siRNAs and their conjugates that inhibit MSTN gene expression and their applications

Abstract

This invention provides an siRNA for inhibiting MSTN gene expression, its conjugates, and their applications. The siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II; each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified or unmodified nucleotide. The siRNA, its conjugates, and the pharmaceutical composition provided by this invention exhibit strong inhibitory activity against the MSTN gene, significantly reducing the expression level of MSTN mRNA, and have low drug toxicity.

Description

[0001] This invention is a divisional application of the invention patent filed on December 31, 2024, entitled "siRNA that inhibits MSTN gene expression and its conjugates and applications", with the parent application number "CN02411976821.9". Technical Field

[0002] This invention belongs to the field of biomedicine and relates to siRNA and its conjugates that inhibit MSTN gene expression and their applications. Background Technology

[0003] Obesity is a chronic, relapsing disease caused by the excessive accumulation or distribution of adipose tissue and its abnormal function, resulting from a combination of genetic and environmental factors. Currently, more than one-third of adults worldwide are classified as overweight or obese, and the total number of obese children, adolescents, and adults exceeds one billion, making the obesity problem extremely serious. Obese individuals face a range of chronic complications, including abnormal glucose metabolism, dyslipidemia, hypertension, polycystic ovary syndrome, and obstructive sleep apnea syndrome.

[0004] Myopathy is a non-inflammatory disease originating in skeletal muscle or at the neuromuscular junction. It is mainly characterized by decreased or absent muscle contractility and muscle atrophy. Neuromuscular diseases are a group of muscle disorders characterized by weakness and atrophy of both muscular and bulbar muscles. Patients will experience symptoms such as muscle atrophy, changes in muscle strength, and neuromuscular junction lesions. Spinal muscular atrophy (SMA) is a single-gene recessive genetic disorder. Typical SMA manifestations include generalized muscle weakness, low muscle tone, and muscle atrophy, limiting normal standing and walking abilities. It is often accompanied by lung infections, joint contractures, and hip subluxation. Severely affected children often die from respiratory failure. The carrier rate of the pathogenic gene for SMA is very high, with one in 40-50 people being a carrier, and the incidence rate in newborns is approximately 1 in 10,000.

[0005] Myostatin (MSTN), belonging to the transforming growth factor β (TGFβ) superfamily, is a key regulator of skeletal muscle mass. MSTN controls muscle volume by inhibiting muscle cell proliferation, leading to reduced muscle mass and thus regulating skeletal muscle growth. MSTN levels may be elevated in many disease conditions, such as muscular dystrophy and obesity. Studies have found that MSTN knockout mice exhibit increased muscle mass, reduced fat deposition, improved insulin sensitivity, enhanced fatty acid oxidation, and anti-obesity characteristics. Research has also shown that MSTN can affect lipid metabolism, reducing obesity by inhibiting fat deposition. Increased MSTN mRNA and protein levels are observed in the muscle and adipose tissue of leptin-deficient obese mice and wild-type mice on a high-fat diet. Therefore, MSTN is currently an important target for obesity, musculoskeletal diseases, and neuromuscular diseases.

[0006] Traditional weight-loss drugs can cause nausea, vomiting, and gastric paralysis; in severe cases, they can even lead to pancreatitis and intestinal obstruction. While GLP-1 inhibitors are highly effective at reducing weight, they also come with numerous side effects. Besides common side effects such as nausea and bloating, there can be weight rebound after discontinuation, muscle loss, and even more serious side effects. Therefore, increasing muscle mass and reducing fat mass to treat obesity has become the primary goal of research and development for next-generation weight-loss drugs.

[0007] Treatment for muscle diseases requires addressing muscle atrophy by increasing muscle mass and strength, but related medications are not yet ideal for treating muscle atrophy. Summary of the Invention

[0008] The purpose of this invention is to provide a siRNA molecule that can inhibit MSTN gene expression, in order to provide a new treatment for diseases including MSTN-related obesity, musculoskeletal disorders, and neuromuscular disorders.

[0009] In a first aspect, the present invention provides an siRNA for inhibiting MSTN gene expression, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence I, and the antisense strand comprising a nucleotide sequence II; each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified or unmodified nucleotide; nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; nucleotide sequence I is substantially identical to a first nucleotide sequence, the first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length in the mRNA expressing the MSTN gene, preferably, the first nucleotide sequence being a nucleotide sequence of 15 to 25 nucleotides in length in the mRNA expressing the MSTN gene, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides;

[0010] Preferably, the mRNA expressing the MSTN gene is as shown in NCBI refseqID NM_005259.3; specifically, the mRNA sequence is as shown in SEQ ID NO:1.

[0011] In some embodiments, the nucleotide sequence I differs from the first nucleotide sequence by one nucleotide; preferably, the difference of one nucleotide is Z1 located at the 3' end, Z1 being optionally A, U, C or G, but different from the nucleotide at the corresponding position of the first nucleotide sequence; even more preferably, the nucleotide sequence II has Z2 at the 5' end, which is complementary to Z1.

[0012] In some implementations, the nucleotide sequence II in the above-described siRNA is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the first nucleotide sequence.

[0013] In some embodiments, in any of the siRNAs described above, the sense and antisense strands may be of the same or different lengths, with the sense strand being 16-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. In some embodiments, the length ratio of the sense to antisense strands of the siRNA is 19 / 21, 21 / 23, or 19 / 24.

[0014] In some embodiments, in any of the above-described siRNAs, the nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the first nucleotide segment.

[0015] In some embodiments, in any of the above-described siRNAs, the nucleotide sequence I comprises at least 15 consecutive nucleotides as shown in SEQ ID NO:2-109, such as at least 15, 16, 17, 18, 19, 20, or 21 nucleotides; preferably, the nucleotide sequence I is as shown in SEQ ID NO:2-109.

[0016] In some embodiments, in any of the above-described siRNAs, the nucleotide sequence II comprises at least 15 consecutive nucleotides as shown in SEQ ID NO:110-217, such as at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides; preferably, the nucleotide sequence II is as shown in SEQ ID NO:110-217.

[0017] In some embodiments, in any of the siRNAs described above, the sense strand comprises the sense strand of any of the siRNAs shown in Table 1 of this document, and the antisense strand comprises the antisense strand of the corresponding siRNA in Table 1.

[0018] Preferably, in any of the siRNAs described above, the sense strand is substantially identical to the sense strand of any siRNA shown in Table 1, and the antisense strand is substantially identical to the antisense strand of the corresponding siRNA in Table 1. Preferably, "substantially identical" means that the 3' end of the antisense strand or the 5' end of the sense strand differs from the corresponding sequence in Table 1 by no more than three bases.

[0019] Preferably, in any of the above-described siRNAs, the sense strand is of the same length as the sense strand of any of the siRNAs shown in Table 1 and differs by one nucleotide; preferably, the difference of one nucleotide is Z1 located at the 3' end, Z1 being optionally A, U, C or G; even more preferably, in any of the above-described siRNAs, the antisense strand has Z2 at the 5' end that is complementary to Z1.

[0020] In some embodiments, in any of the siRNAs described above, each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified nucleotide, wherein the modified nucleotide is a fluorinated nucleotide or a non-fluorinated nucleotide.

[0021] In some embodiments, the fluorinated nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2'-position of the ribosyl group with fluorine, having the structure shown in formula (1); the non-fluorinated nucleotide refers to a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2'-position of the ribosyl group with a non-fluorinated group. In some embodiments, each non-fluorinated nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'-position of the ribosyl group with a non-fluorinated group. These nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'-position of the ribosyl group with a non-fluorinated group are well known to those skilled in the art, and these nucleotides or nucleotide analogs may be selected from 2'-alkoxy-modified nucleotides or nucleotide analogs, 2'-substituted alkoxy-modified nucleotides or nucleotide analogs, 2'-alkyl-modified nucleotides or nucleotide analogs, 2'-substituted alkyl-modified nucleotides or nucleotide analogs, 2'-amino-modified nucleotides or nucleotide analogs, 2'-substituted amino-modified nucleotides or nucleotide analogs, and 2'-deoxynucleotides. In some embodiments, the 2′-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2′-OMe), as shown in formula (2). In some embodiments, the 2′-substituted alkoxy-modified nucleotide may be, for example, a 2′-O-methoxyethyl-modified nucleotide (2′-MOE), as shown in formula (3). In some embodiments, the 2′-amino-modified nucleotide (2′-NH2) is shown in formula (4). In some embodiments, the 2′-deoxynucleotide (DNA) is shown in formula (5).

[0022]

[0023] Nucleotide analogs are groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine ribonucleotide. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleotides, or acyclic nucleotides.

[0024] A bridging nucleotide is a restricted or inaccessible nucleotide. Bridging nucleotides can contain a five-membered, six-membered, or seven-membered ring with a fixed C3-endoglucan condensation. In some embodiments, the bridging nucleotide can be LNA, ENA, cET BNA, etc.; wherein LNA is shown in formula (6), ENA is shown in formula (7), and cET BNA is shown in formula (8).

[0025]

[0026] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, acyclic nucleotides can be unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA), where UNA is shown in formula (9) and GNA is shown in formula (10).

[0027]

[0028] In formulas (9) and (10) above, R is selected from H, OH or alkoxy (O-alkyl).

[0029] Isonucleotides are compounds formed by altering the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide can be a compound formed by moving a base from the 1' position to the 2' or 3' position on the ribose ring, as shown in formula (11) or formula (12).

[0030]

[0031] In formulas (11) and (12), R is selected from H, OH, F or non-fluorine groups as described above.

[0032] In equations (1) to (12), Base represents a base.

[0033] In some embodiments, in any of the above-described siRNAs, one or more nucleotides at positions 5, 7, 8, 9, 10, and 11 of nucleotide sequence I are fluorinated nucleotides, and one or both nucleotides at positions 8 and 10 are deoxyribonucleotides; and one or more nucleotides at positions 2, 6, 8, 9, 12, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, in the direction from 5' end to 3' end.

[0034] Preferably, the nucleotides at positions 7, 9, 10, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or

[0035] Preferably, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or

[0036] Preferably, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides in the direction from the 5' end to the 3' end; and the nucleotides at positions 2, 14, and 16 of nucleotide sequence II are fluorinated nucleotides in the direction from the 5' end to the 3' end.

[0037] Alternatively, preferably, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides in the direction from the 5' end to the 3' end; and the nucleotides at positions 2, 14, and 16 of nucleotide sequence II are fluorinated nucleotides in the direction from the 5' end to the 3' end.

[0038] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, and the nucleotide at position 10 is a deoxyribonucleotide; and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides; or

[0039] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides, and the nucleotide at position 8 is a deoxyribonucleotide; and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides; or

[0040] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, and the nucleotide at position 10 is a deoxyribonucleotide; and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 14, and 16 of nucleotide sequence II are fluorinated nucleotides; or

[0041] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides, and the nucleotide at position 8 is a deoxyribonucleotide; and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 14, and 16 of nucleotide sequence II are fluorinated nucleotides; or

[0042] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, and the nucleotide at position 10 is a deoxyribonucleotide; and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 12, 14, and 16 of nucleotide sequence II are fluorinated nucleotides; or

[0043] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, and 9 of the nucleotide sequence I are fluorinated nucleotides, and the nucleotide at position 8 is a deoxyribonucleotide; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 12, 14, and 16 of the nucleotide sequence II are fluorinated nucleotides.

[0044] In some embodiments, at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA are phosphate ester groups with modifying groups. In some embodiments, the phosphate ester group with modifying groups is a thiophosphate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom. In some embodiments, the phosphate ester group with modifying groups is a thiophosphate group having the structure shown in formula (13).

[0045]

[0046] In some embodiments, in any of the above-described siRNAs, the thiophosphate group linkage is present at least one of the following positions: between the first and second nucleotides of the sense and / or antisense strands; between the second and third nucleotides of the sense and / or antisense strands; between the 19th and 20th nucleotides of the antisense strand; between the 20th and 21st nucleotides of the antisense strand; between the 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination thereof.

[0047] In some embodiments, in any of the above-described siRNAs, the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, as shown in formulas (14), (15) and (16).

[0048]

[0049] In a second aspect, the present invention provides an siRNA conjugate comprising any of the siRNAs described above and a conjugating group conjugated to the siRNA. In some embodiments, the siRNA conjugate may be a steroid. Exemplary steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, saturated, unsaturated, substituted hydrocarbons, or combinations thereof. In some cases, the steroid is cholesterol. In some cases, the binding moiety is cholesterol. In some embodiments, the conjugation site of the siRNA to the conjugating group may be at the 3' or 5' end of the siRNA's sense strand, at the 3' end of the antisense strand, or within the internal sequence of the siRNA.

[0050] In some embodiments, the conjugating group in the above-described siRNA conjugate is cholesterol, with the structure shown below:

[0051]

[0052] The siRNA conjugate has its positive strand selected from the nucleotide sequence shown in the following formula: 5′-XmsXmsXmXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3′, or 5′-XmsXmsXmXmXmXmXfXmXmXfXmXmXmXmXmXmXmXmXm-Cholesteryl-3′, or 5′-XmsXmsXmXmXfXmXfXmXmXmXmXmXmXmXmXm-Cholesteryl-3′, or 5′-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXm-Cho lesteryl-3'; or 5'-XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'; XdXfXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'; or 5'-XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'.

[0053] The antisense strand is selected from the nucleotide sequence shown in the following formula: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmfXmXfXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmfXmXfXmXmXmXmXmXmsXms-3', or 5'-VPXmsXfsXmXmXmXmXfXmXmXmXmXmXmXmXmXmXmXmXm-3', or 5'-VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXmXfXm ... mXmXmsXmsXm-3'; or 5'-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXm XmsXm-3'; or 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsX msXm-3'; or 5'-VPXms XmXmXfXmXfXmXfXmXm

[0054] Wherein, Xm represents any nucleotide modified with 2'-methoxy, such as 2'-methoxy modified C, G, U, A, T; Xf represents any nucleotide modified with 2'-fluoride, such as 2'-fluoride modified C, G, U, A, T; Xd represents any nucleotide modified with 2'-deoxy, such as dC, dG, dU, dA, dT; the lowercase letter s indicates that the two nucleotides adjacent to the letter s on the left and right are linked by thiophosphate subunits; VP indicates that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinylphosphonate (5'-(E)-vinylphosphonate, E-VP).

[0055] In some implementations, the justice chain and the antisense chain are combined as follows:

[0056] M-1: (21 / 23)

[0057] Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXm-Cholestery l-3'

[0058] Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXmXfXmXmXmXmXmXmXmsXmsXm-3';

[0059] Or M-1'(19 / 21)

[0060] Justice chain: 5'-XmsXmsXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3′

[0061] Antonym chain: 5′-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXmXmXmXmXmXmXmsXmsXm-3′; or M-2: (21 / 23)

[0062] Justice chain: 5′-XmsXmsXmXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm-Cholester yl-3′

[0063] Antisense strand: 5′-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsX m-3';

[0064] Or M-2'(19 / 21)

[0065] Justice Chain: 5′-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3′ Antisense Chain: 5′-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXms-3′; or M3: (21 / 23)

[0066] Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholester yl-3′

[0067] Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXmXfXmXmXmXmXmXmXmXmsXmsXm-3';

[0068] Or M3'(19 / 21)

[0069] Justice chain: 5′-XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3′

[0070] Antonym chain: 5′-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm-3′; or M4: (21 / 23)

[0071] Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholester yl-3′

[0072] Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXfXmXmXmXmXmXmXm-3';

[0073] Or M4'(19 / 21)

[0074] Justice chain: 5′-XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3′

[0075] Antonym chain: 5′-VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXmXmXmXmXmXm-3′; or M5: (21 / 23)

[0076] Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholester yl-3′

[0077] Antonyms: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXfXmXmXfXmXmXmXmXmXmXmXmsXmsXm-3'; or M5'(19 / 21)

[0078] Justice chain: 5′-XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3′

[0079] Antonym chain: 5′-VPXmsXfsXmXmXmXfXmXfXmXfXmXmXfXmXmXfXmXmXmXmXmsXmsXm-3′.

[0080] In some embodiments, the siRNA conjugate comprises a sense strand and an antisense strand, wherein the sense strand comprises the sense strand of any of the siRNA conjugates shown in Tables 2-6 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate.

[0081] In some embodiments, the siRNA conjugate is YG-95M2, YG-96M2, YG-97M2, YG-105M2, YG-106M2; or YG-95M3, YG-96M3, YG-97M3, YG-105M3, YG-106M3; or YG-95M4, YG-96M4, YG-97M4, YG-105M4, YG-106M4; or YG-95M5, YG-96M5, YG-97M5, YG-105M5, YG-106M5.

[0082] In a third aspect, the present invention provides a composition comprising any of the siRNAs or siRNA conjugates described above.

[0083] In some embodiments, the composition is a pharmaceutical composition and further includes a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carriers or excipients involved in this invention include, but are not limited to, water for injection, sodium hydroxide, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, phosphoric acid, sodium chloride, potassium chloride, hydrochloric acid, anhydrous potassium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, PEG2000, PEG6000, cholesterol, distearate, 1,2-dimyristate glyceryl, and dimethyl adipic acid.

[0084] In a fourth aspect, the present invention provides the use of any of the above-described siRNAs, siRNA conjugates, or pharmaceutical compositions in the preparation of a medicament for the prevention and / or treatment of diseases associated with MSTN expression.

[0085] In a fifth aspect, the present invention provides a method for preventing or treating diseases associated with MSTN expression, the method comprising administering to a subject the siRNA, siRNA conjugate, or pharmaceutical composition of the present invention.

[0086] In some implementations, the diseases associated with elevated MSTN levels include obesity and obesity-related diseases, muscle diseases such as musculoskeletal diseases and neuromuscular diseases.

[0087] In some implementations, the diseases associated with elevated MSTN levels include obesity and obesity-related diseases, and muscle diseases. The obesity-related diseases include, but are not limited to, abnormal blood glucose, abnormal blood lipids, hypertension, metabolic-associated fatty liver disease, obstructive sleep apnea syndrome, polycystic ovary syndrome, and cardiovascular diseases. The muscle diseases include, for example, spinal muscular atrophy, metabolic myopathy, Duchenne muscular dystrophy, mitochondrial myopathy, polymyositis, Guillain-Barré syndrome, and myasthenia gravis.

[0088] The pharmaceutical compositions involved in this invention can be used alone for the treatment of obesity, musculoskeletal disorders, and neuromuscular diseases, or in combination with standard oral weight loss, musculoskeletal disorders, and neuromuscular diseases, providing experimental support for achieving diversified treatment options for the aforementioned patients.

[0089] Based on the amount of siRNA contained therein, the generally suitable dosage range of the MSTN gene expression inhibitory siRNA, siRNA conjugate or pharmaceutical composition involved in this invention will be from about 0.1 mg / kg to about 30.0 mg / kg, preferably from about 5 mg / kg to about 15.0 mg / kg.

[0090] The administration routes involved in this invention include intravenous administration, subcutaneous administration, intrathecal injection, intramuscular administration, transdermal administration, airway administration (aerosol), ocular administration, nasal administration, rectal administration, pulmonary administration, and local administration (including oral administration and sublingual administration).

[0091] The siRNA, its conjugates, and pharmaceutical compositions of the present invention can specifically target muscle, complementarily pair with the muscle MSTN mRNA sequence, induce MSTN mRNA degradation, thereby inhibiting the synthesis of the MSTN gene in muscle, resulting in a sustained reduction of MTN protein and exerting a sustained muscle-building effect, while avoiding off-target effects and exhibiting low cytotoxicity. They can fundamentally prevent or treat MSTN-related obesity, musculoskeletal diseases, neuromuscular diseases, and other related diseases.

[0092] In addition, compared with traditional small molecule drugs and antibody drugs, small nucleic acid drugs can directly regulate the expression of upstream genes and are relatively less likely to develop drug resistance; moreover, small nucleic acid drugs have a long half-life in the body, so the frequency of administration is low (they can be given once every six months), and patients have good compliance.

[0093] Beneficial technical effects

[0094] The siRNA, its conjugates, and pharmaceutical compositions provided by this invention exhibit strong inhibitory activity against the MSTN gene, significantly reducing MSTN mRNA expression levels, and possess low drug toxicity. Therefore, the siRNA, its conjugates, and pharmaceutical compositions of this invention can effectively prevent and / or treat diseases associated with MSTN expression, providing patients with more effective, safe, and convenient therapeutic drugs, and have promising prospects as pharmaceutical products. Attached Figure Description

[0095] Figure 1 The results of the siRNA conjugate cytotoxicity experiment in Example 4 of this invention;

[0096] Figure 2 The results of the siRNA conjugate inhibiting MSTN gene expression in vivo in Example 5 of this invention (D7 and D14);

[0097] Figure 3 The results of the siRNA conjugate inhibiting MSTN gene expression in vivo in Example 5 of this invention (D7, D14 and D28);

[0098] Figure 4 This is the result of the siRNA conjugate inhibiting MSTN protein expression in vivo in Example 6 of the present invention;

[0099] Figure 5 This is the result of the siRNA conjugate inhibiting MSTN protein expression in vivo in Example 7 of the present invention;

[0100] Figure 6 This is the result of the siRNA conjugate inhibiting MSTN protein expression in vivo in Example 8 of the present invention;

[0101] Figure 7 This is the result of the siRNA conjugate inhibiting MSTN protein expression in vivo in Example 9 of the present invention;

[0102] Figure 8 This is the result of the siRNA conjugate inhibiting MSTN protein expression in vivo in Example 10 of the present invention. Detailed Implementation

[0103] definition

[0104] Unless otherwise specified, in the preceding and following text, uppercase letters C, G, U, and A represent cytosine, guanine, uracil, and adenine nucleotides, respectively; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter d indicates that the nucleotide adjacent to the left of letter d is a deoxynucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by a thiophosphate subunit; the letter combination VP indicates that the nucleotide adjacent to the right of letter combination VP is a vinylphosphonate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide; Cholesteryl has the structure of formula (I) and is linked to the 3' end of the positive chain via a phosphate ester bond.

[0105] In the preceding and following text, "fluorinated nucleotides" refers to nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosome with fluorine, and "non-fluorinated nucleotides" refers to nucleotides or nucleotide analogs formed by replacing or deoxygenating the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group. "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids but whose structure differs from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides. "Methoxylated nucleotides" refers to nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosome with a methoxy group.

[0106] In the context of this document, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired complementaryly with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, "mismatch" in the art means, in a double-stranded nucleic acid, that the bases at corresponding positions are not paired complementaryly.

[0107] As used herein, “includes” is intended to be used interchangeably with the phrase “includes but is not limited to”. The term “or” is used herein to mean “and / or” and is used interchangeably with that term unless the context clearly indicates otherwise.

[0108] Unless otherwise specified, in the foregoing and hereinafter, "substantially anticomplementary" means that there are no more than three base mismatches between the two nucleotide sequences involved; "substantially anticomplementary" means that there are no more than one base mismatch between the two nucleotide sequences; and "completely anticomplementary" means that there are no base mismatches between the two nucleotide sequences. In the foregoing and hereinafter, particularly in describing the preparation methods of the siRNA, pharmaceutical compositions, or siRNA conjugates of the present invention, unless otherwise specified, the nucleoside monomer refers to the modified or unmodified nucleoside phosphoramidites (sometimes also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for the synthesis of siRNA. All nucleoside monomers used in the present invention are commercially available.

[0109] In the context of this invention, the term "corresponding siRNA" refers to the same siRNA mentioned above. For example, when it is stated that "the sense strand comprises the sense strand of any of the siRNAs shown in Table 1 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA," it means that the included sense strand and antisense strand are from the same siRNA shown in Table 1 herein. For example, when the sense strand comprises 5'-GUGUUUAUAUUUACCUGUUUA-3' (SEQ ID NO: 2), the antisense strand comprises 5'-UAAACAGGUAAAUAUAAACACAG-3' (SEQ ID NO: 110). Similarly, the term "corresponding siRNA conjugate" refers to the same siRNA conjugate mentioned above. For example, when it is stated that "the sense strand comprises the sense strand of any of the siRNA conjugates shown in Table 2 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate," it means that the included sense strand and antisense strand are from the same siRNA conjugate shown in Table 2 herein. Furthermore, in these contexts, "comprising" includes cases where the sequences consist of these sequences.

[0110] Example

[0111] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of the invention) are given for illustrative purposes only, as various changes and modifications made within the spirit and scope of the invention will become apparent to those skilled in the art upon reading this detailed description.

[0112] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to 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 specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0113] Example 1. siRNA Design and Synthesis

[0114] 1.1 siRNA Design

[0115] A set of siRNAs targeting the human MSTN gene (human MSTN gene: NCBI refseq ID NM_005259.3; NCBI Gene ID: 2660) was designed online using OligoWalk. The human NM_005259.3REFSEQ mRNA has a length of 2819 bases. Sequences similar to the human gene were also excluded to avoid any toxicity.

[0116] Human NM_005259.3 REFSEQ mRNA (NCBI refseq ID NM_005259.3):

[0117]

[0118]

[0119] 1.2 siRNA sequence synthesis

[0120] siRNA was synthesized according to a standard oligonucleotide solid-phase synthesis protocol, including a negative control siRNA (siCtrl).

[0121] Oligonucleotide solid-phase synthesis protocol: Commercially available 5'-DMT-2'-TBDMS-rU phosphoramide monomers, 5′-DMT-2'-TBDMS-rA(Bz) phosphoramide monomers, 5′-DMT-2'-TBDMS-rC(Ac) phosphoramide monomers, and 5′-DMT-2'-TBDMS-rG(iBu) phosphoramide monomers were used. RNA was synthesized at a synthesis scale of 500 nM. A phosphoramide solution was prepared at a concentration of 50 mM, and 0.3 M benzylthiotetrazole (BTT) acetonitrile solution was used as an activator. During synthesis, a 0.1 M oxidizing agent (pyridine:THF:water = 20:78:2) was used to convert trivalent phosphorus to pentavalent phosphorus to stabilize the phosphate backbone. After synthesis, the sequence was ammonolyzed from the solid support and precipitated. The 2'-2'-tert-butyldimethylsilyl protecting group was removed with triethylamine trihydrofluoric acid.

[0122] For the synthesized RNA sequence, ammonolysis was performed at 55°C for 40 minutes using an ammonia:methylamine ratio of 1:1. After ammonolysis, the solid support CPG powder was removed, and the supernatant was dried. A protecting group remover was added, and the reaction was carried out at 60°C for 2 hours. Then, n-butanol was added at a 1:5 ratio, and the mixture was allowed to stand at -20°C for 30 minutes. The precipitate was collected by centrifugation. The precipitate was dissolved in RNase-free water and purified using reversed-phase chromatography (0.1M triethylamine acetic acid (TEAA) and acetonitrile). The purified sample was desalted by ultrafiltration with PBS and annealed to obtain siRNA. Verification of the obtained siRNA confirmed successful preparation of the target siRNA.

[0123] 1.3 siRNA sequence modification and conjugate synthesis

[0124] Modified siRNAs are synthesized according to oligonucleotide solid-phase synthesis schemes. The modified nucleotide groups can be introduced into the siRNAs of this invention using correspondingly modified nucleoside monomers. Methods for preparing correspondingly modified nucleoside monomers are well known to those skilled in the art. Cholesterol is conjugated to siRNA to synthesize siRNA conjugates, referring to the synthesis method disclosed in WO2019217459A1.

[0125] The structure of the conjugated group is shown below:

[0126]

[0127] Annealing of oligonucleotides to produce siRNA conjugates: The RNA oligomers to be annealed were prepared into a 200 μM solution using sterile RNase-free water. The annealing reaction system was set up as follows: 100 μL of the above solution (10 nM duplex concentration) was placed in a 95°C water bath for 10 minutes (≥100 nM requires 20 minutes at high temperature) → immediately cooled in a 60°C water bath → the annealed solution was stored at 4°C. Equimolar amounts of RNA solution were combined to mix complementary strands. The siRNA molecule was confirmed to be correctly constructed. The siRNA solution was prepared into a dry powder for later use.

[0128] The sequences of the synthesized siRNA molecules are shown in Table 1 below:

[0129] Table 1. siRNA sequence listing targeting MSTN

[0130]

[0131]

[0132]

[0133]

[0134] Table 2. Sequence listing of siRNA conjugates targeting MSTN (M1 or M1' modification pattern)

[0135]

[0136]

[0137]

[0138]

[0139] Note 1: YG-96M1 and YG-97M1 in the table are M1' modified patterns, and the rest are M1 modified patterns. Note 2: siCtrlM1 justice chain:

[0140] XmsXmsXmXmXmXmXfXm

[0141] Table 3. Sequence listing of siRNA conjugates targeting MSTN (M2 or M2' modification pattern)

[0142]

[0143]

[0144]

[0145]

[0146] Note 1: In the table, YG-96M2 and YG-97M2 are M2' modified patterns, and the remaining sequences are M2 modified patterns.

[0147] Note 2: siCtrlM2 justice chain: XmsXmsXmXmXmXmXfXmXfXmXmXfXmXmXmXmXmXmXmXmdTdT-Cholesteryl; antisense chain: VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmdTdT.

[0148] Table 4. Sequence listing of siRNA conjugates targeting MSTN (M3 or M3' modification pattern)

[0149]

[0150]

[0151]

[0152]

[0153] Note 1: In the table, YG-96M3 and YG-97M3 are M3' modification patterns, and the remaining sequences are M3 modification patterns.

[0154] Note 2: siCtrlM3 justice chain: XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmdTdT-Cholesteryl; antisense chain: VPXmsXfsXmXmXmXfXmXmXmXmXmXmXfXmXmXmXfXmXmsXmsXmdTdT.

[0155] Table 5. Sequence listing of siRNA conjugates targeting MSTN (M4 or M4' modification pattern)

[0156]

[0157]

[0158]

[0159]

[0160] Note 1: In the table, YG-96M4 and YG-97M4 are M4' modified patterns, while the remaining sequences are M4 modified patterns.

[0161] Note 2: siCtrlM4 justice chain: XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmdTdT-Cholesteryl; antisense chain: VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXmXmXfXmXmsXmsXmdTdT.

[0162] Table 6. Sequence listing of siRNA conjugates targeting MSTN (M5 or M5' modification pattern)

[0163]

[0164]

[0165]

[0166]

[0167] Note 1: In the table, YG-96M5 and YG-97M5 are M5' modification patterns, and the remaining sequences are M5 modification patterns.

[0168] Note 2: siCtrlM4 justice chain: XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmdTdT-Cholesteryl; antisense chain: VPXmsXfsXmXmXmXfXmXfXmXmXmXfXmXmXfXmXmXmXmsXmsXmdTdT.

[0169] In this context, uppercase letters C, G, U, A, and T represent cytosine, guanine, uracil, adenine nucleotide, and thymine deoxynucleotide, respectively; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is methoxy-modified; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is fluorinated; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by thiophosphate subunits; Cholesteryl has the structure of formula (I) and is linked to the 3' end of the positive strand via a phosphate ester bond; dT represents 2'-deoxythymine nucleotide.

[0170] Example 2. In vitro activity screening of siRNA in RH30 cell lines

[0171] 2.1 Experimental Procedure

[0172] 2.1.1 Cell Culture

[0173] RH30 cells (China Center for Type Culture Collection, GDC0611) were cultured in IMDM medium (Eallbio, with 10% FBS and 1% PS) at 37°C and 5% CO2. When the confluence reached 80%–90%, the cells were digested with trypsin, centrifuged, resuspended in DMEM high-glucose medium (Eallbio, with 10% FBS), counted, and transfected.

[0174] 2.1.2 Preparation of siRNA dilution buffer

[0175] (1) The dry powder of the siRNA to be tested was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM siRNA stock solution.

[0176] (2) Prepare 200 nM siRNA dilution solution Y:

[0177] a) Take 50 μl of the 100 μM siRNA stock solution obtained in step (1) above, add 50 μl of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 50 μM.

[0178] b) Take 2 μl of the 50 μM siRNA dilution solution obtained in step a) and add 18 μl of ultrapure distilled water to obtain siRNA stock solution X with a final concentration of 5 μM.

[0179] c) Take 2 μl of the prepared siRNA stock solution X and add 48 μl of Opti-medium to obtain a 200 nM siRNA dilution solution Y.

[0180] 2.1.3 RH30 cell transfection

[0181] Pick 0.6 μl of RNAiMAX (ThermoFisher, 13778150) transfection reagent was added to 10 μl of Opti-medium to obtain... RNAiMAX transfection reagent dilution solution; The RNAiMAX transfection reagent diluent and the 200 nM siRNA diluent Y prepared in step 2.1.2 were mixed at a 1:1 volume ratio to prepare a transfection mixture. After standing for 5 minutes, 10 μl of the transfection mixture was added to a 96-well plate, along with 90 μl of RH30 cells cultured in step 2.1.1 (final volume 100 μl / well, cell number 20,000 / well, siRNA concentration in this system 10 nM). The plate was then cultured for 24 hours after transfection.

[0182] 2.1.4 RNA Extraction

[0183] Total RNA was extracted from RH30 cells obtained in step 2.1.3 according to the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit product instructions.

[0184] 2.1.5 Quantitative Real-Time PCR

[0185] The extracted total RNA was analyzed by reverse transcription and real-time PCR using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit.

[0186] 2.1.6 Results Analysis

[0187] (1) Use the software of the 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher) to automatically calculate the Ct value;

[0188] (2) Calculate the relative expression level of the gene using the following formula:

[0189] ΔCt1=Ct(MSTN group)–Ct(MSTN group's ACTIN)

[0190] ΔCt2=Ct(siCtrl group)–Ct(siCtrl group's ACTIN)

[0191] ΔCt = ΔCt1 (MSTN group) - ΔCt2 (siCtrl group), where the siCtrl group is the negative control group;

[0192] mRNA expression relative to the siCtrl group = 2 -ΔΔCt

[0193] Inhibition rate (%) = (1 - mRNA expression relative to siCtrl group) × 100%.

[0194] 2.2 Experimental Results

[0195] The inhibitory effect of the siRNA of the present invention is shown in Table 7 below.

[0196] Table 7. Results of in vitro screening of siRNA RH30 cell lines

[0197]

[0198]

[0199]

[0200]

[0201] The results showed that some of the siRNAs of the present invention could significantly inhibit the expression of the MSTN gene at 10 nM.

[0202] Example 3. IC50 assay of siRNA in RH30 cell line

[0203] 3.1 Experimental Procedure

[0204] Cell culture and transfection were performed in a manner similar to that in Example 2. The concentrations of transfected siRNA were 0.0032 nM, 0.016 nM, 0.08 nM, 0.4 nM, 2 nM, 10 nM, and 50 nM, respectively. Then, the IC50 of siRNA inhibiting MSTN gene expression was measured.

[0205] 3.2 Results Analysis

[0206] (1) Use the software of the 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher) to automatically calculate the Ct value;

[0207] (2) Calculate the relative expression level of the gene using the following formula:

[0208] ΔCt1=Ct(MSTN group)–Ct(MSTN group's ACTIN)

[0209] ΔCt2=Ct(siCtrl group)–Ct(siCtrl group's ACTIN)

[0210] ΔCt = ΔCt1 - ΔCt2, where the siCtrl group is the negative control group;

[0211] mRNA expression relative to the siCtrl group = 2 -ΔΔCt

[0212] Inhibition rate (%) = (1 - mRNA expression relative to siCtrl group) × 100%.

[0213] Using the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, the dose-response curve was fitted using the "log(inhibitor) vs. normalized response - Variable slope" function module of the analysis software GraphPadPrism 8, thereby obtaining the IC50 value of each siRNA.

[0214] The fitting formula is: Y = 100 / (1 + 10^((LogIC50 - X) × HillSlope))

[0215] Where HillSlope represents the slope of the percentage inhibition rate curve.

[0216] 3.3 Experimental Results

[0217] The IC50 assay results of the siRNA of this invention are shown in Table 8 below:

[0218] Table 8. IC50 assay results of siRNA in RH30 cell line

[0219]

[0220]

[0221] The results of IC50 assays using the RH30 cell line showed that some of the siRNAs of this invention can significantly inhibit MSTN gene expression, and these sequences all have good in vitro activity.

[0222] Example 4. Cytotoxicity of siRNA conjugates

[0223] 4.1 Experimental Procedure

[0224] 4.1.1 Cell Culture

[0225] RH30 cells were cultured in IMDM medium (Eallbio, with 10% FBS and 1% PS) at 37°C and 5% CO2. When the confluence reached 80%–90%, the cells were digested with trypsin, centrifuged, resuspended in DMEM high-glucose medium (Eallbio, with 10% FBS), counted, and transfected.

[0226] 4.1.2 RH30 cell transfection

[0227] Based on the above results regarding cell viability in RH30 cells, 17 siRNA conjugates were selected for cytotoxicity assays. RH30 cells were transfected using a method similar to that in Example 2, with 15,000 RH30 cells seeded per well. After 72 hours of culture, the cytotoxicity of each siRNA conjugate was measured by determining the cell viability / cytotoxicity ratio in each sample, with the transfected siRNA conjugate concentrations being 50 nM and 5 nM, respectively. Cell viability was measured by determining intracellular ATP content using a CellTiter-Glo (Promega, catalog number G7570) assay, according to the manufacturer's protocol. ToxiLight was used according to the manufacturer's protocol. TM (Lonza, catalog number LT07-217) Measures cytotoxicity in the supernatant.

[0228] 4.2 Experimental Results

[0229] The cytotoxicity of siRNA conjugates can be found in [link to documentation]. Figure 1 .

[0230] The results showed that some of the siRNA conjugates of the present invention exhibited low cytotoxicity and good cell compatibility.

[0231] Example 5. Screening of siRNA conjugates for in vivo inhibition of target gene activity in muscle

[0232] 5.1 siRNA sequence modification

[0233] Based on the in vitro screening results of Examples 2-4 above, some siRNA sequences with good in vitro activity were selected from Table 2, and their conjugates were used to verify their in vivo activity.

[0234] 5.2 Experimental Procedure

[0235] Six- to eight-week-old wild-type male mice (C57BL / 6J) were purchased from SPAF (Beijing) Biotechnology Co., Ltd., with six mice in each group. Mice were injected with 15 mg / kg of the siRNA conjugate on day 0, while the negative control group received an equal volume of siCtrlM1. Quadriceps muscle samples were collected on days 7 (D7) and 14 (D14) after drug administration, and the total RNA obtained was analyzed by qRT-PCR. Based on the qPCR results, combined with the results of cytotoxicity and immunogenicity assays, the optimal siRNA conjugate was selected, and quadriceps muscle samples were collected on day 28 (D28). The total RNA obtained from these mice was then analyzed by qRT-PCR.

[0236] 5.3 Experimental Results

[0237] The in vivo activity screening results of siRNA conjugates are shown in the figure. Figure 2 .

[0238] Depend on Figure 2 The results showed that within 14 days of injection of the siRNA conjugate drug, the knockdown effects of YG-54M1, YG-95M1, YG-96M1, YG-97M1, YG-105M1, and YG-106M1 were significant in mice. Mice in this group were further observed for up to 28 days, and the relative expression level of the MSTN gene in the quadriceps femoris muscle was measured. The results are as follows: Figure 3 As shown. By Figure 3 The results showed that the knockdown of YG-95M1, YG-96M1, YG-97M1, YG-105M1, and YG-106M1 remained very effective in mice on day 28.

[0239] Example 6. In vivo inhibition of serum hMSTN expression levels by siRNA conjugates with modified patterns M1 or M1'.

[0240] Based on the in vivo screening results of Example 5, siRNA conjugate sequences with good in vivo activity were selected, and the serum content of the target protein hMSTN was detected.

[0241] 6.1 Experimental Procedure

[0242] Six male mice aged 6-8 weeks were administered the conjugate at a dose of 15 mg / kg subcutaneously. Blood was collected from the orbital venous plexus of the mice on days 14 (D14) and 28 (D28) after administration, and serum was separated. The expression level of hMSTN in the serum was detected by an ELISA kit (R&D Systems, DGDF80).

[0243] 6.2 Experimental Results

[0244] The results of siRNA conjugate inhibiting serum hMSTN expression levels are shown in the figure. Figure 4 .

[0245] The results showed that YG-95M1, YG-96M1, YG-97M1, YG-105M1, and YG-106M1 could significantly inhibit the expression level of hMSTN in serum.

[0246] Example 7. In vivo inhibition of serum hMSTN expression levels by siRNA conjugates with modified patterns M2 or M2' 7.1 Experimental Procedure

[0247] Following the experimental steps in Example 6, in vivo activity tests were performed on some of the siRNA conjugate sequences in Table 3.

[0248] 7.2 Experimental Results

[0249] The results of the inhibition of serum hMSTN expression levels by siRNA conjugates are shown in the figure. Figure 5 .

[0250] The results showed that YG-95M2, YG-96M2, YG-97M2, YG-105M2, and YG-106M2 could also significantly inhibit the expression level of hMSTN in serum, and their inhibitory effect was better than that of the corresponding M1 modified siRNAs.

[0251] Example 8. In vivo inhibition of serum hMSTN expression levels by siRNA conjugates with modified patterns M3 or M3' 8.1 Experimental procedures

[0252] Following the experimental steps in Example 6, in vivo activity tests were performed on some of the siRNA conjugate sequences in Table 4.

[0253] 8.2 Experimental Results

[0254] The results of the inhibition of serum hMSTN expression levels by siRNA conjugates are shown in the figure. Figure 6 .

[0255] The results showed that YG-95M3, YG-96M3, YG-97M3, YG-105M3, and YG-106M3 could also significantly inhibit the expression level of hMSTN in serum, and their inhibitory effect was better than that of the corresponding M1 modified siRNAs.

[0256] Example 9. In vivo inhibition of serum hMSTN expression levels by siRNA conjugates with modified patterns M4 or M4' 9.1 Experimental Procedure

[0257] Following the experimental steps in Example 6, in vivo activity tests were performed on some of the siRNA conjugate sequences in Table 5.

[0258] 9.2 Experimental Results

[0259] The results of the inhibition of serum hMSTN expression levels by siRNA conjugates are shown in the figure. Figure 7 .

[0260] The results showed that YG-95M4, YG-96M4, YG-97M4, YG-105M4, and YG-106M4 could also significantly inhibit the expression level of hMSTN in serum, and their inhibitory effect was better than that of the corresponding M1 modified siRNAs.

[0261] Example 10. In vivo inhibition of serum hMSTN expression levels by siRNA conjugates with modified M5 or M5' patterns. 10.1 Experimental Procedure

[0262] Following the experimental steps in Example 6, in vivo activity tests were performed on some of the siRNA conjugate sequences in Table 6.

[0263] 10.2 Experimental Results

[0264] The results of the inhibition of serum hMSTN expression levels by siRNA conjugates are shown in the figure. Figure 8 .

[0265] The results showed that YG-95M5, YG-96M5, YG-97M5, YG-105M5, and YG-106M5 could also significantly inhibit the expression level of hMSTN in serum, and their inhibitory effect was better than that of the corresponding M1 modified siRNAs.

Claims

1. A siRNA for inhibiting MSTN gene expression, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising nucleotide sequence I, and the antisense strand comprising nucleotide sequence II; each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified or unmodified nucleotide; nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; nucleotide sequence I is substantially identical to a first nucleotide sequence, the first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length of the mRNA expressing the MSTN gene, the mRNA expressing the MSTN gene being as shown in SEQ ID NO:

1.

2. The siRNA for inhibiting MSTN gene expression as described in claim 1, wherein the nucleotide sequence II is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the first nucleotide sequence.

3. The siRNA for inhibiting MSTN gene expression as described in claim 2, wherein nucleotide sequence I comprises at least 15 consecutive nucleotides of SEQ ID NO:2-109; and nucleotide sequence II comprises at least 15 consecutive nucleotides of SEQ ID NO:110-217.

4. The siRNA for inhibiting MSTN gene expression as described in claim 3, wherein nucleotide sequence I comprises at least 15 consecutive nucleotides of the following nucleotide sequences: SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:106, or SEQ ID NO:107; and nucleotide sequence II comprises at least 15 consecutive nucleotides of the following nucleotide sequences: SEQ ID:204, SEQ ID NO:205, SEQ ID:206, SEQ ID NO:214, or SEQ ID NO:

215.

5. The siRNA for inhibiting MSTN gene expression as described in claim 3, wherein the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence SEQ ID NO: 2-109 or has no more than 1, 2, or 3 base differences, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence SEQ ID NO: 110-217 or has no more than 1, 2, or 3 base differences; the sense strand is 16-23 nucleotides in length, and the antisense strand is 19-26 nucleotides in length.

6. The siRNA for inhibiting MSTN gene expression as described in claim 5, wherein the sense strand comprises at least 19 consecutive nucleotides of the nucleotide sequence SEQ ID NO: 2-109 or has no more than 1, 2 or 3 base differences, and the antisense strand comprises at least 19 consecutive nucleotides of the nucleotide sequence SEQ ID NO: 110-217 or has no more than 1, 2 or 3 base differences; the lengths of the sense and antisense strands of the siRNA are 19 / 21 or 21 / 23.

7. The siRNA for inhibiting MSTN gene expression as described in claim 1 or 2, wherein each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified nucleotide, and the modified nucleotide is a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

8. The siRNA for inhibiting MSTN gene expression according to claim 7, wherein the non-fluorinated modification is a methoxylated nucleotide or a deoxyribonucleotide.

9. The siRNA for inhibiting MSTN gene expression as described in claim 1 or 2, wherein at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA are phosphate ester groups with modifying groups; wherein the phosphate ester group with modifying groups is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom.

10. The siRNA for inhibiting MSTN gene expression as described in claim 1 or 2, wherein the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analogue.

11. An siRNA conjugate comprising the siRNA of claim 1 or 2 and a conjugation group conjugated to the siRNA, the conjugation group targeting muscle cells.

12. The conjugate of claim 11, wherein the conjugation site of the siRNA and the conjugate group is at the 3' end of the positive strand of the siRNA, and the structure of the conjugate group is as follows:

13. The siRNA conjugate as described in claim 12, The positive strand of the siRNA conjugate is selected from the nucleotide sequence shown in the following formula: 5′-XmsXmsXmXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3′, or 5′-XmsXmsXmXmXmXmXfXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3′, or 5′-XmsXmsXmXmXfXmXfXmXmXmXmXmXmXmXmXm-Cholesteryl-3′, or 5′-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXm-Cho lesteryl-3'; or 5'-XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'; XdXfXm The antisense strand in the siRNA conjugate is selected from the nucleotide sequence shown in the following formula: 5'-VPXmsXfsXmXmXmXm XmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXmXmXmXm XmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXfXmXfXfXmXmXmXmXf -3', or 5' -VPXmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmsXmsXm XmXfXmXfXmXmXmXmXmXmsXmsXm-3'; or 5'-VPXms sXfsXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm-3'; or 5'-VPXms mXmXmsXmsXm-3'; or 5'-VPXms Xm-3'; in, Xm represents any nucleotide modified with 2'-methoxy, Xf represents any nucleotide modified with 2'-fluoride, lowercase s indicates that the two nucleotides adjacent to s are linked by thiophosphate subunits, VP indicates that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinylphosphonate (5'-(E)-vinylphosphonate, E-VP); Cholesteryl represents the structure shown in Formula I.

14. The siRNA conjugate of claim 13, wherein the sense and antisense strands in the conjugate are a combination of the following: M-1: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXm-Cholestery l-3' Antisense chain: 5′-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXmXfXmXmXmXmXmXmXmXmsXmsXm-3′; or M-1' Sense strand: 5′-XmsXmsXmXmXf Or M-2: Justice chain: 5′-XmsXmsXmXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm-Cholester yl-3′ Antisense strand: 5′-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsX m-3'; or M-2' Justice Chain: 5′-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3′ Antisense Chain: 5′-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXmsXm-3′; or M3: Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholester yl-3′ Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXmXfXmXmXmXmXmXmXmXmsXmsXm-3'; or M3' Justice Chain: 5′-XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXm-Cholesteryl-3′ Antisense Chain: 5′-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXmXfXmXmXmXmXm-3′; or M4: Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholester yl-3′ Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXfXmXmXmXmXmXmXm-3'; or M4' Justice Chain: 5′-XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXm-Cholesteryl-3′ Antisense Chain: 5′-VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXmXmXm-3′; or M5: Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholester yl-3' Antonyms: 5′-VPXmsXfsXmXmXmXfXmXfXmXmXmXfXmXmXfXmXmXmXmXmXmXmsXmsXm-3′; or M5′ Sense strand: 5′-XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3′Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXfXf 15. A composition comprising the siRNA of claim 1 or 2 or the siRNA conjugate of claim 11 or 12.

16. Use of the siRNA of claim 1 or 2, or the siRNA conjugate of claim 11 or 12, or the composition of claim 15 in the preparation of a medicament for the prevention and / or treatment of diseases associated with MSTN gene expression.

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