MSTN RNAi agents, their conjugates, and applications

Abstract

The present application relates to the field of biomedical technology, and in particular to a MSTN RNAi agent, a conjugate thereof and application. The present application provides an RNA interference agent targeting MSTN, comprising a first nucleotide sequence, wherein the first nucleotide sequence comprises any one of SEQ ID NO: 1 to SEQ ID NO: 25 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto. The RNAi agent of the present application can achieve efficient and specific MSTN gene silencing, thereby achieving the effect of reducing fat deposition while increasing muscle mass.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to an MSTN RNAi agent, its conjugates, and its applications. Background Technology

[0002] Obesity, a global public health crisis, affects over a billion people and is often accompanied by metabolic disorders, cardiovascular disease, and muscle atrophy. While current mainstream GLP-1 (glucagon-like peptide-1) weight-loss drugs can significantly reduce weight, they have serious drawbacks: weight loss may partially come from lean body mass (including muscle), exacerbating metabolic disorders and increasing the risk of rebound weight gain, while also causing gastrointestinal motility suppression and pancreatitis. Traditional muscle atrophy treatments struggle to simultaneously address the dual pathologies of muscle loss and abnormal fat deposition.

[0003] Myostatin (MSTN), a core member of the transforming growth factor β superfamily, is a key negative regulator of skeletal muscle mass. By inhibiting myoblast proliferation and differentiation, it directly leads to muscle atrophy and is abnormally overexpressed in obese pathological states, accelerating lipid deposition and reducing insulin sensitivity. Experiments have demonstrated that MSTN gene knockout animal models exhibit a dual positive effect of increased muscle mass and reduced fat, with significantly enhanced fatty acid oxidation capacity. This unique "fat reduction and muscle gain" biological characteristic makes MSTN an ideal target for treating obesity and related muscular atrophy diseases.

[0004] MSTN silencing strategies based on RNA interference technology achieve etiological treatment by specifically degrading target gene mRNA. This not only effectively reduces fat deposition but also simultaneously increases muscle mass to boost basal metabolic rate, fundamentally avoiding muscle loss caused by GLP-1 inhibitors. Therefore, there is an urgent need to develop a highly effective RNAi agent targeting MSTN. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides an RNAi agent targeting MSTN, its conjugates, and their applications, achieving efficient and specific MSTN gene silencing, thereby reducing fat deposition while increasing muscle mass.

[0006] To achieve the above objectives, the first aspect of the present invention provides an RNA interference agent targeting MSTN, comprising a first nucleotide sequence, the first nucleotide sequence comprising any one of the nucleotide sequences in SEQ ID NO: 1 to SEQ ID NO: 25 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with it, the first nucleotide sequence hybridizing to at least 8 consecutive bases of the mRNA of the MSTN gene.

[0007] Furthermore, the RNA interference agent also includes a second nucleotide sequence that hybridizes with the first nucleotide sequence.

[0008] Furthermore, the second nucleotide sequence includes any one of the nucleotide sequences in SEQ ID NO: 26 to SEQ ID NO: 50 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with it.

[0009] Furthermore, the RNA interference agent is siRNA, the first nucleotide sequence is the antisense strand of the siRNA, and the second nucleotide sequence is the sense strand of the siRNA.

[0010] Furthermore, the antisense strand and the sense strand each independently contain 8 to 50 nucleotides.

[0011] More preferably, the antisense strand and the sense strand each independently contain 10 to 30 nucleotides.

[0012] Furthermore, the antisense strand and the sense strand contain at least one modified nucleotide and at least one modified nucleotide linker.

[0013] Furthermore, the modified nucleotide is selected from at least one of nucleotides modified with 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA), locked nucleic acids, ethylene nucleic acids, and ethylene glycol nucleic acids.

[0014] Furthermore, the antisense strand and the sense strand comprise 5% to 100% of modified nucleotides, specifically including 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of modified nucleotides.

[0015] Furthermore, the modified nucleotide linkages are thiophosphate linkages or dithiophosphate linkages.

[0016] Furthermore, the antisense strand includes any one of the nucleotide sequences in SEQ ID NO: 51 to SEQ ID NO: 75 or SEQ ID NO: 101 to SEQ ID NO: 138, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0017] Furthermore, the positive chain includes any one of the nucleotide sequences in SEQ ID NO: 76 to SEQ ID NO: 100 or SEQ ID NO: 139 to SEQ ID NO: 176, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it.

[0018] A second aspect of the present invention provides a conjugate comprising the above-described RNA interference agent and a conjugated antibody, wherein the conjugated antibody is attached to the 5' end of the sense strand of the RNA interference agent.

[0019] Furthermore, the RNA interference agent used for conjugation with the conjugated antibody includes any nucleotide sequence of SEQ ID NO: 177 and SEQ ID NO: 178 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with it.

[0020] Furthermore, the conjugated antibody is a human anti-transferrin receptor antibody.

[0021] Furthermore, the human anti-transferrin receptor antibody specifically binds to human transferrin receptor 1.

[0022] Furthermore, the binding ratio of the RNA interference agent to the conjugated antibody is 1:1 to 16:1. Specifically, the ratio of RNA interference agent to conjugated antibody (DAR ratio) is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, or 16:1.

[0023] A third aspect of the present invention provides a kit comprising the aforementioned RNA interference agent or the aforementioned conjugate.

[0024] The present invention also provides the use of the aforementioned RNA interference agent or conjugate in the preparation of a drug, wherein the drug is used to reduce the expression of MSTN mRNA or protein in mammals.

[0025] Furthermore, the drug is used to promote skeletal muscle growth, increase muscle mass, inhibit muscle atrophy, or reduce fat deposition.

[0026] Furthermore, the drug is used to lower lipids and increase muscle mass.

[0027] Compared with existing technologies, the MSTN-targeting RNA interference agent provided by this invention achieves highly efficient and specific recognition and binding to MSTN gene mRNA through specific sequence design (SEQ ID NO: 1-50). Simultaneously, the introduction of chemically modified nucleotides and nucleotide linkages significantly enhances the activity of the RNA interference agent in vivo. Furthermore, this invention also provides a conjugate formed by coupling the RNA interference agent with an anti-transferrin receptor antibody. This conjugate utilizes receptor-mediated endocytosis to achieve highly efficient delivery of the RNA interference agent, significantly improving therapeutic efficacy. The RNA interference agent and its conjugate described in this invention can effectively reduce MSTN gene expression, thereby promoting skeletal muscle growth, increasing muscle mass, inhibiting muscle atrophy, or reducing fat deposition, providing an effective treatment for diseases with abnormal MSTN mRNA or protein expression. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The non-denaturing IP-RP diagram for the preferred sequence; Figure 2 For the non-denatured IP-RP graph of the modified sequence; Figure 3 For modifying the temperature-dependent IP-RP plot of the sequence; Figure 4 The image shows the effect of antibody-siRNA conjugate on MSTN knockdown in wild-type CD1 mice; Figure 5 The image shows the effect of antibody-siRNA conjugate on MSTN knockdown in wild-type CD1 mice; Figure 6 The graph shows the effect of antibody-siRNA conjugate on the body fat ratio of DIO mice. Figure 7 This is a weighing diagram showing the endpoint of the antibody-siRNA conjugate experiment on muscle tissue from DIO mice. Figure 8 This is a weighing chart showing the endpoint of the antibody-siRNA conjugate assay on the quadriceps femoris, diaphragm, and myocardium of DIO mice. Detailed Implementation

[0030] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0031] The RNAi agent for inhibiting MSTN gene expression described in this invention (referred to herein as the MSTN RNAi agent) inhibits or knocks down MSTN expression in vitro and / or in vivo through the biological process of RNA interference (RNAi) after delivery to cells expressing the MSTN gene. As used herein, unless otherwise specifically indicated, MSTN may refer to the MSTN gene, MSTN mRNA, or MSTN protein where appropriate. RNAi agents include, but are not limited to, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), and short hairpin RNA (shRNA).

[0032] The first aspect of this invention provides an RNA interference agent targeting MSTN, comprising a first nucleotide sequence, the first nucleotide sequence comprising any one of SEQ ID NO: 1 to SEQ ID NO: 25 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with it, the first nucleotide sequence being capable of hybridizing to at least 8 consecutive bases of the mRNA of the MSTN gene.

[0033] In one embodiment, the RNA interference agent further includes a second nucleotide sequence that can hybridize with the first nucleotide sequence.

[0034] In one embodiment, the second nucleotide sequence comprises any one of the nucleotide sequences in SEQ ID NO: 26 to SEQ ID NO: 50 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it.

[0035] In one embodiment, the RNA interference agent is siRNA, the first nucleotide sequence being the antisense strand of the siRNA, and the second nucleotide sequence being the sense strand of the siRNA.

[0036] In one embodiment, the antisense and sense strands of the MSTN RNAi agent form a double-stranded structure. The antisense and sense strands of the MSTN RNAi agent are partially, largely, or completely complementary to each other. Within the complementary double-stranded region, the antisense strand is at least 90% or 100% complementary to the sense strand.

[0037] In one implementation, the antisense strand and / or the sense strand may, in addition to their complementary sequences, optionally and independently contain 1, 2, 3, 4, 5, or 6 additional nucleotides (extensions) at the 3' end, 5' end, or both the 3' and 5' ends. If the antisense strand contains additional nucleotides, it may be partially complementary to or not complementary to the corresponding sequence in the MSTN mRNA. If the sense strand contains additional nucleotides, it may be identical to or different from the corresponding sequence in the MSTN mRNA. If the antisense strand contains additional nucleotides, it may be complementary to or not complementary to the additional nucleotides of the corresponding sense strand.

[0038] In one embodiment, the MSTN RNAi agent comprises a 3' extended antisense strand having a length of 1, 2, 3, 4, 5, or 6 nucleotides. In other embodiments, the MSTN RNAi agent comprises a 3' extended antisense strand having a length of 1 or 2 nucleotides. In one embodiment, one or more antisense strand extended nucleotides comprise uracil or thymidine nucleotides or nucleotides complementary to the corresponding MSTN mRNA sequence.

[0039] In one implementation, the antisense strand and the sense strand each independently contain 8 to 50 nucleotides.

[0040] In some preferred embodiments, the antisense strand and the sense strand each independently contain 10 to 30 nucleotides.

[0041] In one embodiment, the antisense and sense strands of the RNA interference agent are independently 19, 20, 21, 22, or 23 nucleotides long. The antisense and sense strands may be the same length or they may be different lengths. In one embodiment, the antisense strand is 19 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 23 nucleotides long.

[0042] In one embodiment, the antisense strand and the sense strand comprise at least one modified nucleotide and at least one modified nucleotide linker.

[0043] The MSTN RNAi agents of this invention include the aforementioned unmodified nucleic acids and nucleic acids modified to improve efficacy. Unmodified nucleic acids refer to those in which the sugar, base, and phosphate ester structural components are the same as or substantially the same as those of natural components, preferably natural components found in the human body. Modified RNAi, as used in this invention, refers to nucleic acid components, i.e., one or more components of the sugar, base, and phosphate ester structure, that are different from natural components, preferably different from natural components produced in the human body. Two main purposes of modifying RNAi agents are to obtain their stability against degradation in the biological environment and to improve their pharmacological properties, such as pharmacodynamic properties. RNAi agents may contain non-naturally occurring bases or non-naturally occurring sugars, such as non-carbohydrate cyclic carrier molecules, for the typical characteristics of non-naturally occurring sugars in RNAi reagents. RNAi reagents may include bonds between nucleotides (e.g., chiral thiophosphate bonds) to increase nuclease resistance. RNAi reagents may also contain, or optionally contain, riboside analogs to increase nuclease resistance.

[0044] In one embodiment, the modified nucleotide is at least one of 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA), locked nucleic acid, ethylene nucleic acid, or ethylene glycol nucleic acid.

[0045] In one embodiment, the antisense strand and the sense strand comprise 5% to 100% of modified nucleotides, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of modified nucleotides.

[0046] In one embodiment, one or more nucleotides of the MSTN RNAi agent are linked by non-standard bonding. In one embodiment, the modified internucleotide link is a non-phosphate-containing covalent nucleoside link. Modified internucleotide links include 5'-thiophosphate groups (represented in this application as lowercase 's' before nucleotides) with normal 3'-5' bonding, chiral thiophosphates, thiophosphates, dithiophosphates, triphosphates, aminoalkyl-phosphophosphates, methyl phosphonates, and other alkyl esters including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, aminophosphates including 3'-aminophosphates and aminoalkylaminophosphates, thiocarbonylaminophosphates, thiocarbonylalkyl-phosphonates, thiocarbonylalkyl phosphates, morpholino linkages, and boron phosphates. In other embodiments, the modified internucleotide link does not contain a phosphorus atom. The linkages between modified nucleotides without phosphorus atoms include, but are not limited to, intra-linkage of short-chain alkyl or cycloalkyl sugars, intra-linkage of mixed heteroatoms and alkyl or cycloalkyl sugars, and intra-linkage of one or more short-chain heteroatoms or heterocyclic sugars.

[0047] In some preferred embodiments, the modified nucleotide linkages are thiophosphate linkages or dithiophosphate linkages.

[0048] In one embodiment, the antisense strand comprises any one of the nucleotide sequences of SEQ ID NO: 51 to SEQ ID NO: 75 or SEQ ID NO: 101 to SEQ ID NO: 138, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0049] In one embodiment, the positive strand comprises any one of the nucleotide sequences of SEQ ID NO: 76 to SEQ ID NO: 100 or SEQ ID NO: 139 to SEQ ID NO: 176, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it.

[0050] A second aspect of the present invention provides a conjugate comprising the above-described RNA interference agent and a conjugated antibody, wherein the conjugated antibody is linked to the 5' end of the sense strand of the RNA interference agent.

[0051] In one embodiment, the RNA interference agent used for conjugation with the conjugated antibody includes any one of the nucleotide sequences in SEQ ID NO: 177 and SEQ ID NO: 178 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it.

[0052] In one embodiment, the conjugated antibody alters the distribution, targeting, or half-life of the RNA interference agent it binds to. In another embodiment, the conjugated antibody promotes transport, hybridization, and specificity properties, enabling it to promote nuclease resistance to the obtained natural or modified oligonucleotides, or to promote nuclease resistance to multimeric molecules containing any of the monomers and / or conjugates of natural or modified ribonucleotides described in this application.

[0053] In one embodiment, the conjugated antibody is a human anti-transferrin receptor antibody.

[0054] Furthermore, the human anti-transferrin receptor antibody specifically binds to human transferrin receptor 1.

[0055] In one embodiment, the binding ratio of the RNA interference agent to the conjugated antibody is 1:1 to 16:1, that is, the ratio of RNA interference agent to conjugated antibody (DAR ratio) is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 or 16:1.

[0056] A third aspect of the present invention provides a kit comprising the aforementioned RNA interference agent or the aforementioned conjugate.

[0057] The present invention also provides the aforementioned RNA interference agent or conjugate for use in the preparation of a drug, wherein the drug is used to reduce the expression of MSTN mRNA or protein in mammals.

[0058] In one embodiment, the drug is used to promote skeletal muscle growth, increase muscle mass, inhibit muscle atrophy, or reduce fat deposition.

[0059] In one implementation, the drug is used to lower lipids and increase muscle mass.

[0060] The following description is based on specific embodiments: Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available, and techniques not described in detail were performed according to standard methods well known to those skilled in the art.

[0061] Example 1: Synthesis of siRNA

[0062] siRNAs are initially designed to consist of perfectly complementary sense and antisense strands, each 19 or 21 nucleotides in length. The antisense strand must perfectly match the target mRNA to exert its catalytic activity. In some cases, to increase cellular preference for the antisense strand, a two-nucleotide overhang can be added to the 3' end of the antisense strand, which is unpaired with the sense strand. Therefore, 19 / 21mer can be used to describe siRNA designs that add two nucleotides to the 3' end of the antisense strand of an initially 19-nucleotide siRNA, where 19 and 21 refer to the lengths of the sense and antisense strands, respectively; 21 / 23mer can be used to describe siRNA designs that add two 2-nucleotide overhangs to the 3' end of the antisense strand of an initially 21-nucleotide siRNA, where 21 and 23 refer to the lengths of the sense and antisense strands, respectively.

[0063] In some cases, the base sequence at the 3' overhang of the antisense strand can perfectly match the target mRNA, or be composed of other unrelated dinucleotide sequences, such as "UU," without affecting the activity of the siRNA. In some cases, when the antisense strand is loaded into Ago2, the first base at the 5' end binds to Ago2 without binding to the target mRNA, and "U" is thermodynamically optimal for binding to Ago2. Therefore, all antisense strands can replace the first base at the 5' end with "U," and correspondingly, all sense strands can replace the last base at the 3' end (position 19) with "A" without affecting the activity of the siRNA.

[0064] Both single strands of siRNA were synthesized separately on a solid-phase support using standard phosphoramide chemistry and purified by HPLC, with the purity of each single strand controlled to be above 95%. Subsequently, the two complementary single strands were annealed and paired at a close molar ratio to form a double-stranded siRNA, which was then lyophilized for later use.

[0065] Table 1. Unmodified 19 / 21mer siRNA sequence information

[0066] Example 2: In vitro evaluation method for siRNA (DualGlo reporter plasmid system)

[0067] A DualGlo reporter plasmid (Promega, psiCHECK™-2 Vector) was designed for screening siRNAs. This plasmid contains the coding sequence (ENST00000260950.5) of human MSTN mRNA in the 3'-UTR of the reporter luciferase.

[0068] Human 293T cells were grown in DMEM supplemented with a mixture of 10% v / v heat-inactivated fetal bovine serum and 1% v / v penicillin-streptomycin. For transfection, cells were seeded at a density of 4,000 cells / well in 96-well opaque cell culture plates and transfected within 24 hours.

[0069] Cells were co-transfected using MSTN-DualGlo reporter plasmid and siRNA at the specified dilution concentrations, mixed with Lipofectamine® 3000 (ThermoFisher) according to the manufacturer's recommendations. In the two-dose selection, the siRNA concentrations were 100 pM and 8 pM. Transfected cells were incubated at 37°C in 5% CO2 for 2 days. In the multiple-dose assay, the siRNA concentrations were 100,000 pM, 10,000 pM, 1,000 pM, 100 pM, 10 pM, 1 pM, 0.1 pM, 0.01 pM, and 0.001 pM. Transfected cells were incubated at 37°C in 5% CO2 for 2 days. Using the Dual-Glo® luciferase assay (Promega) and the Dual-Glo® Stop & Glo® assay, the appropriate reagents were added sequentially according to the manufacturer's instructions, and the corresponding fluorescence readings for Firefly luminescence and Renilla luminescence were measured. In the screening experiments, Renilla luminescence values ​​were normalized relative to Firefly luminescence values ​​within each sample. The quantification of MSTN mRNA downregulation was calculated using the manufacturer's recommended analytical method. All experiments were performed in triplicate, and the mean of three replicates was calculated. In the two-dose screening, the residual amount of MSTN mRNA was determined by comparing the experimental group with the control group. In the multi-dose validation experiments, KDmax was calculated using a 4-parameter dose-response inhibition function (GraphPad Prism 8.4.3) via nonlinear regression analysis, and IC50 was calculated by inserting a 50% threshold into the function.

[0070] Example 3: Screening of modified siRNA

[0071] We designed 25 siRNAs targeting the human MSTN transcript ENST00000260950.5 and synthesized the modified siRNAs using a chemical modification method. The modified sequence information is shown in Table 2. Using the in vitro evaluation method with two doses of siRNA described in Example 2, we transfected 293T cells with the pre-defined concentrations of siRNA and the MSTN-DualGlo plasmid. The MSTN mRNA content was calculated according to the method description, as shown in Table 3. The values ​​are expressed as the percentage of the mRNA content compared to the control group, along with the coefficient of variation.

[0072] Table 2. Preliminary modified 19 / 21mer siRNA sequence information

[0073] Where N = deoxyribonucleic acid; Nm = 2'-O-methyl; Nf = 2'-deoxy-2'-fluoro; Vp / = vinyl phosphate linker; / = Thiophosphate bond; / = Phosphodiester bond

[0074] Table 3. Results of two-dose screening of preliminarily modified siRNAs

[0075] Where ND = Not Detected

[0076] Based on the in vitro dual-concentration screening experiment, we selected 5 sequences for further activity evaluation. Using the in vitro evaluation method for multiple doses of siRNA described in Example 2, we transfected 293T cells with a predetermined amount of siRNA and the MSTN-DualGlo plasmid. The IC50 and KDmax of the siRNA were calculated according to the method description, as shown in Table 4.

[0077] Table 4. Results of multi-dose screening of preferred siRNAs

[0078] Example 4: Validation of the modified preferred siRNA sequence

[0079] Non-variable IP-RP data indicates that, as Figure 1 Based on the common platform modifications in Table 1, the five preferred sequences CGBN-0187, CGBN-0189, CGBN-0192, CGBN-0200, and CGBN-0209 showed risks of unwinding and insufficient stability. Therefore, we further designed new sequences near their respective transcripts, modified some bases, or adopted entirely new modification patterns to improve stability while preserving activity. The optimized sequence information is shown in Table 5, and the stability of siRNA was assessed using non-denaturing IP-RP and temperature-dependent IP-RP. Figure 2 and Figure 3 As shown in Table 6, for the sequences with better stability, the pre-defined amounts of siRNA and MSTN-DualGlo plasmid were transfected into 293T cells using the in vitro evaluation method for multiple doses of siRNA described in Example 2. The IC50 and KDmax of the siRNA were calculated according to the method description.

[0080] Table 5 Novel Modification Information for Optimal Sequences

[0081] Where d = deoxynucleoside.

[0082] Table 6. Results of multi-dose in vitro detection of the novel modified sequence

[0083] Example 5: Conjugation of modified siRNA and antibody

[0084] Conjugation of cysteine ​​mutant antibodies and siRNA (site-directed conjugation process)

[0085] The conjugation of cysteine-mutant antibodies and siRNA employs a site-directed conjugation process. Specifically, the antibody's cysteine ​​conjugation site (which varies depending on the specific antibody sequence and includes, but is not limited to, S239C, S238C, S245C) and interchain disulfide bonds are first reduced. Then, the interchain disulfide bonds are oxidized while preserving the reduced state of the cysteine ​​site. Finally, the siRNA is conjugated to the 5' end of the siRNA's positive strand via a SMCC linker, forming an antibody-siRNA conjugate. For the linker-siRNA used in this experiment, the single strand was fully assembled on a solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well-described in the RNAi field were used to optimize the stability of the duplex. The siRNA's positive strand contains a C6-NH2 conjugate stalk at its 5' end, which is connected to the siRNA's positive strand via a phosphodiester at the terminal base. In some implementations, the conjugation process is generally applicable to different antibodies and sequences; differences in antibody targets or sequences, as well as differences in nucleic acid sequences, often do not lead to significant changes in the conjugation process.

[0086] The MSTN siRNA used in the experiment to synthesize the antibody-siRNA conjugate is shown in Table 7, and the TfR1 antibody used is shown in Table 8. The CGB-1003 antibody-siRNA conjugate was synthesized using a site-directed coupling process.

[0087] Table 7. siRNA sequences of MSTN used for antibody conjugation.

[0088] Table 8 Antibody sequences against TfR1

[0089] Example 6: Study on the knockdown of MSTN in the muscle of wild-type CD1 mice by antibody-siRNA conjugate

[0090] The experimental design and dosage are shown in Table 9. Mice were administered PBS (control) and antibody-siRNA conjugates intravenously. Body weight (BW), leg volume, leg area, and / or leg length were measured at days 7, 14, 21, and 28. The results are as follows: Figures 4-5 As shown.

[0091] Table 9. Study protocol for knocking down MSTN in the muscle of wild-type CD1 mice using antibody-siRNA conjugates.

[0092] Example 7: Study on the knockdown of MSTN in DIO mouse muscle by antibody-siRNA conjugate

[0093] The experimental design and dosage are shown in Table 10. Mice were administered PBS (control) and antibody-siRNA conjugates intravenously. Lean and fat weights were collected at days 7, 13, and 21. At the 21-day endpoint, DIO mice were sacrificed, and the weights of the tibialis anterior, extensor digitorum longus (EDL), gastrocnemius, soleus, quadriceps, diaphragm, and cardiac muscle were collected from each group of DIO mice. The experimental results are as follows: Figures 6-8 As shown.

[0094] Table 10. Study protocol for knocking down MSTN in DIO mouse muscle using antibody-siRNA conjugates.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An RNA interference agent targeting MSTN, comprising a first nucleotide sequence, the first nucleotide sequence comprising any one of the nucleotide sequences in SEQ ID NO: 1 to SEQ ID NO: 25 or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with it, the first nucleotide sequence being capable of hybridizing to at least 8 consecutive bases of the mRNA of the MSTN gene.

2. The RNA interference agent according to claim 1, characterized in that, The RNA interference agent further includes a second nucleotide sequence that can hybridize with the first nucleotide sequence. The second nucleotide sequence includes any one of the nucleotide sequences in SEQ ID NO: 26 to SEQ ID NO: 50 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it. The first nucleotide sequence is the antisense strand of the RNA interference agent, and the second nucleotide sequence is the sense strand of the RNA interference agent.

3. The RNA interference agent according to claim 2, characterized in that, The antisense strand and the sense strand each independently contain 8 to 50 nucleotides.

4. The RNA interference agent according to claim 3, characterized in that, The antisense strand and the sense strand each independently contain 10 to 30 nucleotides.

5. The RNA interference agent according to claim 2, characterized in that, The antisense strand and / or the sense strand contain at least one modified nucleotide and at least one modified or unmodified nucleotide linked together.

6. The RNA interference agent according to claim 5, characterized in that, The modified nucleotide is at least one of 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-deoxy, 2'-O-dimethylaminoethyl, 2'-O-dimethylaminopropyl, 2'-O-dimethylaminoethoxyethyl, 2'-ON-methylacetamido, locked nucleic acid, ethylene nucleic acid, or ethylene glycol nucleic acid.

7. The RNA interference agent according to claim 5, characterized in that, The antisense strand and / or the sense strand comprise 5% to 100% modified nucleotides.

8. The RNA interference agent according to claim 5, characterized in that, The modified nucleotide linkages are either thiophosphate linkages or dithiophosphate linkages.

9. The RNA interference agent according to claim 5, characterized in that, The antisense strand comprises any one of the nucleotide sequences SEQ ID NO: 51 to SEQ ID NO: 75 or SEQ ID NO: 101 to SEQ ID NO: 138, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; the sense strand comprises any one of the nucleotide sequences SEQ ID NO: 76 to SEQ ID NO: 100 or SEQ ID NO: 139 to SEQ ID NO: 176, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

10. A conjugate comprising an RNA interference agent according to any one of claims 2-9 and a conjugate antibody, said conjugate antibody being linked to the 5' end of the positive strand of said RNA interference agent.

11. The conjugate according to claim 10, characterized in that, The conjugated antibody is a human anti-transferrin receptor antibody, and the RNA interference agent used to conjugate with the conjugated antibody includes any one of the nucleotide sequences in SEQ ID NO: 177 and SEQ ID NO: 178 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with it.

12. The conjugate according to claim 11, characterized in that, The human anti-transferrin receptor antibody specifically binds to human transferrin receptor 1.

13. The conjugate according to claim 11, characterized in that, The binding ratio of the RNA interference agent to the conjugated antibody is from 1:1 to 16:

1.

14. A kit comprising an RNA interference agent according to any one of claims 1-9, or a conjugate according to any one of claims 10-13.

15. Use of the RNA interference agent according to any one of claims 1-9, or the conjugate according to any one of claims 10-13, for the preparation of a medicament, wherein the medicament is used to reduce the expression of MSTN mRNA or protein in mammals.

16. The use according to claim 15, wherein the drug is used to promote skeletal muscle growth, increase muscle mass, inhibit muscle atrophy, or reduce fat deposition.

17. The use according to claim 16, wherein the drug is used for lowering lipids and increasing muscle mass.

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