

[0147] Each L1 is independently a straight-chain alkylene group having 1 to 40 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more of the groups selected from the group consisting of: C(O), NH, O, S, CH═N, S(O)2, C2-C 10 alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18 heterocycloalkylene and C5-C 10 heteroarylene; and wherein R1 may optionally have one or more substituents selected from the group consisting of: C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10 alkyl, -OC1-C 10 alkylphenyl, -C1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C 10 alkyl, -SC1-C 10 alkylphenyl, -C1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halogen substituents, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 )alkyl(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), -NH(C1-C 10 )alkylphenyl), cyano, -CO2H, C(O)O(C1-C 10 )alkyl), -CON(C1-C 10 )alkyl(C1-C 10 alkyl), -CONH(C1-C 10 )alkyl), -CONH2, -NH C(O)(C1-C 10 )alkyl), -NHC(O)(phenyl), -N(C1-C 10 )alkyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 )alkyl), -N(C1-C 10 )alkyl)C(O)(phenyl), -C(O)C1-C 10 alkyl, -C(O)C1-C10 alkylphenyl, -C(O)C1-C 10 haloalkyl, -OC(O)C1-C 10 alkyl, -SO2(C1-C 10 )alkyl, -SO2(phenyl), -SO2(C1-C 10 )haloalkyl), -SO2NH2, -SO2NH(C1-C 10 alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 )alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 )haloalkyl);

[0148] In some embodiments, L1 is optionally selected from the group consisting of A1-A14 groups or any linked combination thereof, wherein the structures and definitions of A1-A14 are shown below:

[0149]

[0150] wherein each k1 is independently an integer from 1 to 20;

[0151] each k2 is independently an integer from 1 to 20;

[0152] each R c is independently C1-C 10 alkyl;

[0153] each R d is selected from the group consisting of A15-A19 and any combination thereof:

[0154]

[0155] each R e is independently C1-C 10 alkyl; represents the site of covalent attachment of the group.

[0156] Those skilled in the art should understand that although for convenience L1 is defined as a linear alkylene group, it may not be a linear group or have a different name, such as an amine or alkenyl group resulting from the above substitutions and / or replacements. For the purposes of the present disclosure, the length of L1 is the number of atoms in the chain connecting the two attachment points. For this purpose, a ring (such as a heterocycloalkyl or heteroaryl group) obtained by replacing the carbon atoms of the straight-chain alkylene group is counted as one atom.

[0157] M1 represents a targeting group, the definition and optional scope of which are the same as those of the above targeting group. In some embodiments, each M1 is independently selected from one of the ligands having an affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.

[0158] R2 is a group having the structure represented by formula (S1), wherein E1 is OH or SH.

[0159] R1 is selected to effect the attachment of the N atom on the nitrogen-containing backbone to S1. R1 can be any linking group that can attach the S1 group to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, when preparing the siRNA conjugate represented by formula (24), (25), or (26) by solid-phase synthesis, the R1 group needs to contain a linking site for attachment to the N atom on the nitrogen-containing backbone and a linking site for attachment to the P atom in R2. In some embodiments, the site for attachment of R1 to the N atom on the nitrogen-containing backbone forms an amide bond with the N atom, and the site for attachment of R1 to the P atom in R2 forms a phosphate ester bond with the P atom.

[0160] In some embodiments, the siRNA conjugate has the structure represented by 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 are conjugating groups and Nu is the siRNA of the present invention.

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169] In some embodiments, the P atom in formula S1 can be linked to any possible position in the siRNA sequence. For example, the P atom in formula S1 can be linked to any nucleotide in the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula S1 is linked to any nucleotide in the sense strand of the siRNA. In some embodiments, the P atom in formula S1 is linked to the end of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula S1 is linked to the 3'-end of the sense strand of the siRNA. In the case of linking to the above positions of the sense strand of the siRNA, after the siRNA conjugate shown in (24), (25) or (26) enters the cell and unwinds, the separate siRNA antisense strand can be released to block the process of CIDEB mRNA translating into protein and inhibit CIDEB gene expression.

[0170] In some embodiments, the P atom in formula S1 can be linked to any possible position on the nucleotide in the siRNA, such as 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'-position, 3'-position or 5'-position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments, the P atom in formula S1 is linked to the oxygen atom formed after dehydrogenation of the 3'-hydroxyl of the nucleotide at the 3'-end of the sense strand of the siRNA (at this time, the P atom in S1 can also be regarded as the P atom in the phosphate group contained in the siRNA), or the P atom in formula S1 is linked to the nucleotide by substituting the hydrogen in the 2'-hydroxyl of a nucleotide in the sense strand of the siRNA, or the P atom in formula S1 is linked to the nucleotide by substituting the hydrogen in the 5'-hydroxyl of the nucleotide at the 5'-end of the sense strand of the siRNA.

[0171] The siRNA conjugate of the present invention has significantly improved stability in plasma and low off-target effects, and also exhibits high CIDEB mRNA silencing activity. In some embodiments, the siRNA of the present invention can be any one of the siRNAs shown in Table 1 or Table 3. The siRNA conjugates containing these siRNAs exhibit higher CIDEB mRNA silencing activity.

[0172] In the siRNA or siRNA conjugate of the present invention, each adjacent nucleotide is linked by a phosphodiester bond or a phosphorothioate bond, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond carries a negative charge, which can exist in the form of a hydroxyl group or a mercapto group, and the hydrogen ion in the hydroxyl group or mercapto group can also be partially or completely replaced by a cation. The cation can be any cation, such as a metal cation, ammonium ion NH4 +, one of organic ammonium cations. For the consideration of improving solubility, in one embodiment, the cation is selected from one or more of alkali metal ions, ammonium cations formed by tertiary amines, and quaternary ammonium cations. The alkali metal ion can be and / or Na + , and the cation formed by the tertiary amine can be the ammonium ion formed by triethylamine and / or the ammonium ion formed by N,N-diisopropylethylamine. Therefore, the siRNA or siRNA conjugate of the present invention can exist at least partially in the form of a salt. In one mode, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond is at least partially combined with sodium ions, and the siRNA or siRNA conjugate of the present invention exists in the form of a sodium salt or a partial sodium salt.

[0173] Those skilled in the art know that modified nucleotide groups can be introduced into the siRNA of the present invention by using nucleoside monomers with corresponding modifications. The methods for preparing nucleoside monomers with corresponding modifications and the methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers can be commercially available or prepared by known methods.

[0174] Preparation of the siRNA conjugate shown in formula (24), (25), or (26)

[0175] The siRNA conjugate shown in formula (24), (25), or (26) can be prepared by any reasonable synthetic route.

[0176] In some embodiments, the siRNA conjugate shown in formula (24), (25), or (26) can be prepared by the following method, which includes successively connecting nucleoside monomers in the 3' to 5' direction according to the nucleotide types and sequences of the sense strand and antisense strand of the siRNA under the conditions of phosphoramidite solid-phase synthesis. The connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; separating the sense strand and antisense strand of the siRNA and annealing, wherein the siRNA is the siRNA of the present invention as described above; and, the method further includes contacting the compound shown in formula (27), (28), or (29) with the nucleoside monomer or the nucleotide sequence linked to the solid-phase support under the coupling reaction conditions and in the presence of a coupling reagent, so that the compound shown in formula (27), (28), or (29) is linked to the nucleotide sequence through a coupling reaction. Hereinafter, the compound shown in formula (27), (28), or (29) is also called a conjugate molecule.

[0177]

[0178] Wherein:

[0179] R3 is a group capable of binding to the siRNA represented by Nu in the compound shown in formula (24), (25) or (26). In some embodiments, R3 is a group capable of covalently binding to the siRNA represented by Nu. In some embodiments, R3 is a group capable of reacting to conjugate to any functional group of the siRNA represented by Nu via a phosphodiester bond;

[0180] Each T1 is independently a group formed by replacing all active hydroxyl groups in M1 with YCOO- groups, where each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; in some embodiments, Y is methyl. The definition and optional range of L1 are as described above.

[0181] The selection of R3 is to achieve connection with the N atom on the nitrogen-containing backbone and to provide a suitable reaction site for synthesizing the siRNA conjugate shown in formula (24), (25) or (26). In some embodiments, R3 includes an R1 linking group or a protected R1 linking group, and a functional group capable of reacting with the siRNA to form the structure shown in S1.

[0182] In some embodiments, R3 contains a first functional group capable of forming a phosphite ester with a group on the siRNA or nucleoside monomer represented by Nu and a second functional group capable of reacting with a hydroxyl group or an amino group to form a covalent bond or contains a solid support connected by the covalent bond. In some embodiments, the first functional group is phosphoramidite, hydroxyl group or protected hydroxyl group. In some embodiments, the second functional group is phosphoramidite, carboxyl group or carboxylate. In some embodiments, the second functional group is a solid support covalently connected to other parts of the molecule, and the covalent bond is formed by a hydroxyl group or an amino group. In some embodiments, the solid support is connected via a phosphoester bond, a carboxylate ester bond or an amide bond. In some embodiments, the solid support is a resin.

[0183] In some embodiments, the first functional group contains a hydroxyl group, -OR m or the group shown in formula (C3); the second functional group contains the structures shown in formula (C1), (C2), (C3), (C1') or (C3'):

[0184]

[0185] In the formula, q1 is an integer from 1 to 4, X is O or NH, M + is a cation, R m is a hydroxyl protecting group, SPS represents a solid support, represents the site where the group connects to the covalent part.

[0186] In some embodiments, the first functional group contains a phosphoramidite group as shown in formula (C3), and this phosphoramidite group can undergo a coupling reaction with a hydroxyl group at any position on the nucleotide, such as the 2'-hydroxyl group, 3'-hydroxyl group, or 5'-hydroxyl group, to form a phosphite ester, and through oxidation or sulfurization, a phosphodiester bond or phosphorothioate bond as shown in formula S1 is formed, conjugating the conjugate molecule to the siRNA. At this time, even if the second functional group does not exist, the compounds shown in formula (27), (28), or (29) can conjugate to the nucleotide without affecting the acquisition of the siRNA conjugates shown in formula (24), (25), or (26). In this case, after obtaining the sense strand or antisense strand of the siRNA by methods such as phosphoramidite solid-phase synthesis, the compounds shown in formula (27), (28), or (29) are reacted with the hydroxyl group on the terminal nucleotide in the nucleotide sequence, and a phosphodiester bond connection or phosphorothioate bond connection is formed during the subsequent oxidation or sulfurization process, conjugating the compounds shown in formula (27), (28), or (29) to the siRNA.

[0187] In some embodiments, the first functional group contains a protected hydroxyl group. In some embodiments, the second functional group contains a group that can react with a solid-phase support, and this reaction provides a conjugate molecule containing the solid-phase support. In some embodiments, the second functional group contains a carboxyl group, carboxylate, or phosphoramidite as shown in formula (C1), (C2), or (C3). When the second functional group contains a carboxyl group or carboxylate, the compounds shown in formula (27), (28), or (29) undergo an esterification reaction or amidation reaction with the hydroxyl group or amino group on a solid-phase support, such as a resin, to form a conjugate molecule containing the solid-phase support connected by a carboxylic acid ester bond. When the second functional group contains a phosphoramidite functional group, the compounds shown in formula (27), (28), or (29) undergo a coupling reaction with the hydroxyl group on a general solid-phase support, such as a resin, and through oxidation, a conjugate molecule containing the solid-phase support connected by a phosphodiester bond is formed. Subsequently, starting from the product after connecting the solid-phase support as above, nucleoside monomers are sequentially connected according to the phosphoramidite solid-phase synthesis method to obtain the sense strand or antisense strand of the siRNA connected with the conjugate group. During the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then undergoes a coupling reaction with the phosphoramidite group on the nucleoside monomer under the coupling reaction conditions.

[0188] In some embodiments, the first functional group contains a hydroxyl group or a protected hydroxyl group; the second functional group contains a solid support linked by a carboxylic acid ester bond, an amide bond, or a phosphate ester bond, as shown in formula (C1') or (C3'). At this time, the compounds shown in formulas (27), (28), and (29) are used as starting materials instead of the solid support, and nucleoside monomers are sequentially linked according to the phosphoramidite solid-phase synthesis method to obtain the sense strand or the antisense strand of siRNA linked with a conjugate group.

[0189] In some embodiments, each T1 is independently M1. In some embodiments, each S1 is independently a group formed by protecting at least one active hydroxyl group in M1 with a hydroxyl protecting group. In some embodiments, the protected hydroxyl group can be represented by the formula YCOO-, where each Y is independently selected from methyl, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogenated phenyl, and alkylphenyl; in some embodiments, Y is methyl.

[0190] In some embodiments, R m is one or more of MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-dimethoxytriphenylmethyl), and TMTr (4,4',4''-trimethoxytriphenylmethyl). In some embodiments, R m can be DMTr, i.e., 4,4'-dimethoxytrityl.

[0191] Correspondingly, unless otherwise specified, in the following descriptions related to the preparation of conjugates and / or conjugate molecules, when referring to reactions such as "deprotection", "coupling", "capping", "oxidation", and "sulfidation", it should be understood that the reaction conditions and reagents involved in the well-known phosphoramidite nucleic acid solid-phase synthesis method in the art are also equally applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail later.

[0192] As described above, the method for preparing the siRNA conjugate represented by formula (24), (25) or (26) further comprises the following steps: synthesizing the other strand of the siRNA (for example, when the sense strand of the siRNA conjugated with a conjugate molecule is synthesized in the above step, it further comprises synthesizing the antisense strand of the siRNA according to the 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, and at the same time, 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 the corresponding M1 targeting group), obtaining the sense strand (or antisense strand) of the siRNA linked with the conjugate molecule and the corresponding antisense strand (or sense strand), annealing the sense strand and the antisense strand to form a double-stranded RNA structure, and obtaining the siRNA conjugate represented by formula (24), (25) or (26).

[0193] In some embodiments, the method for preparing the siRNA conjugate represented by formula (24), (25) or (26) comprises the following steps: contacting the compound represented by formula (27), (28) or (29) with the first nucleoside monomer at the 3'-end of the sense strand or antisense strand under coupling reaction conditions and in the presence of a coupling reagent, so that the compound represented by formula (27), (28) or (29) is linked to the first nucleotide in the sequence, and under the conditions of phosphoramidite solid-phase synthesis, the nucleoside monomers are sequentially linked in the 3'-to-5' direction according to the expected types and order of the nucleotides of the sense strand or antisense strand to synthesize the sense strand or antisense strand of the siRNA; wherein, the compound represented by formula (27), (28) or (29) is a compound in which the R2 contains a first functional group and a second functional group, the first functional group contains a protected hydroxyl group, and the second functional group has a structure represented by formula (C1') or (C3'), and before being linked to the first nucleoside monomer, the compound represented by formula (27), (28) or (29) is deprotected; the linking of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; obtaining the sense strand or antisense strand of the nucleic acid linked with the conjugate group; under the conditions of phosphoramidite solid-phase synthesis, the nucleoside monomers are sequentially linked in the 3'-to-5' direction according to the types and order of the nucleotides of the antisense strand or sense strand to synthesize the antisense strand or sense strand of the nucleic acid; the linking of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; removing the protecting groups and cleaving from the solid-phase carrier, separating and purifying to obtain the sense strand and the antisense strand, and annealing.

[0194] In some embodiments, the preparation method of the siRNA conjugate shown in formula (24), (25) or (26) comprises the following steps: according to the nucleotide type and sequence of the sense strand or antisense strand in the double-stranded siRNA, the nucleoside monomers are sequentially connected in the 3' to 5' direction to synthesize the sense strand and the antisense strand, and the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization to obtain the sense strand connected to the solid phase carrier and the antisense strand connected to the solid phase carrier; under coupling reaction conditions and in the presence of a coupling reagent, the siRNA shown in formula (27), (28) or (29) is connected to the solid phase carrier. The compound is contacted with a sense strand connected to a solid phase carrier or an antisense strand connected to a solid phase carrier, and the compound represented by formula (27), (28) or (29) is connected to the sense strand or the antisense strand, wherein the compound represented by formula (27), (28) or (29) is a compound represented by formula (27), (28) or (29) wherein R3 contains a first functional group, and the first functional group is a phosphoramidite group; the protecting group is removed and the compound is cut with the solid phase carrier, and the sense strand or the antisense strand of the siRNA is separated and purified respectively, and annealed, wherein the sense strand or the antisense strand of the siRNA is connected to a conjugated group.

[0195] In some embodiments, the P atom in formula S1 is connected to the 3' end of the sense strand in the siRNA, and the method for preparing the siRNA conjugate shown in formula (24), (25) or (26) comprises:

[0196] (1) removing the compound represented by formula (27), (28) or (29) (wherein the compound represented by formula (27), (28) or (29) is a compound in which R3 contains a first functional group and a second functional group, and the first functional group contains a protected hydroxyl group OR m , the second functional group has a hydroxyl protecting group R in the compound having a structure as shown in formula (C1') or (C3') m ; Under coupling reaction conditions and in the presence of a coupling reagent, the deprotected product is contacted with a nucleoside monomer to obtain a nucleoside monomer connected to a solid phase carrier via a conjugated molecule;

[0197] (2) starting with the nucleoside monomer connected to the solid phase carrier via the conjugated molecule, synthesizing the positive strand of the siRNA via a phosphoramidite solid phase synthesis method in the 3'-5' direction;

[0198] (3) synthesizing the antisense strand of siRNA by phosphoramidite solid phase synthesis method;

[0199] (4) Separating the sense strand and antisense strand of siRNA and annealing them to obtain the siRNA conjugate represented by formula (24), (25) or (26).

[0200] After obtaining the conjugate, in some embodiments, methods such as liquid chromatography-mass spectrometry can be used to characterize the synthesized siRNA conjugate represented by formula (24), (25), or (26) by means of molecular weight detection, etc., to determine that the synthesized siRNA conjugate is the siRNA conjugate represented by the target-designed formula (24), (25), or (26), and the sequence of the synthesized siRNA is the sequence of the desired siRNA.

[0201] In some embodiments, the solid support is a solid support well known in the art that can be used for solid-phase nucleic acid synthesis.

[0202] Drug composition

[0203] The present invention also includes drug compositions and formulations comprising the siRNA conjugate of the present invention. In some embodiments, provided herein are drug compositions comprising the siRNA conjugate as described herein and a pharmaceutically acceptable carrier. The drug composition comprising the siRNA conjugate can be used to treat diseases or disorders related to the expression or activity of the CIDEB gene. Such drug compositions are formulated based on the mode of delivery. An example is a composition formulated for systemic administration by parenteral delivery (e.g., by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery). In certain embodiments, the present invention provides compositions formulated for intra-arterial, intratumoral, intradermal, intravitreal injection, topical ocular, ophthalmic (eye drops), nebulization, topical ocular or other topical routes, suppository, or oral administration to an organ of interest (e.g., liver). In a preferred embodiment, the composition is administered subcutaneously.

[0204] The drug composition of the present invention can be administered in a dose sufficient to inhibit the expression of the CIDEB gene. In some examples, the siRNA conjugate is administered at the following doses: about 0.5 mg / kg to 50 mg / kg per dose, or 0.3 mg / kg to 20 mg / kg, or 3 mg / kg to 10 mg / kg, or preferably 3 mg / kg to 10 mg / kg per dose. For example, the siRNA conjugate can be administered at a dose of about 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg per single dose.

[0205] The composition can also be prepared and packaged in a fixed dose for the subject independent of body weight. The exemplary dose level can be calculated by multiplying the body weight per kilogram by the average body weight of the subject. For example, the average body weight of a general adult is considered to be about 70 kg.

[0206] Repeat dose regimens can include administering a therapeutically effective amount of the siRNA conjugate periodically, such as once a month, once every other month, or once every three months. In a preferred embodiment, the siRNA conjugate is administered at a frequency of no more than once a month. After an initial treatment regimen, treatment can be administered at a lower frequency.

[0207] Those skilled in the art will appreciate that certain factors can affect 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 overall health or age of the subject, and the presence of other diseases. In addition, treating a subject with a therapeutically effective amount of the composition can include a single treatment or a series of treatments. As described elsewhere herein, conventional methods or in vivo testing based on the use of appropriate animal models can be used to estimate the effective dose and in vivo half-life of an individual siRNA conjugate encompassed by the present invention.

[0208] A. Excipients

[0209] A "pharmaceutical carrier" or "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 reagents are well known in the art.

[0210] B. Other Components

[0211] The compositions of the present invention can additionally contain other auxiliary components conventionally present in pharmaceutical compositions at levels established in the art. Thus, for example, the composition can contain additional, compatible pharmaceutically active substances, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or can contain additional substances useful for physically formulating the various dosage forms of the composition of the present invention, such as preservatives, antioxidants, and stabilizers. However, when added, such substances should not unduly interfere with the biological activity of the components of the composition of the present invention. The preparation can be sterilized and, if desired, can be admixed with adjuvants that do not interact harmfully with the nucleic acids of the preparation, such as preservatives, stabilizers, wetting agents, emulsifying agents, salts affecting osmotic pressure, or buffers, etc.

[0212] In some embodiments, the pharmaceutical compositions characterized in the present invention comprise (a) one or more siRNA conjugate compounds and (b) one or more agents that function through non-RNAi mechanisms and are useful for treating non-alcoholic fatty liver disease-related disorders.

[0213] As described above, in addition to their administration, the siRNA conjugates characterized herein can be administered in combination with other known agents effective in treating non-alcoholic fatty liver disease. In any case, the administering physician can adjust the amount and timing of siRNA conjugate administration based on the results observed using standard efficacy measures known in the art or described herein.

[0214] Example

[0215] Unless otherwise specified, the reagents and culture media used in the following examples are commercially available products, and operations such as nucleic acid electrophoresis and real-time PCR are carried out according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). The conjugated molecules such as those of formula (27), (28) or (29) are purchased from Nanjing Leizheng Medical Technology Co., Ltd.

[0216] Example 1 Design of siRNA

[0217] An online tool oligowalk was used to design a set of siRNAs targeting the human CIDEB gene (Human: NCBI refseq ID NM_001393338.1; NCBI Gene ID: 27141). The human NM_001393338 REFSEQ mRNA, version 1 has a length of 2482 bases. At the same time, in order to avoid toxicity caused by any sequences, sequences similar to human genes were also excluded.

[0218] The detailed listings of the unmodified CIDEB sense and antisense strand nucleotide sequences are shown in Table 1. The detailed listings of the modified CIDEB sense and antisense strand nucleotide sequences are shown in Table 3.

[0219] Example 2 Preparation of siRNA or siRNA conjugate

[0220] Synthesis: The sense and antisense strand sequences were synthesized according to the phosphoramidite solid-phase synthesis technique, and were synthesized on a Mermade 192 synthesizer (BioAutomation) at a 1 μmol scale using solid support-mediated phosphoramidite chemistry. The solid support is controlled pore glass (CPG) loaded with a customized GalNAc ligand molecule, ) or a general solid support. Auxiliary synthesis reagents, such as 2'-F and 2'-O-methyl RNA phosphoramidites, are all commercially available reagents. The corresponding phosphoramidites are used to introduce 2'-F, 2'-O-methyl, GNA (glycol nucleic acid), 5'-phosphate, and abasic modifications. The synthesis of the 3'-GalNAc-conjugated single strand is carried out on a GalNAc-modified CPG support. The CPG general solid support is used for the synthesis of antisense single strands or 5'-GalNAc-conjugated single strands. Using 5-ethylthio-1H-tetrazole (ETT) as the activator (in acetonitrile, 0.6 M), the coupling time for all phosphoramidites (dissolved in anhydrous acetonitrile, 100 mM) is 5 minutes. A solution of 50 mM 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile / pyridine (v / v = 1 / 1) is used to generate the phosphorothioate bond, and the reaction time is 3 minutes. All sequences are synthesized after the final removal of the DMT group.

[0221] Cleavage and deprotection of the oligomers bound to CPG: After the termination of solid-phase synthesis, the protecting groups are removed by treating with an acetonitrile solution containing 20% diethylamine for 30 minutes, without cleaving the oligonucleotide from the CPG. Subsequently, the dried CPG is treated with concentrated ammonia at 40 °C for 18 hours. After centrifugation, the supernatant is transferred to a new tube and the CPG is washed with ammonia water. The combined solution is concentrated to obtain a solid mixture.

[0222] Purification: Purification is carried out by using NanoQ anion-exchange HPLC. Buffer A is a 10 mM sodium perchlorate solution, 20 mM Tris, 1 mM EDTA, pH 7.4 and contains 20% acetonitrile, and buffer B is 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 and contains 20% acetonitrile. The target product is separated and desalted using a reversed-phase C18 column.

[0223] Annealing of oligoribonucleotides to produce siRNA conjugates: The RNA oligomers to be annealed are formulated into a 200 μΜ solution with sterile RNase Free H2O (without RNase). The annealing reaction system is set as follows. A solution with a total volume of 100 μL of the above solution (the duplex concentration is 10 nmol) is placed in a 95 °C water bath for 10 minutes (≥ 100 nmol requirement requires 20 minutes at high temperature) → quickly placed in a 60 °C water bath and allowed to cool naturally → the solution after annealing is stored at 4 °C. The complementary strands are mixed by combining equimolar RNA solutions.

[0224] Table 4 shows the CIDEB siRNA conjugates synthesized using the above method.

[0225] Table 4. Nucleotide sequences of modified CIDEB siRNA conjugates

[0226]

[0227] Example 3 In vitro Activity Test of siRNA

[0228] Quantitatively detect the CIDEB mRNA content in HepG2 cells by qPCR, and use the IC 50 value of the compound as an index to evaluate the inhibitory activity of the siRNA conjugate against CIDEB.

[0229] Experimental Materials and Reagents:

[0230] Cell line: HepG2 cells (provided by the Stem Cell Bank of the Chinese Academy of Sciences)

[0231] HepG2 cell culture medium (DMEM, Invitrogen - 11330032; 10% serum, Invitrogen - 10099141; 100 units / mL penicillin and 100 μg / mL streptomycin, Hyclone - SV30010; 1% non - essential amino acids, Invitrogen - 11140050; 2 mM L - glutamine, Invitrogen - 25030081; 1 mM sodium pyruvate, Gibco - 11360 - 070; 500 μg / mL Geneticin, Invitrogen - 10131027).

[0232] Reagents: Trypsin (Invitrogen - 25300062); DMSO (Sigma - D2650 - 100ML); transfection reagent Lipofectamine RNAiMAX (Invitrogen - 13778 - 150); MEM Medium (HyClone - SH30024.01); 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 gDNA Eraser (takara - RR047A); ChamQ Universal SYBR qPCR MasterMix (vyzme - Q711 - 02).

[0233] Consumables and Instruments: 48 - well cell culture plate (Coming - 3599); CO2 incubator (HERA - CELL - 240); Microplate (Axygen-PCR-96-FLT-C); qPCR equipment (QIANGE).

[0234] Experimental procedures:

[0235] siRNA or siRNA conjugate is transfected into HepG2 cells as follows: Take HepG2 cells, wash them with PBS first, then add trypsin for digestion, adjust the cells to an appropriate density, and after 24 h, transfer siRNA into CIDEB cells using the transfection reagent Lipofectamine RNAiMax, and seed the cells at a density of 10,000 cells per well into a 48-well plate, with 500 μL of HepG2 cell medium in each well. The cells are cultured in an incubator at 5% CO2 and 37 °C for 48 h. 48 hours after transfection, collect the cells, extract RNA, and detect the total CIDEB-RNA in the cells by RT-PCR.

[0236] The tested siRNA is tested at 2 concentration points with 3 replicates. The control is set as NM_001393338.1 and is tested at 4 concentration points with 2 replicates.

[0237] The steps for detecting CIDEB RNA are briefly described as follows: Use the trizol method to extract the total RNA in the cells, refer to the instruction manual of the reverse transcription kit (takara), add random primers to reverse transcribe into cDNA, and then detect the CIDEB cDNA in the samples by qPCR. At the same time, GAPDH primers and probes are used to specifically detect GAPDH cDNA.

[0238] The PCR reaction program is: 95 °C for 2 minutes, then enter the cycling mode, 95 °C for 10 seconds, followed by 60 °C for 30 seconds, for a total of 40 cycles. Calculate the content of CIDEB RNA in the samples based on the Ct values of each sample.

[0239] The PCR primers are as follows:

[0240] Human CIDEB-Forward 5-CAGCGACCTTTCCGTGTCT-3;

[0241] Human CIDEB-Reverse 5-GGGTCTCCAATGCTTTGGCT-3.

[0242] Human GAPDH-Forward 5-GGAGCGAGATCCCTCCAAAAT-3;

[0243] Human GAPDH-Reverse 5-GGCTGTTGTCATACTTCTCATGG-3。

[0244] The expression level of the target gene CIDEB mRNA in each sample was calculated by the ΔΔCt relative quantification method. The relative expression of the target gene was expressed using 2 -ΔΔCT and the calculation formula was as follows:

[0245] a) The Ct value was automatically calculated according to the default settings of the Quant Studio 7 software. The Ct value was exported as an Excel file.

[0246] b) The relative expression of the gene was calculated using the following formula:

[0247] ΔCt = Ct(target gene) - Ct(gapdh)

[0248] ΔΔCt = ΔCt(test sample) - ΔCt(Mock)

[0249] mRNA expression relative to Mock = 2 -ΔΔCt

[0250] where Mock represents the negative control with an equal concentration of Lipofectamine RNAiMax but without siRNA.

[0251] The inhibition rate was calculated as follows: (1 - (2 -ΔΔCt )) * 100%. Table 5 shows the inhibitory activity of the siRNA of the present invention against CIDEB.

[0252] Table 5. Inhibition rate of the siRNA of the present invention against CIDEB mRNA

[0253]

[0254] Transfected into HepG2 cells using the same method as above, and the inhibitory activity (IC 50 ) of the siRNA conjugate of the present invention against CIDEB was determined. The IC 50 value was obtained by data fitting using GraphPad. Table 6 shows the inhibitory activity (IC 50 ) of the siRNA conjugate of the present invention against CIDEB.

[0255] Table 6. Inhibitory activity (IC 50 ) of the siRNA conjugate of the present invention against CIDEB

[0256]

[0257] As can be seen from Table 6, the siRNA conjugate provided by the present invention has high CIDEB inhibitory activity in HepG2 cells.

[0258] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. An siRNA, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein, The sense strand contains nucleotide sequence I, and the antisense strand contains nucleotide sequence II. Nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. Nucleotide sequence I and nucleotide sequence II are selected from one of the sequences shown in (i)-(iii) below: (i) Nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO.1 and optionally has no more than 3 nucleotide differences, and nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO.2 and optionally has no more than 3 nucleotide differences: SEQ ID NO.1: 5’-ACUGCUGAAUGGAGUGCUAUU-3’; SEQ ID NO.2: 5’-UAGCACUCCAUUCAGCAGUUU-3’; (ii) Nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO.3 and optionally has no more than 3 nucleotide differences, and nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO.4 and optionally has no more than 3 nucleotide differences: SEQ ID NO.3: 5’-CCUUUGACGUGUACAAGCAUU-3’; SEQ ID NO.4: 5’-UGCUUGUACACGUCAAAGGUU-3’; (iii) Nucleotide sequence I has the same length as the nucleotide sequence shown in SEQ ID NO.5 and optionally has no more than 3 nucleotide differences, and nucleotide sequence II has the same length as the nucleotide sequence shown in SEQ ID NO.6 and optionally has no more than 3 nucleotide differences: SEQ ID NO.5: 5’-CUCUAUGAGUUGUGACUUUUU-3’; SEQ ID NO.6: 5’-AAAGUCACAACUCAUAGAGUU-3’.

2. The siRNA according to claim 1, wherein Optionally, there is no more than 1 nucleotide difference between nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO.1, and / or optionally, there is no more than 1 nucleotide difference between nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO.2; or, optionally, there is no more than 1 nucleotide difference between nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO.3, and / or optionally, there is no more than 1 nucleotide difference between nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO.4; or, optionally, there is no more than 1 nucleotide difference between nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO.5, and / or optionally, there is no more than 1 nucleotide difference between nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO.

6.

3. The siRNA according to claim 2, wherein At least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group with a modifying group.

4. The siRNA according to claim 3, wherein Each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

5. The siRNA according to claim 4, wherein Each non-fluorinated modified nucleotide is independently selected from a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose group of a nucleotide with a non-fluorine group or a nucleotide analogue.

6. The siRNA according to claim 5, wherein The nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose group of the nucleotide with a non-fluorine group is selected from one of 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-deoxynucleotides; The nucleotide analogue is selected from a locked nucleic acid, a bridged nucleotide or an acyclic nucleotide; The locked nucleic acid is a compound formed by moving the base from the 1'-position of the ribose ring to the 2'-position or 3'-position; The bridged nucleotide is selected from one of LNA shown in formula (6), ENA shown in formula (7), and cET shown in formula (8), The acyclic nucleotide is selected from one of UNA shown in formula (9) and GNA shown in formula (10), Among them, in the above formulas (6) to (10), Base represents a nucleic acid base, and R a is selected from H, OH or C1-C 10 alkoxy group.

7. The siRNA according to any one of claims 4-6, characterized in that, Each non-fluorinated modified nucleotide is a methoxy-modified nucleotide.

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

9. The siRNA according to claim 8, wherein The connection of the phosphorothioate group exists at least at one place in the group consisting of the following positions: 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; Alternatively, the connection of the phosphorothioate group exists at all the above positions except at the 5'-end of the sense strand, Alternatively, the connection of the phosphorothioate group exists at all the above positions except at the 3'-end of the sense strand.

10. The siRNA according to claim 3, wherein The 5'-terminal nucleotide of the antisense strand is a 5'-phosphonucleotide or a nucleotide modified with a 5'-phosphate analogue; The 5'-phosphonucleotide is a nucleotide containing a 5'-phosphate modification shown in formula (14), The nucleotide modified with a 5'-phosphate analogue is a nucleotide containing a vinyl phosphate modification, as shown in formula (15), or a phosphorothioate modification, as shown in formula (17), wherein, R is selected from Η, OH, methoxy, fluorine; Base represents a nucleic acid base, selected from A, U, C, G or T.

11. The siRNA according to any one of claims 1-10, characterized in that, The siRNA is selected from the following table:

12. An siRNA conjugate, characterized in that, The siRNA conjugate contains the siRNA according to any one of claims 1-11 and a conjugating group conjugated to the siRNA; the conjugating group contains a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are covalently or non-covalently connected in sequence.

13. The siRNA conjugate according to claim 12, wherein The linker has a structure shown in formula (19): wherein, m is an integer from 1 to 3; L A is a chain-like moiety containing an amide bond and having a structure represented by formula (20), each of said Ls A is connected to one of said targeting groups and said L moiety at its two ends respectively C via an ether bond: L B is a chain portion containing N-acylpyrrolidine having a structure represented by the formula (21), the chain portion having a carbonyl group at one end and being linked to the L C portion by an amide bond and having an oxygen atom at the other end and being linked to the siRNA by a phosphate bond: L C is a 2-4 valent linking group based on tris(hydroxymethyl)aminomethane, bis(tris(hydroxymethyl)aminomethane) or tris(tris(hydroxymethyl)aminomethane), said L C is connected to each of the said L A parts via an oxygen atom through an ether bond, and is connected to the said L B parts via a nitrogen atom through an amide bond; The linker is connected to the 3'-end or 5'-end of the sense strand of the siRNA.

14. The siRNA conjugate according to claim 13, wherein The conjugate has a structure shown in formula (24), (25) or (26): Among them, R2 is a group having the structure shown in formula (S1): Among them, E1 is OH or SH; Nu is the siRNA described in any one of claims 1-12; R1 is a straight-chain or cyclic alkylene group having 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more of the groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18 heterocycloalkylene and C5-C 10 heteroarylene; and wherein R1 optionally has a substituent of any one or more of the groups consisting of: C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10 alkyl, -OC1-C 10 alkylphenyl, -C1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C 10 alkyl, -SC1-C 10 alkylphenyl, -C1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), -NH(C1-C 10 alkylphenyl), -CON(C1-C 10 alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 alkyl, -C(O)C1-C 10 alkylphenyl, -C(O)C1-C 10 haloalkyl, -OC(O)C1-C 10 alkyl, -SO2(C1-C 10 alkyl), -S(O)2(phenyl), -(C1-C 10 haloalkyl), -NH2, -NH(C1-C 10 alkyl), -NH(phenyl), -NH(C1-C 10 alkyl), -NH(phenyl) and -NH(C1-C 10 haloalkyl); Each L1 is independently a straight-chain alkylene group having 1 to 40 carbon atoms, wherein one or more of the carbon atoms are optionally replaced by any one or more of the groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18 heterocycloalkylene, and C5-C 10 heteroarylene; and wherein R1 may optionally have one or more substituents selected from the group consisting of: C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10 alkyl, -OC1-C 10 alkylphenyl, -C1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C 10 alkyl, -SC1-C 10 alkylphenyl, -C1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halogen substituents, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, -CO2H, C(O)O(C1-C 10 alkyl), -CON(C1-C 10 alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 alkyl, -C(O)C1-C 10 alkylphenyl, -C(O)C1-C 10 haloalkyl, -OC(O)C1-C 10 alkyl, -SO2(C1-C 10 alkyl), -(phenyl), -(C1-C 10 haloalkyl), -NH2, -NH(C1-C 10 alkyl), -NH(phenyl), -NH(C1-C 10 alkyl), -NH(phenyl) and -NH(C1-C 10 haloalkyl); M1 represents a targeting group; Indicates the site of covalent attachment of the group.

15. The siRNA conjugate according to claim 14, wherein Each of said L1s is independently selected from the group consisting of groups A1 - A 14 and any combination thereof: Among them, each k1 is independently an integer from 1 to 20; Each k2 is independently an integer from 1 to 20; Each R c independently is C1-C 10 alkyl; Each R d is selected from the group consisting of A15 - A19 and any combination thereof: Each R e independently is C1-C 10 alkyl; represents the site at which the group is covalently linked.

16. The siRNA conjugate according to claim 15, wherein, L1 is a linking combination of at least two of groups A1, A4, A8, A10, and A11; the length of L1 is 3 to 20 atoms; 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.

17. The siRNA conjugate according to claim 14, wherein The R1 simultaneously has a binding site connected to the N atom on the nitrogen-containing backbone and a binding site connected to the P atom in R2; The site on R1 connected to the N atom on the nitrogen-containing backbone forms an amide bond with N, and the site connected to the P atom on R2 forms a phosphoester bond or a phosphorothioate bond with P.

18. The siRNA conjugate according to claim 17, wherein, The P atom in the formula (S1) is connected to the 2', 3' or 5' position of the terminal nucleotide of the sense strand or antisense strand of the siRNA through a phosphodiester bond.

19. The siRNA conjugate according to claim 12, wherein Each of the targeting groups is independently selected from D-galactose, L-galactose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine.

20. The siRNA conjugate according to claim 12, wherein, The siRNA conjugate has a structure shown in 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 described in any one of claims 1-11.

21. The siRNA conjugate according to claim 12, wherein, The siRNA conjugate has a structure shown in formula (23): Among them, the double-stranded helical structure represents the siRNA described in any one of claims 1-11.

22. A plasmid or host cell containing the siRNA described in any one of claims 1-11 and / or the siRNA conjugate described in any one of claims 12-21.

23. A pharmaceutical composition, characterized in that, Containing the siRNA described in any one of claims 1-11 and / or the siRNA conjugate described in any one of claims 12-21 and a pharmaceutically acceptable carrier. Use of the siRNA according to any one of claims 1-11, and / or the siRNA conjugate according to any one of claims 12-21, and / or the pharmaceutical composition according to claim 23, in the preparation of a medicament for treating a disease caused by the expression of the cell death-inducing DFFA-like effector B gene.

25. The application according to claim 24, wherein The disease caused by the expression of the cell death-inducing DFFA-like effector B gene is a disease associated with non-alcoholic fatty liver disease; the diseases associated with non-alcoholic fatty liver disease include non-alcoholic hepatic steatosis, non-alcoholic steatohepatitis, non-alcoholic cirrhosis, liver cancer, obesity, type 2 diabetes, cardiovascular disease, chronic kidney disease, dyslipidemia, or extrahepatic malignancy.

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