Small interfering nucleic acids, short antisense oligonucleotides targeting the plin2 gene and uses thereof

Abstract

The present invention provides short antisense oligonucleotides targeting the PLIN2 gene and uses thereof. Also provided are RNAi agents that inhibit the PLIN2 gene and compositions comprising the same. The present invention further relates to uses of the short antisense oligonucleotides, RNAi agents, and compositions comprising the same, in reducing lipid deposition in a subject, and in treating or preventing nonalcoholic fatty liver disease (NAFLD), including simple steatosis and nonalcoholic steatohepatitis (NASH). The present invention further relates to methods of treating NAFLD, comprising administering to a subject in need thereof, a short antisense oligonucleotide targeting the PLIN2 gene.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene therapy in the field of biomedicine. In particular, the present application relates to a short antisense oligonucleotide and an RNAi agent targeting the PLIN2 gene, and a composition comprising the RNAi agent. The present application also relates to the use of the short antisense oligonucleotide and the RNAi agent for treating or preventing a disease associated with abnormal liver lipid metabolism. BACKGROUND

[0002] With the rising incidence, non-alcoholic fatty liver disease (NAFLD) has become a worldwide health problem, affecting 1 / 4 of the world's population, and threatening the life and health of nearly 2 billion people worldwide. When the fat content in the liver of a patient exceeds 5% of the total weight of the liver, and the patient's weekly alcohol consumption is less than 140g, it is diagnosed as NAFLD. NAFLD includes simple steatosis without progression and non-alcoholic steatohepatitis (NASH) with progression. If not intervened in time, NASH will gradually progress to liver fibrosis, cirrhosis and liver cancer, and eventually cause the patient to die.

[0003] The pathogenesis of NAFLD is very complex. Obesity, drugs, malnutrition, etc. are important predisposing factors of NAFLD. In addition, environmental factors and genetic background can also affect the development of NAFLD and cause different patients to respond differently to specific therapeutic drugs. For example, it has been shown in a large number of studies that PNPLA3 (patatin-like phospholipase domain containing 3 protein) I148M variant is the most relevant mutation to NAFLD, and 50% of NAFLD patients carry at least one mutant PNPLA3 148M allele. It has been reported that the PNPLA3 I148M variant can induce the liver retention of newly synthesized triglycerides and affect the secretion of very low density lipoprotein in the liver, thereby affecting the plasma triglyceride level. It is believed that in the context of PNPLA3 I148M, the effectiveness of many NAFLD treatment drugs (such as ACCi, FASi or DGAT2i) is greatly reduced.

[0004] Most of the preclinical experiments of current NAFLD drugs only consider the factor of diet, ignoring the different genetic backgrounds of patients in actual situations. In addition, when using experimental animals for pre-screening of drugs, it is necessary to go from "effective on experimental animals" to "effective on humans"; or when exploring other uses of existing drugs, a large number of animal-level experiments are needed to go from "effective on humans" to "effective on experimental animals". Due to the differences between the genes of experimental animals and humans, this "migration" is hindered, consuming a large amount of manpower, material resources and animal life. This may be an important reason why many NAFLD treatment drugs in recent years have good effects in animal models, but have not successfully passed clinical trials.

[0005] The current first-line treatment for NAFLD mainly includes weight loss through dietary intervention combined with exercise. For these therapies, patients inevitably have poor compliance and other problems. So far, only one drug, Elafibranor, from GENFIT company has been approved for marketing in India, which is a PPAR agonist. There is no NAFLD treatment drug approved for marketing by the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA), indicating that there is still a huge clinical demand for NAFLD treatment drugs.

[0006] Exploring the mechanism of NAFLD disease to design treatment drugs will help solve the current problems of drugs.

[0007] As an important place for liver cells to store fat, the accumulation of lipid droplets is an early feature common to NAFLD. Through years of research in the field of lipid droplet homeostasis, the inventors identified and analyzed a series of key proteins and signaling pathways involved in the regulation of lipid droplet homeostasis. They found that perilipin-2 (PLIN2) is an important lipid droplet protective protein in hepatocytes, and its protein level significantly increases during the pathogenesis of NAFLD, showing strong clinical relevance. In the case of LD accumulation, PLIN2 expression increases, inhibiting the entry of adipose triglyceride lipase (ATGL) into the core of LD, preventing the normal breakdown of fat, and promoting steatosis.

[0008] For the relationship between Plin2 and NAFLD, corresponding to the above clinical relevance, it has been reported that Plin2 knockout mice can resist NAFLD caused by obesity or high-fat diet, showing healthy physiological indicators and exercise capacity.

[0009] There is no treatment targeting Plin2-related proteins or pathways for diseases related to abnormal liver lipid metabolism. It is necessary to further study how to design drugs targeting Plin2-related proteins or pathways, hoping that the drugs can alleviate or correct abnormal liver lipid metabolism in vivo, and are safe and have no side effects during administration, especially hoping that the treatment drugs are still effective when the subjects have a genetic background of PNPLA3 I148M. SUMMARY

[0010] As a new drug development direction, the inventors envisage reducing the fat content in the liver by reducing the protein level of PLIN2 in hepatocytes, and especially being able to treat diseases related to abnormal liver lipid metabolism such as NAFLD.

[0011] The inventors successfully screened a pair of short antisense oligonucleotides targeting the PLIN2 gene of humans and mice in vitro cultured hepatocytes, which targets the same position of the PLIN2 gene in humans and mice, and only has 1 or 2 base differences in humans and mice. It is proved by experiments that siRNA containing the human short antisense oligonucleotide in the above-mentioned pair reduces fat deposition in the liver of humanized mice fed with high-fat diet.

[0012] On this basis, the short antisense oligonucleotide is acetylgalactosamine modified, and the obtained GalNAc-PLIN2 short antisense oligonucleotide has liver delivery targeting and enhanced stability.

[0013] It is proved by experiments that the GalNAc-PLIN2 short antisense oligonucleotide can reduce the liver-weight ratio, reduce the liver fat content, significantly reduce the NAFLD score, reduce liver damage and inflammation and fibrosis, and obtain a significant treatment effect of liver fat deposition in wild-type mice and humanized mice with human PLIN2 gene. In addition, when the GalNAc-PLIN2 short antisense oligonucleotide is administered to mice, no adverse reactions such as immune response and organ damage are caused, and it has good safety.

[0014] Therefore, the inventors provide a short antisense oligonucleotide targeting PLIN2, and particularly relate to a method for reducing liver lipid deposition and treating NAFLD using the short antisense oligonucleotide. In particular, the inventors provide a GalNAc-PLIN2 short antisense oligonucleotide, and its use as a NAFLD prevention or treatment drug, and its use for correcting liver steatosis and lipid accumulation in other tissues under other pathological conditions, thereby completing the present application.

[0015] In a first aspect, the present application provides a short antisense oligonucleotide, wherein the short antisense oligonucleotide targets a nucleotide encoding a PLIN2 protein.

[0016] In a specific embodiment of the first aspect, the short antisense oligonucleotide comprises, or consists of, a nucleotide sequence selected from the group consisting of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or a nucleotide sequence differing by 1 or 2 nucleotides therefrom.

[0017] In a second aspect, the present application provides an RNAi agent comprising the short antisense oligonucleotide of the first aspect.

[0018] In a specific embodiment of the second aspect, the RNAi agent comprises a sense strand and an antisense strand, the antisense strand being the short antisense oligonucleotide targeting the PLIN2 gene of the first aspect, and the sense strand being at least partially complementary to the short antisense oligonucleotide.

[0019] In a specific embodiment of the second aspect, the sense strand and the antisense strand preferably have a modification selected from the group consisting of:

[0020] (a) 3’-terminal N-acetylgalactosamine (GalNAc) modification, i.e. N-acetylgalactosamine (GalNAc modification) having affinity to asialoglycoprotein receptor, to achieve delivery targeting the liver;

[0021] (b) sugar ring modification, such as interval modification using 2’-fluoro (2’-F) and 2’-methoxy (2’-OMe); and

[0022] (c) backbone modification, such as phosphorothioate modification at the 5’ end of the antisense strand, to enhance siRNA nucleic acid drug stability and to increase the action period of the siRNA nucleic acid drug.

[0023] In a third aspect, the present application provides a composition, preferably a pharmaceutical composition, for inhibiting PLIN2 expression, comprising (a) the short antisense oligonucleotide of the first aspect or the RNAi agent of the second aspect, and (b) a pharmaceutically acceptable excipient.

[0024] In a fourth aspect, the present application provides a method for treating a disease related to abnormal lipid metabolism, such as NAFLD, comprising administering to a subject having NAFLD or considered to have a possibility of developing NAFLD (such as having hyperglycemia, hyperlipidemia, obesity, etc.) a therapeutically effective amount of the RNAi agent of the second aspect or the composition of the third aspect.

[0025] In a fifth aspect, the present application provides a method for treating or preventing liver steatosis in other pathological conditions and lipid accumulation in other tissues, comprising administering to a subject in need thereof a therapeutically effective amount of the RNAi agent of the second aspect or the composition of the third aspect.

[0026] In a sixth aspect, the present application provides use of the RNAi agent of the second aspect in the manufacture of a medicament for treating or preventing a disease associated with abnormal lipid metabolism, such as NAFLD, or liver steatosis in other pathological conditions and lipid accumulation in other tissues.

[0027] The present application has at least the following advantages.

[0028] Firstly, the present application innovatively uses the PLIN2 gene as a target of small interfering nucleic acid for treating or preventing NAFLD, and it is proved that RNAi targeting the PLIN2 gene can improve NAFLD. In particular, the RNAi agent of the present application targeting the PLIN2 gene is still effective when the subject has a genetic background of PNPLA3 I148M.

[0029] Secondly, the PLIN2 siRNA of the present application has GalNAc-modification and 2'-fluoro (2'-F) and 2'-methoxy (2'-OMe) spacer modification, phosphorothioate modification at the 5' end of the antisense strand, etc. The above-mentioned modifications can prevent the cleavage of PLIN2 siRNA by nucleases, improve the stability and half-life of nucleic acid drugs, reduce the immunogenicity of nucleic acid drugs, and improve the safety of nucleic acid drugs. Thus, the siRNA drug of the present application is liver-targeting, has good drug stability and half-life, and is low in immunogenicity and high in safety.

[0030] Thirdly, in terms of base composition, the human PLIN2 short antisense oligonucleotide of the present application is extremely similar to the base composition of the RNAi site corresponding to the mouse PLIN2 gene, only having a difference of 1 or 2 bases, and ensuring the effective work of siRNA. This design overcomes the difference in codon bias between two species, greatly improves the effect of the short antisense oligonucleotide in a mouse model on the guiding significance of the effect in humans, and greatly reduces the species difference when moving the drug test from an animal model to humans. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The structure diagram of the siRNA nucleic acid drug of the present application targeting PLIN2 with modification (GalNAc-siRNA) is shown.

[0032] Figure 2Statistical histograms of the reduction of mouse or human Plin2 mRNA levels for different siRNAs. Figure 2 A, Figure 2 B is the effect of Plin2 mRNA 1-5 (without chemical modification) on Plin2 mRNA levels in mice, humans, respectively. Figure 2 C is the result of Plin2 mRNA 2-3 (without chemical modification), Plin2 mRNA 2-3 ST (with chemical modification for improved stability) in mice, Figure 2 D is the mouse Plin2 mRNA levels and IC 50 .

[0033] Figure 3 Results for the administration of murine GalNAc-siRNA targeting the mouse Plin2 gene to non- modeled mice, left side: histograms of the mRNA expression levels of Plin2 in the liver, muscle and heart of non-modeled mice and Western blots, right side: body weight and blood lipid levels of the administration group and the control group (wild-type mice).

[0034] Figure 4 Results for the administration of murine GalNAc-siRNA targeting the mouse Plin2 gene to NAFLD model mice, left side: liver / body weight ratio, triglyceride content in the liver of mice in the administration group and the control group, middle: liver sections (H&E staining, oil red O staining, and TUNEL staining for cell apoptosis marker), right side: NAFLD score, fat content represented by the oil red O staining positive area, and liver damage degree represented by the proportion of TUNEL positive cells.

[0035] Figure 5 Schematic diagram for the construction of humanized mice of the PLIN2 gene, showing part of the alleles.

[0036] Figure 6 Results for the administration of human GalNAc-siRNA targeting the human PLIN2 gene to humanized mice, from left to right: histograms of the Plin2 mRNA expression levels in the liver of mice in the administration group and the control group, Western blots, body weight, and blood triglyceride content.

[0037] Figure 7 Results for the administration of human GalNAc-siRNA to NAFLD model mice of humanized mice, left side: liver / body weight ratio, triglyceride content in the liver of mice in the NAFLD control group and the NAFLD administration group, middle: liver sections (H&E staining, oil red O staining, and TUNEL staining for cell apoptosis marker), fat content represented by the oil red O staining positive area, right side: NAFLD score, and liver damage degree represented by the proportion of TUNEL positive cells.

[0038] Figure 8 Graphs of physiological indicators after 8 weeks of humanized Plin2 mice fed with western diet (WD) and subcutaneously injected with GalNAc-siRNA targeting human Plin2 gene. Left: liver / body weight ratio, triglyceride content in liver, right: H&E staining, oil red O staining, and TUNEL staining of liver sections.

[0039] Figure 9 Graphs of inflammatory cytokines and fibrosis-related gene expression after 8 weeks of humanized Plin2 mice fed with western diet (WD) and subcutaneously injected with GalNAc-siRNA targeting human PLIN2 gene.

[0040] Figure 10 Graphs of Sirius red staining and Masson staining of liver sections after 8 weeks of humanized Plin2 mice fed with western diet (WD) and subcutaneously injected with GalNAc-siRNA targeting human PLIN2 gene.

[0041] Figure 11 Relative lipid content after oil red O staining for control group without siRNA treatment, control RNA, and Plin2 siRNA 2ST groups in human HepG2 cells overexpressing PNPLA3 I148M.

[0042] Figure 12 Fluorescent staining graphs for oil red O and Plin2 siRNA 2ST groups in human cells overexpressing PNPLA3 I148M.

[0043] Figure 13 Oil red and H&E staining graphs of liver tissue sections after administration of Plin2 siRNA 2 or different drugs in PNPLA3-I148M mice modeling NAFLD.

[0044] Figure 14 Statistical results of multiple organ / body weight ratios after administration of Plin2 siRNA 2 (GalNAc-siRNA) in healthy young mice.

[0045] Figure 15 Histograms of mRNA expression levels of TNF-a, IFN-a, liver function physiological indicators, and lipid indicators in blood after administration of Plin2 siRNA 2 (GalNAc-siRNA) in healthy young mice. DETAILED DESCRIPTION

[0047] I. DEFINITIONS

[0048] Unless otherwise indicated, the practice of some of the methods disclosed herein employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art.

[0049] The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean ranges approximately 20% to 10%, to 5%, or to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean an order of magnitude up to, preferably within 5-fold, and more preferably within 2-fold. Where particular values are described in the application and claims, unless otherwise stated the term "about" means within an acceptable error range for the particular value.

[0050] The term "nucleotide" generally refers to an alkali-sugar-phosphate combination. Nucleotides can include analogs or derivatives of nucleotides as well as synthetic nucleotides. Nucleotides can be labeled for detection by known techniques. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels. "Nucleotide" includes DNA and RNA in the context of the present application.

[0051] In the term "isolated nucleic acid molecule", "isolated" means that the nucleic acid molecule has been removed from its natural state and exists in a state that is no longer in the chromosome genome, e.g., as a separate molecule.

[0052] The term "expression" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (e.g., transcribed into mRNA or other RNA transcript) and / or the mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as "gene product." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. "Up-regulation" or "down-regulation" of expression refers generally to an increase or decrease, respectively, in the level of expression of a polynucleotide (e.g., RNA, e.g., mRNA) and / or polypeptide sequence relative to its expression level in a wild-type state.

[0053] The term "modulate" with respect to expression or activity means to alter the level of expression or activity. Modulation can occur at the level of transcription and / or translation.

[0054] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, such as a mouse or a human. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0055] The term "treatment" refers herein to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. For example, treatment can include administering a therapeutically effective amount of an siRNA molecule or composition of the present application against PLIN2. Therapeutic benefit refers to the presence of any treatment-related improvement in the disease or symptoms being treated.

[0056] The term "prevention" refers herein to the administration of an siRNA molecule or composition of the present application against PLIN2 to a subject at risk of developing a particular disease or symptoms, or to a subject who exhibits one or more physiological indications of the disease, prior to the onset of the particular disease, to produce a prophylactic benefit, even though the disease or symptoms can not yet be evident.

[0057] The term "effective amount" or "therapeutically effective amount" refers to the amount of a composition, e.g., a composition comprising an siRNA molecule of the present application, which is sufficient to effect a desired activity or effect, e.g., an effect of reducing fat content (deposition) in the liver, when administered to a subject in need thereof.

[0058] As a double-stranded RNA consisting of 19 to 21 base pairs, siRNA can specifically target the target mRNA and promote its degradation, thereby inhibiting the transcriptional expression of the target gene, providing a new solution to the drug-making problem of many difficult target points.

[0059] II. Short antisense oligonucleotides targeting PLIN2

[0060] Perilipin-2 (PLIN2), also known as Adipose Differentiation-Related Protein (ADRP). The CDS sequences of human and murine PLIN2 are shown in SEQ ID NO: 36 and SEQ ID NO: 34, respectively, which have 82% sequence similarity.

[0061] The present application provides short antisense oligonucleotides and siRNA molecules targeting the PLIN2 gene, comprising a sense strand and an antisense strand, the antisense strand comprising a short antisense oligonucleotide targeting the PLIN2 gene, the sense strand being at least partially complementary to the short antisense oligonucleotide. The short antisense oligonucleotides or siRNA molecules of the present application can induce the degradation of PLIN2 mRNA by acting on PLIN2 mRNA.

[0062] Specific examples of double-stranded siRNA molecules targeting human PLIN2 include siRNA molecules having an antisense strand as set forth in SEQ ID NO: 12, 14, 16, 18, 20, preferably SEQ ID NO: 14, 16, more preferably SEQ ID NO: 14. Specific examples of double-stranded siRNA molecules targeting mouse PLIN2 include siRNA molecules having an antisense strand as set forth in SEQ ID NO: 2, 4, 6, 8, 10, preferably SEQ ID NO: 4, 6, more preferably SEQ ID NO: 4.

[0063] However, it will be understood by those skilled in the art that the scope of the present application is not limited to these specific siRNA molecules, but encompasses any siRNA molecule targeting PLIN2 mRNA and capable of acting on PLIN2 mRNA to produce an effect of reducing the level of PLIN2 mRNA.

[0064] On the basis of the short antisense oligonucleotide of the present application, a terminal can be introduced with a group that facilitates membrane permeation.

[0065] Thus, the present application provides an antisense oligonucleotide targeting the PLIN2 gene, and a double-stranded RNAi agent targeting the PLIN2 gene.

[0066] The basic method for designing antisense oligonucleotides and double-stranded RNAi agents against known sequences is known. Nonetheless, in a preferred embodiment of the present application, a design of siRNA with two strands that are not completely complementary is employed. Specifically, the exemplary siRNA of the present application has two strands whose lengths comply with the "19+2 base overhang principle". "19+2 base overhang" or "19+2" means that, of two strands each 21 nucleotides in length, in addition to 19 completely paired nucleotides, there are two nucleotides each that do not participate in pairing, thereby forming a 2-nucleotide overhang at each end. Such siRNA also complies with the definition of "the sense strand is at least partially complementary to the short antisense oligonucleotide" in the foregoing. The siRNA of the present application thus designed is capable of effectively inhibiting the expression of the target Plin2, and has desirable specificity.

[0067] On this basis, another unique aspect of the siRNAs of the present application is that the siRNAs have counterparts in both humans and mice. For example, for the short antisense oligonucleotides or siRNAs targeting mouse Plin2 in the present application, short antisense oligonucleotides and siRNAs with desired inhibitory activity and specificity can also be designed on the region of the orthologous human PLIN2 genomic sequence corresponding to the genomic region of mouse Plin2 that they target; and vice versa. Such siRNAs or short antisense oligonucleotides targeting the same region of Plin2 in the human and mouse genomes, respectively, are referred to as a set of counterparts. The set of counterpart short antisense oligonucleotides in humans and mice that meet the above definition include the following sets in the present application: SEQ ID NO: 12 and SEQ ID NO: 2, SEQ ID NO: 14 and SEQ ID NO: 4; SEQ ID NO: 16 and SEQ ID NO: 6; SEQ ID NO: 18 and SEQ ID NO: 8; SEQ ID NO: 20 and SEQ ID NO: 10. Each short antisense oligonucleotide in each set, when used as an antisense strand, forms a 19+2 base overhang double strand with the respective sense strand.

[0068] In the sets of counterparts, the smaller the sequence difference between the siRNA targeting the human gene and the counterpart sequence of the siRNA targeting the mouse gene, the more desirable it is, provided that the siRNA function is ensured. For example, the siRNA targeting the human gene and the counterpart sequence of the siRNA targeting the mouse gene differ by no more than 3 nucleotides, preferably differ by no more than 2 nucleotides, more preferably differ by no more than 1 nucleotide, and most preferably are completely identical. This is because when the siRNAs in the same set target the same gene region in humans and mice, and the sequences are highly similar, the data obtained in mice will have higher reference value for predicting the effect of the counterpart in humans. Therefore, such siRNA counterparts will be more convenient when conducting research or drug development.

[0069] III. Modifications

[0070] The siRNA agents can be modified (e.g., modified nucleotides or linked to other groups) to impart certain properties, such as improved silencing efficiency of the target gene, tissue / cell specificity, etc. In addition, since natural double-stranded RNA is susceptible to attack by nucleases, the siRNA can be chemically modified to enhance stability, thereby improving the therapeutic effect of the siRNA nucleic acid drug in vivo.

[0071] The short antisense oligonucleotides and siRNAs of the present application can have one or more modifications as described below.

[0072] Sugar ring modifications

[0073] One or more nucleotides in the siRNA of the application can be sugar ring modified to improve the stability of the siRNA.

[0074] In some embodiments, the sugar ring modification can be a 2'-methoxy modification, i.e. the 2'-OH position of the pentose is modified with a methoxy group, abbreviated as "2'-O-Methyl" or "2OMe". In specific embodiments, the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th position from the 5' end of the sense strand is 2OMe. In specific embodiments, the 1st, 3rd, 4th, 5th, 7th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st position from the 5' end of the antisense strand is 2OMe.

[0075] In some embodiments, the sugar ring modification can be a 2'-fluoro modification, i.e. the 2'-OH position of the pentose is modified with a fluoro group (2F). In specific embodiments, the 5th, 7th, 8th, 9th position from the 5' end of the sense strand is 2F. In specific embodiments, the 2nd, 6th, 8th, 9th, 14th, 16th position from the 5' end of the antisense strand is 2F.

[0076] In preferred embodiments, the siRNA of the application has both 2OMe and 2F modifications as described in the two paragraphs above on both the sense strand and the antisense strand.

[0077] sugar ring modifications

[0078] The nucleotide backbone of the siRNA of the application can be modified to improve the stability of the siRNA.

[0079] In some embodiments, the two phosphodiester linkages at the end of the sense strand or the antisense strand are replaced with phosphorothioate linkages.

[0080] In specific embodiments, the 1st and 2nd phosphodiester linkages at the 5' end of the sense strand are replaced with phosphorothioate linkages; and / or the 1st and 2nd phosphodiester linkages at the 5' end and the 1st and 2nd phosphodiester linkages at the 3' end of the antisense strand are replaced with phosphorothioate linkages.

[0081] 3' terminal N-acetylgalactosamine (GalNAc) modifications

[0082] Naked siRNA molecules are easily degraded by nucleases in vivo, and have poor membrane permeability, off-targeting, and immunogenicity, and thus need to be combined with an siRNA delivery system. As a transmembrane glycoprotein specifically expressed in hepatocytes, the asialoglycoprotein receptor (ASGPR) is a C-type lectin widely distributed on the surface of hepatocyte membranes and is highly expressed on the surface of hepatocytes (0.5-1 M receptors / cell). The extracellular segment exhibits a strong affinity for N-acetylgalactosamine (GalNAc) ligands. The affinity of a single GalNAc residue for the receptor is relatively low, while several GalNAc residues interact synergistically as a multimer to exhibit much higher affinity for ASGPR than a single GalNAc.

[0083] GalNAc trimer (trivalent) modification can drive selective and efficient oligonucleotide delivery to hepatocytes using the asialoglycoprotein receptor (ASGPR). As a preferred embodiment of the present application, by conjugating GalNAc in the form of a trimer to an siRNA molecule, GalNAc-siRNA can bind to ASGRP on the surface of hepatocyte membranes, enter hepatocytes through endocytosis, and achieve silencing of target genes.

[0084] 3' terminal N-acetylgalactosamine (GalNAc) modification methods are known in the art. As a GalNAc-modified siRNA, for example, it can be a structure as shown in Figure 1

[0085] IV. Compositions

[0086] The present application provides a composition comprising the short antisense oligonucleotide and the RNAi agent of the present application.

[0087] The short antisense oligonucleotide molecules of the present application can be used alone or in combination. Therefore, the present application provides a pharmaceutical composition comprising one or more, for example, 2, 3, 4, or more short antisense oligonucleotide molecules targeting PLIN2.

[0088] In the pharmaceutical composition of the present application, the short antisense oligonucleotide of the present application can also be combined with other pharmaceutically acceptable excipients, carriers for RNAi, such as encapsulated with nanocarriers (e.g., liposomes), exosomes, macromolecules, lipid molecules, or combined with novel carriers such as viruses, ligands, etc.

[0089] V. Delivery and modes of administration

[0090] The short antisense oligonucleotide and the RNAi agent or composition of the present application can be delivered into a subject by various means.​

[0091] In preferred embodiments, the RNAi agent or composition of the application is delivered to a subject in a tissue-specific manner, preferably in a liver-specific manner.

[0092] The short antisense oligonucleotides and RNAi agents or compositions of the application can be administered to a subject in a variety of modes of administration including, but not limited to, intraperitoneal administration, intravenous administration, subcutaneous / local administration, and the like. When the short antisense oligonucleotides and RNAi agents have GalNAc-modification, subcutaneous injection is preferred.

[0093] In one embodiment, the RNAi agent or composition is administered by subcutaneous injection. In mice, a dosage of 2-10 mg / kg, preferably 4 mg / kg, of siRNA, once in 4-6 weeks can be exemplified.

[0094] VI. Therapeutic uses

[0095] The agents, compositions, methods or systems for inhibiting PLIN2, antisense oligonucleotides, RNAi agents and compositions of the application can be used for preventing or treating a disease or symptoms associated with abnormal lipid metabolism, in particular a disease associated with liver fat deposition, more particularly non-alcoholic fatty liver disease (NAFLD), which includes simple fatty degeneration and NASH.

[0096] The term "non-alcoholic fatty liver disease (NAFLD)", according to the NAFLD Diagnosis and Treatment Guidelines published by the Chinese Medical Association Hepatology Branch Fatty Liver and Alcoholic Liver Disease Group in 2006, NAFLD is a clinical and pathological syndrome caused by excluding alcohol and other clear liver damaging factors, with diffuse large cell vacuolar fatty degeneration as the main feature, including simple fatty liver and fatty liver (nonalcoholic steatohepatitis, NASH) and cirrhosis. For the clinical diagnostic criteria of the disease, see the guidelines.

[0097] In some embodiments, the agents, compositions, methods or systems for inhibiting PLIN2 of the application can reduce the expression of PLIN2 as compared to the expression in the absence of the agents, compositions, methods or systems. Preferably, after the expression of PLIN2 is reduced, the lipid deposition, including liver fat content, lipid accumulation, liver-body weight ratio, can be reduced. For example, the expression of PLIN2 can be reduced to 95% or less, 90% or less, 80% or less, for example 70% or less, 60% or less, 50% or less, of the expression in the absence of the treatment. The reduction in expression can be determined by the reduction in mRNA and / or protein content.

[0098] The short antisense oligonucleotide and the RNAi agent of the present application can also be used in the research field. Since the siRNA of the present application can achieve sustained inhibition of PLIN2, for example, the inhibition time can be up to 4 weeks or more, therefore, by using the siRNA of the present application, an animal model with defective PLIN2 expression, such as a mouse model with defective Plin2 expression, or a humanized mouse model with defective PLIN2 expression, can be constructed, which can be used for the research of related metabolic diseases.

[0099] The present application specifically includes the following items.

[0100] 1. A short antisense oligonucleotide targeting a nucleotide sequence encoding a PLIN2 protein, specifically targeting a mRNA sequence encoding a PLIN2 protein.

[0101] 2. The short antisense oligonucleotide of the above item, wherein the PLIN2 is human PLIN2 (SEQ ID NO: 35) or mouse PLIN2 (SEQ ID NO: 33).

[0102] 3. The short antisense oligonucleotide of item 2, which is a short antisense oligonucleotide targeting mouse PLIN2, and comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID Nos: 2, 4, 6, 8, or 10.

[0103] 4. The short antisense oligonucleotide of item 3, which is a short antisense oligonucleotide targeting human PLIN2, and comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID Nos: 12, 14, 16, 18, or 20.

[0104] 5. An RNAi agent for inhibiting the expression of a PLIN2 gene, comprising the short antisense oligonucleotide of any one of items 1-4 as an antisense strand, and a sense strand which is at least partially complementary to the short antisense oligonucleotide, wherein the sense strand is at least 85% complementary to the short antisense oligonucleotide within the length range of the short antisense oligonucleotide.

[0105] 6. The RNAi agent of item 5, wherein the sense strand and the antisense strand are complementary in 19 nucleotides in length.

[0106] 7. The RNAi agent of item 5 or 6, comprising:

[0107] an antisense strand selected from the group consisting of the nucleotide sequences set forth as SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, and / or

[0108] the sense strand is selected from the group consisting of the nucleotide sequence as set forth in SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19, or a nucleotide sequence formed by deleting 2 nucleotides from the 3’ end of the nucleotide sequence as set forth in SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19.

[0109] 8. The RNAi agent of item 7, having the sense strand and antisense strand pairs as shown in Table 1.

[0110] 9. The RNAi agent of any one of items 5 to 9, wherein the sense strand or antisense strand comprises one or more modifications selected from the group consisting of:

[0111] (a) sugar ring modifications, such as 2’-fluoro modifications and / or 2’-methoxy modifications; and

[0112] (b) backbone modifications, such as phosphorothioate.

[0113] 10. The RNAi agent of item 9, the modification(s) increasing the stability of the RNAi agent.

[0114] 11. The RNAi agent of item 9 or 10, having the sense strand and antisense strand pairs as shown in Table 3.

[0115] 12. The RNAi agent of any one of items 5-11, wherein the RNAi agent further comprises the following modification:

[0116] (c) the 3’ end of the sense strand is modified with N-acetylgalactosamine (GalNAc).

[0117] 13. The RNAi agent of item 12, having the sense strand and antisense strand pairs as shown in Table 4.

[0118] 14. A composition comprising

[0119] (a) the short antisense oligonucleotide of any one of items 1 to 4 or the RNAi agent of any one of items 5-13, and

[0120] (b) a pharmaceutically acceptable excipient.

[0121] 15. A method for treating or preventing a disease associated with abnormal liver lipid metabolism in a subject, comprising administering to the subject a therapeutically effective amount of the RNAi agent of any one of items 5-13 or the composition of item 14.

[0122] 16. The method of item 15, the disease being non-alcoholic fatty liver disease (NAFLD).

[0123] 17. The method of item 16, wherein the NAFLD is selected from simple steatosis or progressive nonalcoholic steatohepatitis (NASH).

[0124] 18. The method of any one of items 15 to 17, wherein the subject has a PNPLA3 I148M mutation.

[0125] 19. The method of any one of items 15 to 18, wherein the inhibition of PLIN2 gene expression is performed in a liver tissue-specific manner. DETAILED DESCRIPTION

[0126] For a more complete understanding of and for further appreciation of the application, the application will be described in detail below with reference made to the embodiments and drawings. The following examples are intended to illustrate the application and are not intended to limit the scope of the application. The scope of the application is defined by the claims appended hereto.

[0127] EMBODIMENTS

[0128] Example 1. Screening of siRNAs targeting the same region of human PLIN2 and mouse Plin2

[0129] In this example, the inventors screened 5 groups of siRNAs targeting the same region of human PLIN2 and mouse Plin2 and verified their effects in cells.

[0130] 1.1 Software score screening

[0131] To screen siRNAs targeting the human PLIN2 gene (PLIN2 gene ID (Human): 123) or the mouse Plin2 gene (Plin2 gene ID (Mouse): 11520), the inventors designed hundreds of candidate siRNAs by software. On this basis, based on the results of two online calculation software (RNAhybrid (http: / / bibiserv.cebitec.uni-bielefeld.de / rnahybrid / ) and siDirect (http: / / sidirect2.rnai.jp / )), the top 15 siRNAs were selected from the candidate siRNAs targeting mouse Plin2 and the candidate siRNAs targeting human PLIN2, respectively, in terms of the comprehensive score of the knockdown efficiency and specificity of siRNAs in the form of “19+2”. Among these siRNAs, five groups of candidate siRNAs targeting the corresponding regions of orthologs in human and mouse were finally obtained by alignment, and the sequences thereof are specifically shown in Table 1. http: / / sidirect2.rnai.jp / ; http: / / biodev.extra.cea.fr / DSIR / DSIR.html To screen siRNAs targeting the human PLIN2 gene (PLIN2 gene ID (Human): 123) or the mouse Plin2 gene (Plin2 gene ID (Mouse): 11520), the inventors designed hundreds of candidate siRNAs by software. On this basis, based on the results of two online calculation software (RNAhybrid (http: / / bibiserv.cebitec.uni-bielefeld.de / rnahybrid / ) and siDirect (http: / / sidirect2.rnai.jp / )), the top 15 siRNAs were selected from the candidate siRNAs targeting mouse Plin2 and the candidate siRNAs targeting human PLIN2, respectively, in terms of the comprehensive score of the knockdown efficiency and specificity of siRNAs in the form of “19+2”. Among these siRNAs, five groups of candidate siRNAs targeting the corresponding regions of orthologs in human and mouse were finally obtained by alignment, and the sequences thereof are specifically shown in Table 1.

[0132] For example, in the sequences in Table 1, mouse Paire.1 and human Paire.1 are a group, mouse Paire.2 and human Paire.2 are a group, and so on.

[0133] Table 1. siRNAs targeting the same region of human and mouse plin2

[0134]

[0135] 1.2 Cell Experiments

[0136] Five groups of siRNAs (Suzhou Beixin Biotechnology Co., Ltd.) listed in Table 1 were synthesized and RNAi experiments were performed in mouse cells and human cells, respectively.

[0137] Mouse AML12 cell cultures were divided into five groups and treated with 50 nM mouse-Paire.1 to mouse-Paire.5 for 24 hours. The control group was treated with PBS. After treatment, cells were collected for qPCR to calculate the target knockdown efficiency (knockdown efficiency = plin2 expression level in experimental group / plin2 expression level in control group × 100%). The results are presented as follows. Figure 2 A.

[0138] Similarly, human HepG2 cell cultures were divided into five groups and treated with 50 nM human-Paire.1 to human-Paire.5 for 24 hours. The control group was treated with PBS. After treatment, cells were collected for qPCR to calculate the target knockdown efficiency, and the results are presented as follows. Figure 2 B.

[0139] like Figure 2 A and Figure 2 The results in B showed that mouse Paire.1, mouse Paire.2, and mouse Paire.3 (Plin2siRNA-2, Plin2 siRNA-2, and Plin2 siRNA-3), and the corresponding human Paire.1, human Paire.2, and human Paire.3 (PLIN2 siRNA-1, PLIN2 siRNA-2, and PLIN2 siRNA-3) had higher RNAi efficiency. Figure 2 A and Figure 2 B). Considering that although mouse Paire.1 and human Paire.1 target the corresponding orthologous regions, there are four differences after alignment, Paire.2 and Paire.3, which have differences of 1 and 2 positions respectively, were selected for further research (Table 2).

[0140] Table 2. Sequence differences between human and mouse siRNAs

[0141]

[0142] 1.3 Effects of stability modification

[0143] Paire.2 and Paire.3 were modified in the same way as the human siRNAs to improve stability, including:

[0144] (i) 2'-methoxy modification (2OMe) of the nucleotides at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 of the sense strand, counting from the 5' end;

[0145] (ii) 2'-methoxy modification (2OMe) of the nucleotides at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21 of the antisense strand, counting from the 5' end;

[0146] (iii) 2'-fluoro modification (2F) of the nucleotides at positions 5, 7, 8, 9 of the sense strand, counting from the 5' end;

[0147] (iv) 2'-fluoro modification (2F) of the nucleotides at positions 2, 6, 8, 9, 14, 16 of the antisense strand, counting from the 5' end; and

[0148] (v) replacement of the first and second phosphodiester linkages at the 5' end of the sense strand with phosphorothioate linkages; and replacement of the first and second phosphodiester linkages at the 5' end and the first and second phosphodiester linkages at the 3' end of the antisense strand with phosphorothioate linkages. In addition, the overhang of the last two nucleotides at the 3' end of the sense strand was removed, so the modified sense strand is two nucleotides shorter than the original sense strand.

[0149] The eight sequences of the two sets of four siRNAs after modification are shown in Table 3 as SEQ ID NO: 21 to SEQ ID NO: 28.

[0150] Table 3. siRNAs targeting mouse and human Plin2 containing stability modifications

[0151]

[0152] Note: "m" represents a "2'-OMe" modified RNA base, such as "Um"; "f" represents a "2'-F" modified RNA base, such as "Uf"; and "*" represents a phosphorothioate modification between two RNA bases.

[0153] The expression levels of the two pairs of modified mouse siRNAs were determined in mouse AML12 cells using the same method as in 1.2, and are shown in Figure 2 C. The siRNAs with stability modifications exhibited higher inhibitory activity compared to the corresponding unmodified siRNAs.

[0154] 1.4 Experiments using different concentrations of siRNA

[0155] To determine the siRNA with the best performance, cell experiments were performed using different concentrations of mouse Plin2 siRNA-2ST and mouse Plin2 siRNA-3ST, and the results are shown in Figure 2 D.

[0156] As shown in Figure 2 D, the IC50 of mouse Plin2 siRNA-2ST was smaller than that of mouse Plin2 siRNA-3ST, meaning that mouse Plin2 siRNA-2ST had a better inhibitory effect. Therefore, Plin2 siRNA-2ST was selected for subsequent experiments. 50

[0157] Example 2. Effects of mouse GalNAc-siRNA on expression of mouse Plin2 and physiological indicators

[0158] In this example, the effects of modified mouse GalNAc-siRNA in animals were determined, including the effects on the expression levels of Plin2 gene in the liver, muscle and heart of mice, the body weight of animals and blood lipids.

[0159] Mouse Plin2 siRNA-2ST and human Plin2 siRNA-2ST further containing GalNAc modification to improve liver targeting based on stability modification were prepared, and the sequences are shown in SEQ ID NO: 29 to SEQ ID NO: 32 in Table 4.

[0160] Table 4. siRNAs targeting human and mouse Plin2 further containing liver targeting modification

[0161]

[0162] Note: "m" represents a "2'-OMe" modified RNA base, such as "Um"; "f" represents a "2'-F" modified RNA base, such as "Uf"; and "*" represents a phosphorothioate modification between two RNA bases.

[0163] Unmodeled healthy mice (C57BL / 6J, 8 weeks old) were divided into a dosing group and a control group, with half males and half females in each group. The dosing group was administered mouse Plin2 siRNA-2 at 4 mg / kg body weight in a PBS solution, while the control group was administered PBS.

[0164] ​On day 0, 500 μL of a PBS solution of GalNAc-siRNA was administered by subcutaneous injection at the back of the mice in the administration group (the solution was prepared based on the body weight of the mice and the preset administration amount to ensure an administration amount of 4 mg / kg of body weight and a total administration volume). The control mice were administered with the same volume of PBS. The mice were euthanized on day 3, weighed, and blood was drawn, and the liver, gastrocnemius muscle at the medial side of the lower leg, and heart were removed by surgical operation, and after weighing, RNA extraction and protein extraction were performed. The RNA and protein expression levels were determined by real-time fluorescent quantitative PCR and Western blot, and the results are shown in Figure 3 .

[0165] Figure 3 It was shown that the expression level of the Plin2 gene in the liver of the mice was significantly reduced after 3 days of subcutaneous injection of the GalNAc-siRNA nucleic acid drug targeting the Plin2 gene of the mice (mouse Plin2 siRNA-2), while the expression level of the Plin2 gene in the muscle and heart was not affected, indicating good tissue specificity Figure 3 In addition, the mice receiving the nucleic acid drug exhibited similar body weight and blood lipids to wild-type mice, demonstrating the safety of the GalNAc-siRNA nucleic acid drug of the present application Figure 3 .

[0166] Example 3. Effect of mouse GalNAc-siRNA on NAFLD model mice

[0167] In this example, mouse GalNAc-siRNA (mouse Plin2 siRNA) was administered to NAFLD model mice established by a high-fat diet, and the effect of the siRNA of the present application was determined.

[0168] C57BL / 6J male mice 8 weeks old (6 mice each in the administration group and the control group) were fed with high-fat feed for 12 weeks (from week -12 to day 0) to induce the disease (modeling process). The specific composition of the high-fat diet is shown in Table 5.

[0169] Table 5. Proportion of high-fat diet ingredients

[0170] Fat Cholesterol Carbohydrates / fructose / carbohydrates Protein 60 kcal % 0 20 kcal % 20 kcal %

[0171] On day 0, 500 μL of a PBS solution of GalNAc-siRNA was administered by subcutaneous injection at the back of the mice in the administration group (the solution was prepared based on the body weight of the mice and the preset administration amount to ensure an administration amount of 4 mg / kg of body weight and a total administration volume). The control mice were administered with the same volume of PBS. The administration was continued with a high-fat diet.

[0172] The mice were euthanized at the 6th week after administration, weighed, blood was drawn, and the liver was removed by surgical operation, sectioned and stained, and biochemical indicators were detected. Specifically, the effects of the mouse-derived GalNAc-siRNA on the liver / weight ratio, the triglyceride content in the liver, the fat content, and the degree of liver damage of the NAFLD model mice were tested, H&E staining, oil red O staining, and TUNEL staining of the liver sections were performed, and NAFLD scores were obtained. The results are shown in Figure 4 .

[0173] As shown in Figure 4 , compared with the NAFLD control group mice, the liver / weight ratio of the NAFLD model mice injected with the nucleic acid drug of the application was significantly reduced, and the triglyceride content in the liver was reduced by about 60% ( Figure 4 left). The results of H&E staining and oil red O staining of the liver sections showed that the NAFLD of the mice injected with the nucleic acid drug of the application was significantly improved ( Figure 4 right). In addition, the TUNEL staining of the apoptosis marker also showed that the damage to the liver of the mice injected with the nucleic acid drug of the application was also significantly improved ( Figure 4 right). The above results prove that after subcutaneous injection of the GalNAc-siRNA nucleic acid drug targeting the mouse Plin2 gene, mouse Plin2 siRNA-2ST for 6 weeks, the NAFLD caused by high-fat diet feeding for 12 weeks was significantly reversed.

[0174] Example 4. Effects of human-derived GalNAc-siRNA on the expression amount and physiological indicators of humanized mouse Plin2

[0175] In this embodiment, the effect of the human-derived GalNAc-siRNA nucleic acid drug (Human-Plin2 siRNA-2) of the application targeting the human PLIN2 gene was evaluated in mice with the human PLIN2 gene knocked in.

[0176] 4.1 Construction of humanized Plin2 mice

[0177] As shown in Figure 5 , the genomic sequence of the Plin2 gene of the mouse from the first exon to the end of the 3'UTR was completely replaced with the corresponding human Plin2 gene sequence using the CRISPR-CAS9 technology, thereby obtaining the humanized Plin2 mice.

[0178] 4.2 Construction of NAFLD model of humanized Plin2 mice

[0179] The same method and formulation as in Example 3 were used to administer a high-fat diet to the humanized Plin2 mice for a total of 16 weeks (12 weeks before administration, 4 weeks after administration) to obtain a NAFLD model in the humanized Plin2 mice.

[0180] 4.3 Administration of GalNAc-siRNA to humanized mice

[0181] The mice obtained in 4.2 were divided into an administration group and a control group, each group having 6 male mice.

[0182] On day 0, the mice in the administration group were subcutaneously injected at the back with a 500 μL volume of a human GalNAc-siRNA (sense strand, SEQ ID NO: 31, antisense strand, SEQ ID NO: 32) solution in PBS to achieve an siRNA administration amount of 4 mg / kg of the mice. The control group mice were administered with the same volume of PBS. The mice were euthanized 6 weeks after the injection of the drug, weighed, bled, and the liver was surgically removed, weighed, and subjected to RNA extraction and protein extraction, and the results are shown in Figure 5 .

[0183] As shown in Figure 5 , the expression level of the human PLIN2 gene and the human PLIN2 protein level in the mouse liver were significantly reduced 6 weeks after subcutaneous injection of the human GalNAc-siRNA solution targeting the human PLIN2 gene, demonstrating that the administered siRNA effectively silenced the human PLIN2 gene Figure 5 . In addition, the humanized mice that received the nucleic acid drug of the present application exhibited similar body weight and blood lipids to wild-type mice, demonstrating the safety of the nucleic acid drug Figure 5 .

[0184] The liver / body weight ratio, liver triglyceride content, liver sections (H&E staining, oil red O staining, and TUNEL staining for cell apoptosis marker), NAFLD score, fat content, and degree of liver damage were also measured in the humanized mice of the NAFLD model established by a high-fat diet 6 weeks after administration of the human GalNAc-siRNA. Figure 6 .

[0185] The results show that NAFLD caused by 16 weeks of high-fat diet feeding was significantly reversed 4 weeks after subcutaneous injection of the GalNAc-siRNA nucleic acid drug targeting the human PLIN2 gene. The liver / body weight ratio of the mice injected with the nucleic acid drug was significantly reduced, and the liver triglyceride content was reduced by about 60% compared to the NAFLD control group mice Figure 6 .

[0186] H&E staining and Oil Red O staining results of liver sections showed that NAFLD of mice injected with nucleic acid drugs was significantly improved Figure 6 In addition, TUNEL staining of apoptosis markers also showed that the damage to the liver of mice was also significantly improved Figure 6

[0187] 4.4 Construction of NASH model of humanized Plin2 mice

[0188] The humanized Plin2 mice were divided into drug groups and control groups, each group of 6, all male.

[0189] In order to establish a NASH model, all mice were given a high-fat diet for a total of 14 weeks (6 weeks before administration, 8 weeks after administration) combined with 30% fructose drinking water (Western diet, WD). The high-fat diet formula is the same as the high-fat diet formula of Example 3, thereby obtaining a NASH model of humanized Plin2 mice.

[0190] On day 0, the humanized Plin2 mice that had been high-fat fed for 6 weeks were injected subcutaneously on the back with modified human GalNAc-siRNA (Human-Plin2 siRNA-2) at a dose of 4 mg / kg body weight, by dissolving the GalNAc-siRNA in an appropriate amount of PBS, and with an injection volume of 500 μL. The control group was given an equal volume of PBS.

[0191] The second injection was performed at week 4, and the drug and dose were the same as the first injection.

[0192] At week 8, the effect of human GalNAc-siRNA on the expression level of human Plin2 gene in the liver of mice, the body weight of animals and blood lipids was detected. Specifically, at week 8, the mice were euthanized, weighed, bled, and the liver was removed by surgical operation, and the liver / body weight ratio, triglyceride content in the liver, liver sections (H&E staining, Oil Red O staining, and TUNEL staining of apoptosis markers), inflammation, and fibrosis of the NASH model humanized mice were detected. The results are shown in Figure 7 Figure 8 Figure 9 and Figure 10 .

[0193] The results show that after 8 weeks of subcutaneous injection of GalNAc-siRNA nucleic acid drugs targeting the human Plin2 gene, NASH caused by 14 weeks of WD feeding was significantly improved. Compared with NASH control group mice, the liver / body weight ratio of mice injected with nucleic acid drugs was significantly reduced, and the triglyceride content in the liver was reduced by about 40% Figure 7

[0194] ​​​​H&E staining and Oil Red O staining results of liver sections showed that steatosis of mice injected with nucleic acid drugs was significantly improved Figure 7 In addition, TUNEL staining of apoptosis markers showed that the damage to the mouse liver was significantly improved Figure 7 In addition, the expression of inflammatory factors was significantly reduced Figure 8 Left), indicating that inflammation in the mouse liver was alleviated. At the same time, the expression of fibrosis-related genes was significantly reduced Figure 8 Left), Sirius red staining and Masson staining showed that the area of liver fibrosis was reduced Figure 8 Right), indicating that the fibrosis of the mouse liver was alleviated.

[0195] The above results show that the modified human GalNAc-siRNA (Human-Plin2 siRNA-2) of the present application has a good therapeutic effect on the occurrence and development of NASH.

[0196] Example 5. Effect of Plin2 siRNA and PNPLA3 I148M mutation

[0197] PNPLA3 I148M mutation is the most relevant gene mutation to NAFLD discovered so far, and is very common in NAFLD patients, with about 50% of NAFLD patients carrying at least one PNPLA3 I148M.

[0198] Some literature indicates that the efficacy of some NAFLD treatment drugs is weakened in the presence of PNPLA3 I148M in the subject. Considering that the efficacy of most NAFLD drugs is not obvious after entering the clinic, and there is no literature reporting the mechanism of action between Plin2 and PNPLA3 I148M mutation, the inventors detected whether the good therapeutic effect of Plin2 siRNA was affected by PNPLA3 I148M.

[0199] In this example, human HepG2 cells transfected with overexpression plasmids carrying PNPLA3 I148M mutation were divided into three groups. The first group was not treated with siRNA, and the other two groups were added with control siRNA (19+2 type siRNA not targeting any mouse and human genomic sequence) and Human-Plin2 siRNA-2 of the present application (final concentration 30 nM) respectively under the condition of adding oleic acid (0.6 mM oleic acid treatment for 24 hours to induce fat accumulation in HepG2 cells) and overexpression of PNPLA3 I148M, and the relative lipid content and fluorescence staining results are shown in Figure 11 and Figure 12 .

[0200] The results show that Plin2 siRNA can still well reduce lipid accumulation in the background of the test cells having the PNPLA3 I148M mutation.

[0201] Example 6. Effect of Plin2 siRNA in PNPLA3-I148M mouse NAFLD model

[0202] In this example, the inventors first fed PNPLA3-I148M mice high-fat feed with 30% fructose drinking water for 6 weeks, then divided the mice into the following 7 groups and gave corresponding treatments:

[0203] Group 1 is the control group, injected with PBS;

[0204] Groups 2-6 are experimental groups, respectively given 5 kinds of comparative drugs, Bempedoic acid (MCE, HY-12357), AZD3988 (DGAT-1 inhibitor, Aladdin, A286752), TVB2640 (Aladdin, T421492), Firsocostat (Aladdin, F414250), and Obeticholic acid agent (MCE, HY-12222); and

[0205] Group 7 is the drug group of the present application, given Human-Plin2 siRNA-2.

[0206] Specifically, the PNPLA3-I148M mouse is purchased from Nanmou Biological, and the amino acid at position 148 of the mouse Pnpla3 gene is replaced from I to M, establishing the Pnpla3 gene point mutation mouse model.

[0207] From week 0 to week 8, the mice in the 5 comparative drug groups were respectively given Bempedoic acid, AZD 3988, TVB2640, Firsocostat, and Obeticholic acid agent, once a day by gavage. The dose of Obeticholic acid is 10 mg / kg, and the doses of the other 4 drugs are 4 mg / kg.

[0208] From week 0 to week 8, the mice in the Plin2 siRNA group were subcutaneously injected with Human-Plin2 siRNA-2 once every 4 weeks, and the dose of GalNAc-siRNA was 4 mg / kg.

[0209] At the end of week 8, all mice were euthanized, and the liver was surgically removed, prepared into liver tissue sections as described above, and the staining results are shown in Figure 13 .

[0210] The results show that the steatosis treatment effect of Human-Plin2 siRNA-2 is significantly better than that of the other drugs.

[0211] Example 7. Safety test of Plin2 siRNA (male mice)

[0212] In this example, to test whether GalNAc-Plin2 siRNA has obvious adverse reactions on the organism, 8-week-old male C57BL / 6J mice were subcutaneously injected with Human-Plin2 siRNA-2 (4 mg / Kg) on day 0, and then fed with normal feed for 6 weeks. The control group was given the same volume of PBS.

[0213] All mice were euthanized at the end of week 6, and the following body weight and the ratio of organ weight to body weight, inflammatory factors, liver function indicators were detected. The results are shown in Figure 14 and Figure 15 .

[0214] The results show that injection of GalNAc-Plin2 siRNA does not affect the body weight and the ratio of organ weight to body weight, which indicates that there is no obvious damage to the organs Figure 14 ).

[0215] The inventors detected the inflammatory factors Tnf-α and IFN-α in the liver of mice, and the results showed that there was no significant difference in Tnf-α and IFN-α in the liver of the Human-Plin2 siRNA-2 test group compared with the control group, indicating that injection of Human-Plin2 siRNA-2 did not cause an immune response.

[0216] The indicators of liver function in the blood of mice, Albumin, ALT, AST, and the content of triglyceride and cholesterol in the liver were detected, in addition, the serum triglyceride and cholesterol were detected, and the results are shown in Figure 12 .

[0217] The results show that injection of Human-Plin2 siRNA-2 can reduce the content of triglyceride in the liver, and the remaining indicators do not change significantly, indicating that Human-Plin2 siRNA-2 does not have adverse effects on the liver. There is no significant change in serum triglyceride and cholesterol, indicating that Human-Plin2 siRNA-2 does not have adverse effects on systemic lipid metabolism.

Claims

1. Small interfering RNA, which contains an antisense strand and a sense strand, wherein: (a) The nucleotide sequence of the antisense strand is as shown in SEQ ID No: 14, and The nucleotide sequence of the positive strand is the nucleotide sequence shown in SEQ ID No: 13, or a nucleotide sequence formed by deleting the two nucleotides at the 3' end of the nucleotide sequence shown in SEQ ID No: 13; or (b) The nucleotide sequence of the antisense strand is as shown in SEQ ID No: 16, and The nucleotide sequence of the positive strand is the nucleotide sequence shown in SEQ ID No: 15, or the nucleotide sequence formed by deleting the two nucleotides at the 3' end of the nucleotide sequence shown in SEQ ID No:

15.

2. The small interfering RNA according to claim 1, wherein the sense strand or antisense strand comprises one or more modifications selected from the group consisting of: (a) Sugar ring modification; (b) Skeleton modification; (c) The 3' end of the positive chain is modified with N-acetylgalactosamine (GalNAc).

3. The small interfering RNA according to claim 2, wherein the sugar ring modification is a 2'-fluoro modification and / or a 2'-methoxy modification.

4. The small interfering RNA according to claim 2, wherein the backbone modification is a phosphate thioester modification.

5. The small interfering RNA according to claim 1, wherein, The small interfering RNA has the following sense and antisense strand pairs: (a) The nucleotide sequence of the positive strand is the nucleotide sequence Gm*Am*UmUmUfGmAfUfCfUmGmGmUmUmCmAmGmAmAm, as shown in SEQ ID NO:

25. The nucleotide sequence of the antisense strand is as shown in SEQ ID NO: 26: Um*Uf*CmUmGmAfAmCfCfAmGmAmUmCfAmAfAmUmCm*Cm*Um; (b) The nucleotide sequence of the positive strand is the nucleotide sequence shown in SEQ ID NO: 27: Gm*Gm*GmUmGfAmUfGfGfAmCmAmAmGmAmCmCmAmAmAm, and The nucleotide sequence of the antisense strand is as shown in SEQ ID NO: 28: Um*Uf*GmGmUmCfUmUfGfUmCmCmAmUfCmAfCmCmCm*Cm*Um; or (c) The nucleotide sequence of the positive strand is the nucleotide sequence Gm*Am*UmUmUfGmAfUfCfUmGmGmUmUmCmAmGmAmAm-GalNAc, as shown in SEQ ID NO: 31, and The nucleotide sequence of the antisense strand is as shown in SEQ ID NO: 32: Um*Uf*CmUmGmAfAmCfCfAmGmAmUmCfAmAfAmUmCm*Cm*Um. Where m represents a 2'-OMe modified RNA base, f represents a 2'-F modified RNA base, and * represents a phosphate thiophosphate modification between two RNA bases.

6. Use of the small interfering RNA reagent according to any one of claims 1-5 in the preparation of a medicament for the treatment of non-alcoholic fatty liver disease.

7. The use according to claim 6, wherein the non-alcoholic fatty liver disease is simple steatosis or progressive non-alcoholic steatohepatitis.

8. The use according to claim 6, wherein the subject has the PNPLA3 I148M mutation.

9. The use according to claim 7, wherein the subject has the PNPLA3 I148M mutation.

Images