SiRNA for inhibiting MTTP gene expression and application thereof

Abstract

The invention provides siRNA for inhibiting MTTP gene expression and application of the siRNA. Specifically, the invention provides siRNA, a siRNA conjugate and a pharmaceutical composition thereof for inhibiting the expression of a microsome triacylglycerol transferase (MTTP) gene, and a method for reducing the expression of the MTTP gene by using the siRNA, the siRNA conjugate and the pharmaceutical composition thereof. The siRNA, the siRNA conjugate and the pharmaceutical composition of the siRNA can be used for preventing and / or treating MTTP gene mediated diseases or symptoms.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to siRNA for inhibiting MTTP (microsomal triacylglycerol transferase) gene expression and application thereof. Background Art

[0002] Microsomal triglyceride transfer protein (MTTP) plays a crucial role within cells. MTTP regulates lipid metabolism, promoting the assembly and transport of lipid components such as triglycerides, phospholipids, and cholesterol in liver and intestinal cells. In hepatocytes, MTTP is a key protein in lipoprotein synthesis and secretion, participating in the assembly of very low-density lipoprotein (VLDL). VLDL is then secreted into the blood by hepatocytes, where it further binds to the lipoprotein APOB and is converted into LDL-C. Therefore, MTP is a key protein influencing LDL-C levels in the blood.

[0003] Lomitapiprib is the only marketed MTTP inhibitor, effectively lowering LDL-C levels in the blood by inhibiting the assembly and secretion of VLDL particles. Clinically, lomitapiprib is used to treat familial hypercholesterolemia and as an emergency medication for patients with extremely high LDL-C levels to prevent the onset of acute cardiovascular disease. However, the clinical use of lomitapir is associated with numerous limitations and adverse reactions, including severe gastrointestinal toxicity and hepatotoxicity.

[0004] Therefore, there is an urgent need in the art to develop a clinically safer and longer-lasting drug for inhibiting MTTP. Summary of the Invention

[0005] The present invention provides a clinically safer and longer-acting drug for inhibiting MTTP.

[0006] In the first aspect of the present invention, a siRNA for inhibiting MTTP gene expression is provided, wherein the siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, and the antisense strand comprises at least 19 consecutive nucleotides that differ from any antisense strand sequence shown in Table 2 or Table 3 by 0, 1, 2 or 3 nucleotides, and the sense strand and the antisense strand have at least 15, 16, 17, 18, 19, 20 or 21 nucleotides complementary.

[0007] In another preferred example, the sense strand comprises at least 19 consecutive nucleotides that differ from any sense strand sequence shown in Table A or Table B by 0, 1, 2 or 3 nucleotides.

[0008] In another preferred embodiment, the antisense strand has 19-23 bases complementary to the human MTTP transcript (NM_000253.4).

[0009] In another preferred embodiment, the lengths of the sense strand and the antisense strand are independently 19-23 nucleotides.

[0010] In another preferred embodiment, the antisense strand has two nucleotide overhangs at the 3' end relative to the sense strand.

[0011] In another preferred embodiment, the antisense strand is a sequence selected from the following: SEQ ID NO: 2m, and the value of m is any positive integer selected from 1 to 78.

[0012] In another preferred embodiment, the sense strand is a sequence selected from the following: SEQ ID NO: 2n-1, wherein the value of n is any positive integer selected from 1 to 78.

[0013] In another preferred embodiment, the antisense strand comprises any one of the antisense strand nucleotide sequences shown in Table 2, and the sense strand comprises any one of the sense strand nucleic acid sequences shown in Table 2.

[0014] In another preferred embodiment, the siRNA is selected from any siRNA sequence code in Table 2.

[0015] In another preferred embodiment, the sense strand and antisense strand are selected from the sense strand and antisense strand shown in the siRNA sequence code in Table A below:

[0016] Table A

[0017]

[0018]

[0019] In another preferred embodiment, at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.

[0020] In another preferred embodiment, the modified nucleotides are selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, phosphorothioate modified nucleotides, abasic nucleotides and locked nucleotides.

[0021] In another preferred embodiment, the modified nucleotides are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, and phosphorothioate modified nucleotides.

[0022] In another preferred embodiment, the 5' end of the sense strand contains 1 or 2 phosphorothioate-modified nucleotides; and / or the 5' end and 3' end of the antisense strand each independently contain 1 or 2 phosphorothioate-modified nucleotides.

[0023] In another preferred embodiment, the antisense strand comprises any one of the antisense strand nucleotide sequences shown in Table 3, and the sense strand comprises any one of the sense strand nucleic acid sequences shown in Table 3.

[0024] In another preferred embodiment, the siRNA is selected from any siRNA sequence code in Table 3.

[0025] In another preferred embodiment, the sense strand and antisense strand are selected from the sense strand and antisense strand shown in the siRNA sequence code in Table B below:

[0026] Table B

[0027]

[0028] Among them, the letter m indicates that the nucleotide adjacent to the right of m is a 2'-O-methyl modified nucleotide; f indicates that the nucleotide adjacent to the right of the letter f is a 2'-fluoro modified nucleotide; * indicates that the nucleotide adjacent to the left of * is a phosphorothioate modified nucleotide.

[0029] In a second aspect of the present invention, an siRNA conjugate is provided, wherein the siRNA conjugate comprises the siRNA described in the first aspect of the present invention and a targeting group.

[0030] In another preferred embodiment, the conjugate further comprises a linker, and the siRNA, the linker and the targeting group are covalently or non-covalently linked once.

[0031] In another preferred embodiment, the targeting group contains a group derived from carbohydrates, and the carbohydrates include monosaccharides, disaccharides, trisaccharides or polysaccharides.

[0032] In another preferred embodiment, the targeting group is selected from the following monosaccharides: allose, maltose, arabinose, cladinose, brown sugar, erythrose, fructose, D-fucitol, L-fucitol, fucosamine, fucose, fucose, galactosamine, D-galactosaminol, N-acetyllactosamine (GalNAc), galactose, glucosamine, N-acetyl-glucosamine, glucosaminol, glucose, glucose-6-phosphate, glucose glyceraldehyde, L-glycero-D-mannose-heptose, glycerol, glycerol, glucose, iodine, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, quinovose, quinovosamine, rhamnose, rhamnose, ribose, ribulose, heptose, sorbose, tagatose, talose, tartaric acid, threose, xylose and xylose.

[0033] In another preferred embodiment, the targeting group contains a group derived from N-acetylgalactosamine (GalNAc) or a derivative thereof.

[0034] In another preferred embodiment, the conjugate further contains a linker.

[0035] In another preferred embodiment, the conjugate has a structure shown in the following formula (I):

[0036]

[0037] Wherein, the targeting group (GalNAc) n is N-acetylgalactosamine or a derivative thereof, Ln is a linker, Zn is the nucleotide at the 3' end of the sense chain, and the value of n is 2-4.

[0038] In another preferred embodiment, n is 3.

[0039] In another preferred embodiment, when n is 3, the conjugate has a structure shown in the following formula (II):

[0040]

[0041] In another preferred embodiment, the conjugate contains the positive chain shown in SEQ ID NO: 157.

[0042] In another preferred embodiment, the conjugate contains a sense chain as shown in SEQ ID NO: 157 and an antisense chain as shown in SEQ ID NO: 108.

[0043] In a third aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0044] (a) the siRNA according to the first aspect of the present invention, and / or the conjugate according to the second aspect of the present invention; and

[0045] (b) a pharmaceutically acceptable carrier.

[0046] In another preferred embodiment, the pharmaceutical composition is used to inhibit the expression of MTTP gene.

[0047] In another preferred embodiment, the expression inhibition rate can be calculated by the following formula:

[0048]

[0049] In another preferred embodiment, the value of Y is 50% to 90%.

[0050] In the fourth aspect of the present invention, there is provided the use of the siRNA described in the first aspect of the present invention, the conjugate described in the second aspect of the present invention, and the pharmaceutical composition described in the third aspect of the present invention in the preparation of a medicament for preventing and / or treating MTTP-mediated diseases or conditions, for inhibiting the expression of the MTTP gene.

[0051] In another preferred embodiment, the MTTP-mediated disease or disorder is a disease or disorder caused by overexpression of MTTP.

[0052] In another preferred embodiment, the disease or condition includes lipid metabolism disorder.

[0053] In another preferred embodiment, the diseases or conditions include: atherosclerosis, hypercholesterolemia, hypertriglyceridemia, myocardial infarction, and cardiovascular disease.

[0054] In another preferred embodiment, the preparation comprises a laboratory preparation.

[0055] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0056] In another preferred embodiment, the expression inhibition rate can be calculated by the following formula:

[0057]

[0058] In another preferred embodiment, the value of Y is 50% to 90%.

[0059] In a fifth aspect of the present invention, a method for inhibiting MTTP expression in cells in vitro is provided, the method comprising the following steps:

[0060] (z1) Co-culturing cells with an effective amount of the siRNA described in the first aspect of the present invention, the conjugate described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention.

[0061] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0062] In another preferred embodiment, the siRNA described in the first aspect of the present invention, the conjugate described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention enters the cell through endocytosis.

[0063] In the sixth aspect of the present invention, a method for treating MTTP-mediated diseases or conditions is provided, wherein a therapeutically effective amount of the siRNA of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the pharmaceutical composition of the third aspect of the present invention is administered to a subject.

[0064] In another preferred embodiment, the MTTP-mediated disease or condition is a disease or condition caused by overexpression of MTTP.

[0065] In another preferred embodiment, the administered dose is 1 to 20 mg / kg.

[0066] In another preferred embodiment, the disease or condition includes lipid metabolism disorder.

[0067] In another preferred embodiment, the diseases or conditions include: atherosclerosis, hypercholesterolemia, hypertriglyceridemia, myocardial infarction, and cardiovascular disease.

[0068] In another preferred embodiment, the subject is a human or a non-human mammal.

[0069] In another preferred embodiment, the subject is a human.

[0070] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The effects of some GalNAc-modified siRNA compositions on the levels of human MTTP mRNA and protein in the liver of C57BL / 6 mice are shown: (A) Schematic diagram of the experimental process of the in vivo experiment described in Example 3; (B) The level of human MTTP mRNA in the liver of each group of animals after treatment with the specified siRNA composition (5 mg / kg, two subcutaneous injections); (C) The level of human MTTP protein in the liver of each group of animals after treatment with the specified siRNA composition; It can be seen in the figure that after AAV-TBG-hMTTP injection, the level of human MTTP protein in the liver was significantly increased, while the level of human MTTP protein in the liver was significantly decreased after administration of the test drug, and only the background of mouse MTTP protein was visible.

[0072] Figure 2 The effects of some GalNAc-modified siRNA compositions on the level of human MTTP mRNA in the liver of C57BL / 6 mice are shown: (A) Schematic diagram of the experimental flow of the in vivo experiment described in Example 4; (B) is the level of human MTTP mRNA in the liver of each group of animals 30 days after a single subcutaneous injection of the specified siRNA composition (20 mg / kg).

[0073] Figure 3 The effects of some GalNAc-modified siRNA compositions on the levels of human MTTP mRNA and protein in the liver of C57BL / 6 mice are shown: (A) Schematic diagram of the experimental process of the in vivo experiment described in Example 5; (B) The level of human MTTP mRNA in the liver of each group of animals after treatment with the specified siRNA composition (0.3, 1.0, 3.0 mg / kg, single subcutaneous injection); (C) The level of human MTTP protein in the liver of each group of animals after treatment with the specified siRNA composition; It can be seen in the figure that after AAV-TBG-hMTTP injection, the level of human MTTP protein in the liver was significantly increased, while the level of human MTTP protein in the liver was significantly decreased after administration of the test drug, and only the background of mouse MTTP protein was visible. DETAILED DESCRIPTION

[0074] After extensive and in-depth research, the present inventors have developed, for the first time, a class of siRNAs (SEQ ID NOs: 1-98), modified siRNAs (SEQ ID NOs: 99-156), and siRNA conjugates (SEQ ID NO: 157, SEQ ID NO: 108) that can inhibit MTTP expression or reduce its activity. Experimental results demonstrate that the siRNAs, modified siRNAs, and siRNA conjugates of the present invention not only exhibit high cellular stability but also exhibit high MTTP mRNA expression inhibitory activity. This is the basis for the present invention.

[0075] The siRNA provided by the present invention includes a sense chain and an antisense chain. The antisense chain induces the formation of an RNA-induced silencing complex (RISC) and binds to the human MTTP transcript (NM_000253.4) through base complementary pairing, thereby mediating the cleavage of the MTTP gene transcript by the Argonaute 2 protein and reducing the expression of MTTP.

[0076] the term

[0077] As used herein, the abbreviations and meanings of nucleotide monomers used in the present invention (including in the Examples) are shown in Table 1 below.

[0078] Table 1

[0079] abbreviation Nucleotide (full English name) Nucleotide (Chinese) A Adenine ribonucleotide adenosine U Uracil ribonucleotide Uridine G Guanine ribonucleotide Guanosine C Cytosine ribonucleotide Cytidine mA Adenine 2'-O-methyl ribonucleotide 2'-O-methyladenosine mU Uracil 2'-O methyl ribonucleotide 2'-O-methyluridine mG Guanine 2'-O methyl ribonucleotide 2'-O-methylguanosine mC Cytosine 2'-O methyl ribonucleotide 2'-O-methylcytidine fA Adenine 2'-fluoro ribonucleotide 2'-Fluoroadenosine f Uracil 2'-fluoro ribonucleotide 2'-Fluorouridine fG Guanine 2'-fluoro ribonucleotide 2'-Fluoroguanosine fC Cytosine 2'-fluoro ribonucleotide 2'-Fluorocytidine * Phosphorothioate Phosphorothioate bond GalNac Triantennary GalNac(N-acetyl-galactosamine) N-acetylgalactosamine

[0080] It should be noted that the sequences in the sequence listing file of the present invention are all sequences without sequence modification annotations and are for reference only. The sequences containing sequence modifications actually used in this application (including in the Examples) are the sequences annotated in Tables 3 and 4, or Tables B and C of this application. The definitions of mA, mU, mG, mC, fA, fU, fG, fC, *, and GalNac in Tables 3, 4, B, and C are detailed in Table 1.

[0081] As used herein, the term "MTTP" refers to microsomal triacylglycerol transferases from humans, rodents, and primates, including full-length unprocessed precursor forms, mature forms (signal peptide cleavage), and protease-processed MTTP proteins. The human MTTP mRNA transcript is numbered NM_000253.4, and the cynomolgus monkey MTTP mRNA predicted sequence is numbered XM_028848642.1.

[0082] The term "interfering RNA" or "RNAi" or "interfering RNA sequence" includes single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide) or double-stranded RNA (i.e., duplex RNA such as siRNA, dsRNA, shRNA, aiRNA, or precursor miRNA) that can reduce or inhibit the expression of a target gene or sequence when the interfering RNA and the target gene or sequence are in the same cell (e.g., by mediating degradation and inhibiting translation of an mRNA that is complementary to the interfering RNA sequence). Interfering RNA therefore refers to a single-stranded RNA that is complementary to a target mRNA sequence or a double-stranded RNA formed by two complementary strands or by a single self-complementary strand.

[0083] Interfering RNA includes "small interfering RNA" or "siRNA", each strand of the siRNA comprises about 15 to about 60 nucleotides (e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, 17-25, 19-25, 17-23, 17-21, 19-23, or 19-21 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). Ranges and lengths intermediate to the above-recited ranges and lengths are also contemplated as part of the present invention. In a preferred embodiment, the siRNA is chemically synthesized. The siRNA of the present invention is capable of silencing the expression of a target sequence in vitro and / or in vivo. In other embodiments, the siRNA comprises at least one modified nucleotide, for example, the siRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides in the double-stranded region.

[0084] As used herein, the terms "dsRNA," "double-stranded ribonucleic acid," or "precursor RNAi molecule" are intended to include any precursor molecule that is processed by an endonuclease in vivo to generate an active siRNA.

[0085] As used herein, "dsRNA" in the present invention refers to a double helix structure formed by two antiparallel and base-complementary nucleic acid chains, which form two directions of nucleic acid chains, "sense chain" and "antisense chain", relative to the target gene mRNA, depending on whether they are complementary.

[0086] The term "siRNA" refers to a molecule containing an siRNA as that term is defined herein, and which mediates targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. Via a process known as RNA interference (RNAi), the iRNA directs sequence-specific degradation of mRNA. The iRNA regulates (e.g., inhibits) expression of MTTP in a cell (e.g., a cell in a subject such as a mammalian subject).

[0087] Typically, the majority of the nucleotides in each strand of the siRNA are ribonucleotides, but as described in detail herein, each or both of the two strands may also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. In addition, as used in this description, "siRNA" may include ribonucleotides with chemical modifications; siRNA may include substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions, or removals of, for example, a functional group or atom of an internucleoside linkage, a sugar moiety, or a nucleobase. Modifications suitable for use in the present invention include all types of modifications disclosed herein or known in the art.

[0088] As used herein, "G," "C," "A," and "U" refer to nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. "Ribonucleotide" or "nucleotide" may also refer to modified nucleotides.

[0089] The siRNA of the present invention comprises single-stranded RNA that interacts with the MTTP mRNA sequence, thereby guiding the cleavage of the mRNA. Upon entering the cell, the double-stranded siRNA binds to AGO2, unwinding the double helix and then entering the RISC, where it further guides the antisense strand to recognize and complementarily pair with the MTTP mRNA. Ultimately, AGO2 and other endonucleases within the RISC cleave the MTTP mRNA, reducing MTTP mRNA levels.

[0090] As used herein, the term "nucleotide overhang" refers to unpaired nucleotides that protrude from the double helical structure of an siRNA. For example, when the 3' end of the antisense strand of an siRNA extends beyond the 5' end of the sense strand, the nucleotides in the excess are referred to as nucleotide overhangs.

[0091] As used herein, the term "antisense strand" or "guide strand" refers to a nucleotide sequence in a double-stranded siRNA that is complementary to the target sequence (MTTP mRNA). A nucleotide sequence with this complementary characteristic is defined as the antisense strand. The antisense strand and the target mRNA sequence may not completely match; mismatches may occur in the terminal regions.

[0092] As used herein, the term "complementarity" refers to the ability of one nucleotide sequence to hybridize with another nucleotide sequence under certain conditions and form a double-stranded structure. When the two strands of an siRNA are completely base-paired throughout their entire length, this characteristic is called "complete complementarity." If there are 1-3 base mismatches, the term "substantially complementary" is used. The terms "complementarity," "complete complementarity," and "substantially complementary" can be applied to the interior of a double-stranded siRNA; they can also be applied to base pairing between the antisense strand of an siRNA and the target gene mRNA.

[0093] As used herein, the term "inhibit" can be used interchangeably with "reduce," "silence," "downregulate," and similar descriptive language to indicate any level of inhibition. "Inhibiting the expression of MTTP" includes inhibiting the expression of any MTTP gene mRNA and its variant mRNA, MTTP, and its variant protein.

[0094] As used herein, "delivering siRNA into cells" refers to in vitro lipofection, where siRNA is incubated with cells encapsulated in liposomes, thereby delivering the siRNA into target cells. In vivo, siRNA is conjugated to the ligand GalNAc, which binds to the asialoglycoprotein receptor on the surface of liver cells, thereby specifically delivering the siRNA into liver cells.

[0095] As used herein, "lipid metabolism disorder" refers to disorders or diseases or conditions related to lipid metabolism such as hyperlipidemia caused by the disturbance of lipid metabolism and metabolic imbalance, which are mainly manifested as abnormally elevated levels of some lipid metabolites and lipoproteins in the blood. "Lipid metabolism disorder" includes (1) genetic diseases: familial hypertriglyceridemia; (2) acquired disorders, dietary or drug-related abnormalities. "Lipid metabolism disorder" in the general sense is believed to include various types of diseases such as atherosclerosis, dyslipidemia, hypertriglyceridemia, acute pancreatitis related to hypertriglyceridemia, chylomicron syndrome, familial chylomicronemia, hyperlipidemia (including familial combined hyperlipidemia), and hypercholesterolemia. Among cardiovascular diseases, coronary artery disease (ischemic heart disease) and inflammation related to coronary artery disease are also considered to be related to "lipid metabolism disorder".

[0096] The term "conjugate" used in the present invention (sometimes also referred to as conjugate, conjugate, conjugate, conjugate, and sometimes also referred to as conjugate in the literature) corresponds to "conjugate" or "conjugates" in English. A conjugate refers to a new compound generated by covalently linking (coupling) two or more compound molecules through a bivalent or multivalent compound molecule with a linking function. A conjugate can also be generated by directly coupling or condensing two molecules. A common antibody-drug conjugate (ADC) is a conjugate, also known as an antibody-drug conjugate. In some embodiments, the product produced by coupling an siRNA molecule to a targeting group via a linker (or linker) is also a conjugate or siRNA conjugate. In some embodiments, the product produced by coupling siRNA to a targeting group is a siRNA conjugate.

[0097] The term "coupling" as used herein refers to a chemical process in which two or more compound molecules undergo a reaction to form new chemical bonds and new molecules. In certain contexts, "coupling" can be used interchangeably with "connection" or can replace each other.

[0098] As used herein, "inhibiting MTTP gene expression" encompasses any level of MTTP gene inhibition, e.g., at least partial inhibition of MTTP gene expression, such as at least about 20% inhibition. In certain embodiments, inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0099] PCSK9 gene expression can be assessed based on any variable level related to MTTP gene expression, such as MTTP mRNA level or MTTP protein level. MTTP gene expression can also be assessed indirectly based on the level of serum lipids, triglycerides, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C and total cholesterol) or free fatty acids. Inhibition can be assessed by reducing the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from a similar untreated or control-treated subject, cell, or sample (e.g., a buffer-only control or an inert agent control).

[0100] As used herein, "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of the siRNA of the present invention that, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the progression of the disease or condition. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in an improvement in symptoms, e.g., an amount to treat, cure, prevent, or ameliorate a related medical condition or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0101] siRNA of the present invention

[0102] Provided herein are siRNAs for inhibiting MTTP gene expression. Each siRNA comprises a sense strand and an antisense strand. The antisense strand is substantially complementary to the MTTP gene mRNA sequence, and the length of the complementary region is 19-23 nucleotides. The length of the sense strand and the antisense strand can be 19-23 nucleotides respectively. The sense strand and the antisense strand can be the same length, or they can be different lengths. In some embodiments, the length of the sense and antisense strands is independently 19-23 nucleotides. In some embodiments, the length of the sense strand is 19-21 nucleotides, and the length of the antisense strand is 19-23 nucleotides. In some embodiments, the length of the sense strand is about 19 nucleotides, and the length of the antisense strand is about 20 nucleotides. In some embodiments, the length of the sense strand is about 19 nucleotides, and the length of the antisense strand is about 21 nucleotides.

[0103] After the siRNA of the present invention is delivered into cells, the siRNA inhibits the expression of the MTTP gene by at least about 50%.The expression level of the MTTP gene was determined using PCR and Western blotting techniques.

[0104] siRNA can be synthesized by an automated Oligo synthesizer. The siRNA of the present invention is prepared by the following steps: (1) preparing the sense strand and antisense strand of the double-stranded RNA molecule separately; (2) annealing the sense strand and antisense strand at a molar ratio of 1:1 to assemble a double-stranded siRNA composition. The sense strand and antisense strand of the double-stranded RNA molecule are both prepared by solid-phase organic synthesis. The siRNA of the present invention comprises two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand is selected from the sequences provided in Tables 2, 3, and 4, and the antisense strand corresponding to the sense strand is a sequence selected from Tables 2, 3, and 4, and the two sequences are complementary, wherein the antisense strand in the sequence is substantially complementary to the MTTP mRNA sequence.

[0105] The siRNA provided by the present invention may contain one or more mismatches relative to the target gene mRNA sequence, generally containing no more than 3 mismatches. For a 21-nucleotide siRNA, the mismatch is generally not located within the central 2-14 nucleotide region. If the antisense strand of the siRNA contains a mismatch in the target gene mRNA sequence, then the preferred mismatch region is generally confined to the last 5 nucleotides 5' or 3' end of the complementary region. Methods described herein or methods known in the art can be used to determine whether an siRNA containing a mismatch in the target sequence effectively inhibits MTTP gene expression.

[0106] In a preferred embodiment, the sense strand and antisense strand of the siRNA of the present invention are selected from the sense strand and antisense strand shown in the siRNA sequence codes in Table A:

[0107] Table A

[0108]

[0109]

[0110] Modified siRNA of the present invention

[0111] The present invention also provides siRNA chemically modified with RNA, thereby enhancing the stability of siRNA. The nucleic acid characterized by the present invention can be synthesized and modified by methods established in the art. Modifications include: (1) terminal modification, 5' terminal modification (phosphorylation) or 3' terminal modification (covalent binding of conjugates, etc.); morpholino modification at the 5' end of the sense chain, etc.; (2) base modification, replacing the base at a specific site, which can generally be replaced with a stable base, a stable base, or a removed base (non-basic nucleotide); (3) ribose modification: introducing a methoxy group at the 2' position of the ribose, or introducing a fluorine group after deoxygenation; (4) modification of the main chain, including 'terminal modification or the introduction of a thiophosphate at the 3' end. In one embodiment, the 3' end of the sense chain of the siRNA composition is covalently bound to a GalNac ligand, thereby enhancing the distribution of the siRNA in hepatocytes and the ability to target liver tissue.

[0112] In another preferred embodiment, the modified nucleotides are selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, phosphorothioate modified nucleotides, abasic nucleotides and locked nucleotides.

[0113] The GalNAc covalently bound siRNA conjugate provided by the present invention can be conventionally prepared by solid phase synthesis technology, and can also be commissioned to a commercial company with such synthesis technology for preparation.

[0114] In a preferred embodiment, the sense strand and antisense strand of the modified siRNA of the present invention are selected from the sense strand and antisense strand shown in the siRNA sequence codes in Table B:

[0115] Table B

[0116]

[0117]

[0118] In a preferred embodiment, the conjugate provided by the present invention contains the sense strand and antisense strand shown in the siRNA sequence code in Table C below:

[0119] Table C

[0120]

[0121] Pharmaceutical composition

[0122] The present invention also includes pharmaceutical compositions and formulations. These include the siRNA, siRNA conjugates of the present invention, and clinically applicable formulations to form pharmaceutical compositions. The siRNA of the present invention is formulated using a variety of buffer solutions. The buffer solution may include acetate, citrate, carbonate, or phosphate. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The siRNA pharmaceutical composition can be used to treat diseases or disorders associated with MTTP gene expression or activity, including but not limited to lipid metabolism disorders (e.g., hypertriglyceridemia). The pharmaceutical composition provided by the present invention can be administered intravenously (IV) or subcutaneously. The effect of a single dose of the pharmaceutical composition of the present invention on MTTP levels can be sustained over a long period of time, so the pharmaceutical composition can be administered once daily. Alternatively, it can be administered at intervals of 3, 4, or 5 days, or at intervals of no more than 1, 2, 3, or 4 weeks.

[0123] The pharmaceutical composition of the present invention comprises a safe and effective amount of the active ingredient and a pharmacologically acceptable excipient or carrier. "Safe and effective amount" means an amount of the active ingredient sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of active ingredient per dose, more preferably 10-200 mg per dose. Preferably, "one dose" is an injection.

[0124] "Pharmaceutically acceptable carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredient and with each other without significantly reducing the efficacy of the active ingredient. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, etc.), gelatin, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, olive oil, etc.), polyols (such as propylene glycol, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0125] The administration method of the pharmaceutical composition of the present invention is consistent with the application method (or administration method) of the present invention.

[0126] The dosage form of the pharmaceutical composition of the present invention is consistent with the mode of administration, for example (but not limited to): tablets, capsules, powders, pills, granules, syrups, solutions, suspensions, emulsions, suspensions, sprays, aerosols, powder mists, volatile liquids, injections, powder injections, external solutions, lotions, pour-ons, liniments, papules, plasters, rubber plasters, ointments, plasters, pastes, eye drops, nasal drops, eye ointments, gargles, sublingual tablets, suppositories, etc.

[0127] Method of the present invention

[0128] The present invention also provides methods for inhibiting MTTP expression in cells using siRNA, siRNA conjugates, or pharmaceutical compositions containing the same. These methods comprise delivering the siRNA of the present invention into cells and maintaining the cells for a period of time, thereby effectively degrading MTTP mRNA and inhibiting MTTP gene expression in the cells. Reduction in gene expression can be assessed by any method known in the art, including determining MTTP mRNA expression levels by qRT-PCR and determining MTTP protein levels by Western blotting.

[0129] The present invention also provides methods for inhibiting MTTP gene expression in animals. These methods comprise: targeted delivery of a GalNAc-conjugated siRNA conjugate to liver cells via intravenous or subcutaneous injection, and maintaining the animals for a period of time to effectively reduce MTTP expression levels. The reduction in gene expression can be assessed by any method known in the art, including quantification of MTTP mRNA expression by qRT-PCR and determination of MTTP protein levels by Western blotting.

[0130] The present invention further provides methods for treating a subject in need thereof using siRNA or a pharmaceutical composition thereof. These treatment methods include administering an siRNA, siRNA conjugate, or pharmaceutical composition of the present invention to a subject suffering from a condition including, but not limited to, a lipid metabolism disorder (e.g., hypertriglyceridemia). These methods can be used in combination with other drugs and / or other therapeutic approaches, such as cholesterol synthesis inhibitors (statins), bile acid sequestrants, cholesterol acyltransferase inhibitors, and farnesoid X receptor antagonists. Other therapeutic agents include those that increase high-density lipoprotein cholesterol, such as inhibitors of cholesterol ester transfer protein.

[0131] The main advantages of the present invention are:

[0132] 1. The siRNA of the present invention can effectively inhibit the expression of the MTTP gene, thereby effectively treating diseases or conditions caused by overexpression of MTTP.

[0133] 2. The 3' end of the siRNA sense chain of the present invention is coupled to GalNAc, thus achieving liver-specific MTTP silencing without affecting intestinal MTTP expression, theoretically avoiding the intestinal side effects of MTTP small molecule inhibitors, such as abdominal pain and diarrhea.

[0134] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0135] Example 1

[0136] siRNA design:

[0137] Download the annotated human MTTP transcript (NM_000253.4) and cynomolgus macaque MTTP transcript (XM_028848642.1) from the NCBI gene library (http: / / www.ncbi.nlm.nih.gov / gene / ). Simultaneously, human and cynomolgus macaque primary hepatocyte cDNA libraries were prepared and sequenced to obtain the MTTP transcript. These two transcripts were used as references for siRNA design.

[0138] The human MTTP transcript sequence (4043 bp) and the MTTP transcript of the cynomolgus macaque species relevant for toxicology studies were used to rationally design siRNA using the following tools:

[0139] (1) Targeting the full-length MTTP transcript, we used DSIR (http: / / biodev.extra.cea.fr / DSIR / DSIR.html) and combined the siRNA library targeting mammalian genes and their mRNA knockdown effects to search for all possible 19mer sequences.

[0140] (2) Sequence clusters with perfect or near-perfect matches (no mismatches in 2-14 of the antisense strand and ≤2 mismatches in other regions) between humans, cynomolgus macaques, and rhesus macaques were selected;

[0141] (3) The MTTP gene was re-annotated using the cDNA sequencing results of primary human and cynomolgus macaque hepatocytes to obtain the full-length sequences of its 5'UTR, CDS, and 3'UTR.

[0142] (4) Using the siRNAdesigner algorithm (https: / / github.com / anazhmetdin /

[0143] siRNAdesigner) scores siRNA based on GC content, A / U base pair position, and G / C base pair position. It calculates the possible binding sites, number of sites, and mismatch base positions of each siRNA in the target species transcriptome. Based on the number and position of 2-17 and 2-8 mismatches in each siRNA antisense strand, different weights are assigned to the specificity scores and rankings of each sequence. siRNA is selected based on these results.

[0144] Finally, the present invention designed and synthesized a total of 40 siRNAs homologous to humans and cynomolgus monkeys.

[0145] siRNA sequence synthesis:

[0146] Sequence Synthesis: The desired siRNA sequence (single-stranded siRNA) was introduced into an Oligo 48 synthesizer, and the MTTP siRNA sequence was synthesized at a scale of 200-2000 nmol. Using Universal CPG / PS as the vector, the synthesis was performed according to the specified synthesis protocol. Each base introduction required a four-step reaction: demethylation, coupling, oxidation, and capping.

[0147] Nucleoside phosphoramidite monomers with 2'-deoxy-, 2-fluoro-, and 2'-O-methyl modifications were purchased from Shanghai Zhaowei. Single strands were synthesized using nucleotide monomers with 2'-deoxy-, 2-fluoro-, and 2'-O-methyl chemical modifications and terminal phosphorothioate bonds for in vitro screening experiments.

[0148] Free uptake screening and validation experiments used nucleotide monomers with 2'-deoxy, 2-fluoro, 2'-O-methyl chemical modifications and terminal phosphorothioate bonds, while GalNAc was coupled to the 3' end of the sense chain to form a covalent bond.

[0149] In the design of MTTP siRNA, GalNAc is located at the 3' end of the sense strand, and the 5' and 3' ends of the antisense sequence both contain two phosphorothioate bonds.

[0150] Synthesis of a 19mer sense chain and a 21mer antisense chain with GalNAc coupled to the 3' end of the sense chain: The sense chain was synthesized using GalNAc-CPG solid phase synthesis; while the antisense chain was synthesized on Universal CPG / PS at a scale of 200-2000 nmol.

[0151] Synthesis, cleavage and deprotection: For 21mer sequences, universal CPG / PS was used as a solid support, while for GalNAc covalent conjugates, GalNAc-CPG solid support was used for the positive chain. 200-2000nmol scale siRNA sequence synthesis was carried out in a 48-well plate. The phosphoramidite monomer solution was prepared at a concentration of 0.1M, and ethylmercaptotetrazole (0.6M acetonitrile solution) was used as an activator. After the synthesis step, concentrated ammonia was used for cleavage and deprotection. After 16h at 55°C, the reaction was placed in a -20°C refrigerator for cooling for 10min. After centrifugation, the supernatant was transferred to another centrifuge tube, concentrated to dryness, and dissolved in 0.1MTEA for detection using LC-MS, HPLC and UV.

[0152] Purification, Desalting, and Annealing: HPLC purification was performed. The collected fractions were subjected to HPLC, MS, and UV analysis. Qualified fractions were collected, desalted, and lyophilized to obtain single-stranded siRNA. siRNA sequence analysis was performed to determine concentration and purity by A260 absorbance. The complementary sense and antisense strands were then annealed at a 1:1 molar ratio to form a double-stranded siRNA composition.

[0153] The unmodified sense and antisense strand sequences of the MTTP double-stranded siRNA synthesized in this example are shown in Table 2. The target genes of the siRNAs in Table 2 are all MTTP, the species are all human, and the antisense strand position is the position of the antisense strand on the MTTP transcript (NM_000253.4).

[0154] Table 2

[0155]

[0156]

[0157]

[0158] Part of the double-stranded siRNA targeting MTTP was modified to obtain the modified sense and antisense strand sequences of the partial MTTP double-stranded siRNA as shown in Table 3.

[0159] Table 3

[0160]

[0161]

[0162]

[0163] The sequences of the sense and antisense strands of some GalNAc-modified double-stranded siRNAs are shown in Table 4.

[0164] Table 4

[0165]

[0166] Example 2

[0167] 2.1 Cell culture and transfection

[0168] HepG2 cells (Cell Bank, Chinese Academy of Sciences) were cultured in high-glucose DMEM (10% fetal bovine serum, penicillin-streptomycin) in a 37° C., 5% CO 2 incubator. Cells were collected and counted after trypsinization.

[0169] The transfection protocol of siRNA sequence was as follows: add 0.2 μl Lipofectamine RNAiMax (ThermoFisher) to 14.8 μl Opti-MEM, then add 5 μl siRNA sequence solution, mix well and add to 24-well plate, incubate at room temperature for 15 minutes. Then, add 2x10 5 HepG2 cell suspensions were added to 24-well plates at a concentration of 10 nM, 1 nM, and 0.1 nM, respectively. Cultures were then continued for 48 hours. Unless otherwise noted, siRNA screening experiments were performed at final concentrations of 10 nM, 1 nM, and 0.1 nM.

[0170] Total RNA was isolated using Trizol total RNA isolation reagent (Thermofisher). Trizol solution was added to a 24-well plate at a volume of 250 μl per well. After shaking at 1000 rpm for 9 minutes, the cell lysate was transferred to a 1.5 ml centrifuge tube. For every 1 ml of Trizol cell lysate, 0.2 ml of chloroform was added, shaken vigorously for 1 minute, allowed to stand at room temperature for 2 minutes, and then centrifuged at 17,000 rpm for 15 minutes. The supernatant obtained by centrifugation was transferred to a new 1.5 ml centrifuge tube, an equal volume of isopropanol was added, inverted to mix, and allowed to stand at room temperature for 20 minutes. The entire volume of the above mixture was added to the top of the RNA adsorption column, centrifuged at 17,000 rpm for 2 minutes, and the filtrate was discarded. 700 μl of wash buffer was added to the top of the RNA adsorption column, centrifuged at 17,000 rpm for 2 minutes, and the filtrate was discarded. The wash operation was repeated once. Finally, 50 μl of elution buffer was added to the top of the RNA adsorption column, and the column was centrifuged at 17,000 rpm for 2 minutes. The filtrate was collected, quantified for nucleic acid, and stored in a -80°C refrigerator.

[0171] cDNA synthesis was performed using Takara's cDNA reverse transcription kit (Takara). To 10 μl of total RNA solution, 10 μl of reverse transcription mixture (2 μl of 10x buffer, 0.8 μl of 25x dNTPs, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNase inhibitor, and 3.2 μl of water) was added.

[0172] cDNA reverse transcription was performed using an Eppendorf PCR instrument: 16°C for 10 minutes, 42°C for 30 minutes, 85°C for 2 minutes, and maintained at 10°C.

[0173] Implementation of fluorescent quantitative PCR

[0174] 5 μl of cDNA was added to 15 μl of qPCR mix (Fuji Biotechnology, Cat. No. DRT-01021-C2). Each well contained 0.5 μl of GAPDH TaqMan probe (Shanghai Biotechnology) and 0.5 μl of MTTP TaqMan probe (Shanghai Biotechnology). Real-time PCR was performed using the ΔΔCt(RQ) assay in an ABI7500 Fast Real-Time PCR System (ThermoFisher). Two to three independent transfections were performed for each siRNA sequence, and qPCR analysis was performed in duplicate for each transfection.

[0175] 2.2 Results

[0176] (1) The results of transfection of HepG2 cells using the unmodified siRNA sequences in Table 2 are shown in Table 5. RNAiMax was used for delivery of double-stranded siRNA.

[0177] The values ​​in Table 5 represent the MTTP mRNA levels after different concentrations of siRNA were delivered into cells for a period of time. These values ​​were normalized to the blank control, i.e., when no siRNA was added, the MTTP mRNA level was 1.

[0178] Table 5

[0179]

[0180]

[0181]

[0182] (2) The results of transfection of HepG2 cells with the modified siRNA sequences in Table 3 are shown in Table 6. RNAiMax was used for delivery of double-stranded siRNA.

[0183] The values ​​in Table 6 represent the MTTP mRNA levels after different concentrations of siRNA were delivered into cells for a period of time. These values ​​were normalized to the blank control, i.e., when no siRNA was added, the MTTP mRNA level was 1.

[0184] Table 6

[0185]

[0186]

[0187] Example 3 In vivo experiment 1

[0188] 3.1 Test Substance

[0189] hMTTP-B1301.1 covalently bound with GalNAc (sense sequence: mG*mC*mA mGfUfUfGfAmUmUmCmAmAmAmGmUmAmCmA-GalNAc-GalNac; antisense sequence: mU*fG*mUmAmCfUmUfUfGmAmAmUmCfAmAfCmUmGmC*mU*mU); PBS was used as a negative control.

[0190] 3.2 Animal Experiment Procedure

[0191] AAV8-TBG-MTTP adeno-associated virus (AAV) was prepared to express human MTTP mRNA and protein in vivo. TBG is a hybrid promoter of the human thyroid-binding globulin (TBG) promoter and the microglobulin enhancer for liver-specific expression of exogenous genes. This AAV virus was administered to C57BL / 6 mice via tail vein injection at a dose of 2E+11 / mouse. After 14 days, these mice were used to test the MTTP siRNA described above and the effects of the test substances were tested in vivo.

[0192] 3.3 Experimental Results

[0193] The experimental results are as follows Figure 1 shown. Figure 1 Figure A shows the timing, dose, and route of administration of the test substance 14 days after AAV administration; also shown are the time points at which tissue samples were collected from euthanized animals. Figure B shows the levels of human MTTP mRNA in the liver 19 days after two subcutaneous administrations of 5 mg / kg of the test substance (hMTTP-B1301.1). Compared to animals in the PBS group, administration of the test substance significantly reduced human MTTP mRNA levels in the liver. Figure 1 C shows the levels of human and mouse MTTP proteins in the liver after two subcutaneous administrations of 5 mg / kg of the test substance (hMTTP-B1301.1) 19 days later. Compared with the PBS group, administration of the test substance significantly reduced the level of human MTTP protein in the liver.

[0194] Example 4 In vivo experiment 2

[0195] 4.1 Test Substance

[0196] hMTTP-B1301.1 covalently bound with GalNAc (sense sequence: mG*mC*mA mGfUfUfGfAmUmUmCmAmAmAmGmUmAmCmA-GalNAc-GalNac; antisense sequence: mU*fG*mUmAmCfUmUfUfGmAmAmUmCfAmAfCmUmGmC*mU*mU); PBS was used as a negative control.

[0197] 4.2 Animal Experiment Procedure

[0198] AAV8-TBG-MTTP adeno-associated virus (AAV) was prepared to express human MTTP mRNA and protein in vivo. TBG is a hybrid promoter of the human thyroid-binding globulin (TBG) promoter and the microglobulin enhancer for liver-specific expression of exogenous genes. This AAV virus was administered to C57BL / 6 mice via tail vein injection at a dose of 2E+11 / mouse. After 14 days, these mice were used to test the MTTP siRNA described above and the effects of the test substances were tested in vivo.

[0199] 4.3 Experimental Results

[0200] The experimental results are as follows Figure 2 shown. Figure 2 Shown in A are the time, dose, and route of administration of the test substance 14 days after the animals were administered AAV virus; the time points at which the animals were euthanized and tissue samples were collected are also shown. Figure 2 Figure B shows the level of human MTTP mRNA in the liver 30 days after a single subcutaneous administration of 20 mg / kg of the test substance (hMTTP-B1301.1). Compared with the PBS group, administration of the test substance significantly reduced the level of human MTTP mRNA in the liver.

[0201] Example 5 In vivo experiment 3

[0202] 5.1 Test Substance

[0203] hMTTP-B1301.1 covalently bound with GalNAc (sense sequence: mG*mC*mA mGfUfUfGfAmUmUmCmAmAmAmGmUmAmCmA-GalNAc-GalNac; antisense sequence: mU*fG*mUmAmCfUmUfUfGmAmAmUmCfAmAfCmUmGmC*mU*mU); PBS was used as a negative control.

[0204] 5.2 Animal Experiment Procedure

[0205] AAV8-TBG-MTTP adeno-associated virus (AAV) was prepared to express human MTTP mRNA and protein in vivo. TBG is a hybrid promoter of the human thyroid-binding globulin (TBG) promoter and the microglobulin enhancer for liver-specific expression of exogenous genes. This AAV virus was administered to C57BL / 6 mice via tail vein injection at a dose of 2E+11 / mouse. After 14 days, these mice were used to test the MTTP siRNA described above and the effects of the test substances were tested in vivo.

[0206] 5.3 Experimental Results

[0207] The experimental results are as follows Figure 3 shown. Figure 3 Shown in A are the time, dose, and route of administration of the test substance 14 days after the animals were administered AAV virus; the time points at which the animals were euthanized and tissue samples were collected are also shown. Figure 3 Figure B shows the levels of human MTTP mRNA in the liver 14 days after a single subcutaneous administration of the test substance (hMTTP-B1301.1) at 0.3, 1.0, and 3 mg / kg. Compared with the PBS group, administration of the test substance significantly reduced the levels of human MTTP mRNA in the liver. Figure 3 Figure C shows the levels of human and mouse MTTP proteins in the liver after a single subcutaneous administration of the test substance (hMTTP-B1301.1) at 0.3, 1.0, or 3 mg / kg to the PNS. Compared to animals in the PBS group, administration of the test substance significantly reduced the level of human MTTP protein in the liver.

[0208] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An siRNA for inhibiting MTTP gene expression, characterized in that: The siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, the antisense strand comprises at least 19 consecutive nucleotides that differ from any antisense strand sequence shown in Table A or Table B by 0, 1, 2 or 3 nucleotides, and the sense strand is complementary to the antisense strand by at least 15, 16, 17, 18, 19, 20 or 21 nucleotides.

2. The siRNA according to claim 1, wherein The sense strand comprises at least 19 consecutive nucleotides that differ from any of the sense strand sequences shown in Table A or Table B by 0, 1, 2, or 3 nucleotides.

3. The siRNA according to claim 2, wherein The sense strand and antisense strand are selected from the sense strand and antisense strand shown in the following siRNA sequence codes:

4. The siRNA according to claim 1, wherein At least one nucleotide in the sense strand and the antisense strand is a modified nucleotide, and the modified nucleotide is selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, phosphorothioate modified nucleotides, abasic nucleotides and locked nucleotides.

5. The siRNA according to claim 4, wherein The sense strand and antisense strand are selected from the sense strand and antisense strand shown in the following siRNA sequence codes: Among them, the letter m indicates that the nucleotide adjacent to the right of m is a 2'-O-methyl modified nucleotide; f indicates that the nucleotide adjacent to the right of the letter f is a 2'-fluoro modified nucleotide; * indicates that the nucleotide adjacent to the left of * is a phosphorothioate modified nucleotide.

6. A siRNA conjugate, characterized in that The siRNA conjugate comprises the siRNA according to claim 1 and a targeting group.

7. The conjugate according to claim 6, wherein The conjugate further comprises a linker, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a) the siRNA according to claim 1, and / or the conjugate according to claim 2; and (b) a pharmaceutically acceptable carrier.

9. Use of the siRNA according to claim 1, the conjugate according to claim 2, or the pharmaceutical composition according to claim 3 in the preparation of a medicament for preventing and / or treating a disease or condition mediated by MTTP, characterized in that: Used to inhibit the expression of MTTP gene.

10. A method for inhibiting MTTP expression in cells in vitro, characterized in that: The method comprises the following steps: (z1) Co-culturing an effective amount of the siRNA according to claim 1, the conjugate according to claim 2, or the pharmaceutical composition according to claim 3 with cells.

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