siRNA for inhibiting MSTN gene expression and its conjugate and application

Abstract

The present invention provides an siRNA for inhibiting the expression of MSTN gene and its conjugate and application, wherein the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified nucleotide. The siRNA and its conjugate and pharmaceutical composition provided by the present invention have strong ability to inhibit the MSTN gene, can significantly reduce the expression amount of MSTN mRNA, and have low drug toxicity.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to siRNA for inhibiting MSTN gene expression and its conjugate and application. Background Art

[0002] Obesity is a chronic, recurrent disease caused by excessive accumulation or distribution of adipose tissue and abnormal function due to genetic and environmental factors. Currently, more than one-third of adults in the world are classified as overweight and obese, and the total number of obese children, adolescents and adults has exceeded 1 billion. The obesity problem is very serious. The obesity rate of Chinese women has increased from 2.0% in 1990 to 7.8% in 2022; the obesity rate of men has increased from 0.8% to 8.9%. Obese people will face a series of chronic complications, including abnormal glucose metabolism, dyslipidemia, hypertension, polycystic ovary syndrome, obstructive sleep apnea syndrome and other related diseases.

[0003] Myopathy is a non-inflammatory disease originating in skeletal muscle or neuromuscular junction, referred to as myopathy. The main manifestations are decreased or disappeared muscle contraction force and muscle atrophy. Neuromuscular disease is a group of muscle diseases with muscle and bulbar muscle weakness and muscle atrophy as common clinical features. Patients will have related symptoms such as muscle atrophy, muscle strength changes, and neuromuscular head contact lesions. Among them, spinal muscular atrophy (SMA) is a type of single gene recessive genetic disease. The typical manifestations of SMA are generalized muscle weakness, low muscle tension, muscle atrophy, and limited motor functions such as normal standing and walking. It is often accompanied by lung infection, joint contracture, and hip subluxation. Severe children often die of respiratory failure. The pathogenic gene carrier rate of SMA is very high. One in every 40-50 people is a carrier of the pathogenic gene, and the neonatal incidence rate is about 1 / 10000.

[0004] Myostatin (MSTN) belongs to the transforming growth factor β (TGFβ) superfamily and is a key regulator of skeletal muscle mass. MSTN controls muscle volume by inhibiting the proliferation of muscle cells, resulting in a decrease in muscle mass, which in turn regulates skeletal muscle growth. MSTN levels may increase under many disease conditions such as muscular dystrophy and obesity. Studies have found that MSTN knockout mice show increased muscle mass, reduced fat deposition, improved insulin sensitivity, enhanced fatty acid oxidation, and anti-obesity characteristics. Studies have found that MSTN can affect the body's lipid metabolism and can reduce obesity by inhibiting fat deposition. The mRNA and protein levels of MSTN in the muscle and adipose tissue of leptin-deficient obese mice and wild mice on a high-fat diet are increased. Therefore, MSTN has become an important target for obesity, skeletal muscle disease, and neuromuscular disease.

[0005] Traditional weight loss drugs can cause nausea, vomiting, gastric paralysis, and in severe cases, pancreatitis, intestinal obstruction and other side effects. Although GLP-1 weight loss drugs are effective in reducing weight, they are also accompanied by many side effects. In addition to common side effects such as nausea and bloating, there will also be weight rebound and muscle loss after stopping the drug, and even cause blindness without warning, increased suicidal tendencies and other serious side effects. Therefore, increasing muscle mass and reducing fat mass to treat obesity has become the preferred goal of the research and development of a new generation of weight loss drugs.

[0006] The treatment of muscle disease requires increasing muscle mass and muscle strength to target muscle atrophy, but related therapeutic drugs are not yet ideal for the treatment of muscle atrophy. Summary of the invention

[0007] The purpose of the present invention is to provide a siRNA molecule capable of inhibiting the expression of the MSTN gene, in order to provide a new treatment for MSTN-related obesity, skeletal muscle diseases, neuromuscular diseases and the like.

[0008] In a first aspect, the present invention provides an siRNA for inhibiting the expression of the MSTN gene, wherein the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified nucleotide; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complemented to form a double-stranded region; the nucleotide sequence I is substantially identical to a first nucleotide sequence, wherein the first nucleotide sequence is a nucleotide sequence of at least 15 nucleotides in length in the mRNA expressed by the MSTN gene, and preferably, the first nucleotide sequence is a nucleotide sequence of 15 to 25 nucleotides in length in the mRNA expressed by the MSTN gene, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides;

[0009] Preferably, the mRNA expressed by the MSTN gene is as shown in NCBI refseqID NM_005259.3; specifically, the mRNA sequence is as shown in SEQ ID NO: 1.

[0010] In some embodiments, the nucleotide sequence I has a difference of 1 nucleotide with the first nucleotide sequence; preferably, the difference of 1 nucleotide is Z1 located at the 3' end, Z1 is optionally A, U, C or G, but is different from the nucleotide at the corresponding position of the first nucleotide sequence; also preferably, the nucleotide sequence II has Z2 complementary to Z1 at the 5' end.

[0011] In some embodiments, in the above siRNA, the nucleotide sequence II is substantially reverse complementary, essentially reverse complementary, or completely reverse complementary to the first nucleotide sequence.

[0012] In some embodiments, in any of the above-described siRNAs, the sense strand and antisense strand are the same or different in length, the sense strand is 16-23 nucleotides long, and the antisense strand is 19-26 nucleotides long. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21, 21 / 23, or 19 / 24.

[0013] In some embodiments, in any of the above-mentioned 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 section of nucleotides.

[0014] In some embodiments, in any of the above-mentioned siRNAs, the nucleotide sequence I comprises at least 15 consecutive nucleotides 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.

[0015] In some embodiments, in any of the above siRNAs, the nucleotide sequence II comprises at least 15 consecutive nucleotides 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.

[0016] In some embodiments, in any of the above-mentioned siRNAs, the sense strand comprises the sense strand of any one of the siRNAs shown in Table 1 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA in Table 1.

[0017] Preferably, in any of the above siRNAs, 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, the 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 3 bases.

[0018] Preferably, in any of the above siRNAs, the sense strand is equal in length to the sense strand of any of the siRNAs shown in Table 1 and has a difference of 1 nucleotide; preferably, the difference of 1 nucleotide is Z1 located at the 3' end, and Z1 is optionally A, U, C or G. Also preferably, in any of the above siRNAs, the antisense strand has Z2 complementary to Z1 at the 5' end.

[0019] In some embodiments, in any of the above siRNAs, each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified nucleotide, and the modified nucleotide is a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

[0020] In some embodiments, the fluorinated modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is replaced by fluorine, and has a structure shown in the following formula (1); the non-fluorinated modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is replaced by a non-fluorinated group. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is replaced by a non-fluorinated group. These nucleotides or nucleotide analogs in which the hydroxyl group at the 2'-position of the ribose group is replaced by a non-fluorinated group are well known to those skilled in the art, and these nucleotides or nucleotide analogs can be selected from one of 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, for example, can be a 2'-O-methoxyethyl modified nucleotide (2'-MOE), as shown in formula (3). In some embodiments, the 2'-amino modified nucleotide (2'-NH2) is as shown in formula (4). In some embodiments, the 2'-deoxynucleotide (DNA) is as shown in formula (5).

[0021]

[0022] Nucleotide analogs refer to groups that can replace nucleotides in nucleic acids but have structures different from adenine ribonucleoside, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine ribonucleotide. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleotides or acyclic nucleotides.

[0023] A bridging nucleotide refers to a constrained or inaccessible nucleotide. The bridging nucleotide may contain a five-membered ring, a six-membered ring, or a seven-membered ring with a fixed C3-endosugar condensation. In some embodiments, the bridging nucleotide may 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).

[0024]

[0025] Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein UNA is shown in formula (9) and GNA is shown in formula (10).

[0026]

[0027] In the above formulae (9) and (10), R is selected from H, OH or alkoxy (O-alkyl).

[0028] Isonucleotides refer to compounds formed by a change in the position of a base on the ribose ring of a nucleotide. In some embodiments, an isonucleotide may be a compound formed by a base moving from the 1' position to the 2' position or the 3' position of the ribose ring, as shown in formula (11) or formula (12).

[0029]

[0030] In formula (11) and formula (12), R is selected from H, OH, F or the non-fluorine group as described above.

[0031] In formula (1) to formula (12), Base represents a base.

[0032] In some embodiments, in any of the above siRNAs, in the direction from the 5' end to the 3' end, one or more nucleotides at positions 5, 7, 8, 9, 10, and 11 of the nucleotide sequence I are fluorinated modified nucleotides, and one or two nucleotides at positions 8 and 10 are deoxyribonucleotides; and in the direction from the 5' end to the 3' end, one or more nucleotides at positions 2, 6, 8, 9, 12, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides;

[0033] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10 and 11 of the nucleotide sequence I are fluorinated modified nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence II are fluorinated modified nucleotides; or

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

[0035] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9 and 11 of the nucleotide sequence I are fluorinated modified nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 14 and 16 of the nucleotide sequence II are fluorinated modified nucleotides;

[0036] Or 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 modified nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides;

[0037] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9 and 11 of the nucleotide sequence I are fluorinated modified 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 the nucleotide sequence II are fluorinated modified nucleotides; or

[0038] 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 modified 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 the nucleotide sequence II are fluorinated modified nucleotides; or

[0039] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9 and 11 of the nucleotide sequence I are fluorinated modified 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 the nucleotide sequence II are fluorinated modified nucleotides; or

[0040] 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 modified 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 the nucleotide sequence II are fluorinated modified nucleotides; or

[0041] Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9 and 11 of the nucleotide sequence I are fluorinated modified 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 the nucleotide sequence II are fluorinated modified nucleotides; or

[0042] 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 modified 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 modified nucleotides.

[0043] In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand of the siRNA is a phosphate group having a modified group. In some embodiments, the phosphate group having a modified group is a thiophosphate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom. In some embodiments, the phosphate group having a modified group is a thiophosphate group having a structure as shown in formula (13).

[0044]

[0045] In some embodiments, in any of the above siRNAs, the phosphorothioate linkage is present in at least one of the following positions: between the 1st and 2nd nucleotides of the sense strand and / or the antisense strand; between the 2nd and 3rd nucleotides of the sense strand and / or the antisense strand; 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;

[0046] In some embodiments, in any of the above 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 formula (14), formula (15) and formula (16).

[0047]

[0048] In a second aspect, the present invention provides a siRNA conjugate, comprising any of the above-described siRNAs and a conjugated 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 conjugate group may be at the 3' end or 5' end of the siRNA sense strand, or at the 3' end of the antisense strand, or in the internal sequence of the siRNA.

[0049] In some embodiments, in the above siRNA conjugate, the conjugated group is cholesterol, and the structure is shown below:

[0050]

[0051] Formula (I).

[0052] The siRNA conjugate, the sense strand is selected from the nucleotide sequence shown in the following formula: 5'-XmsXmsXmXmXfXmXfXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3', or 5'-XmsXmsXmXmXmXfXmXfXmXmXfXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3', or 5'-XmsXmsXmXmXfXmXfXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'. 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'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXm-3', or 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXm-3', or 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmsXm-3'. mXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXm mXmXfXmXfXmXmXmsXmsXm-3'; or 5'-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmXmXm sXmsXm-3'; or 5'-VPXmsXfsXmXmXmXmXfXmXmXfXmXmXmXm sXfsXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm-3'; or 5'-VPXmsXfsXmXmXmXmXfXmX fXmXmXmXmXmXfXmXfXmXmXmsXmsXm-3'; or 5'-VPXmsXfsXmXmXmXfXmXfXfXmXmXfXmXfXmXfX mXmXmXmXmsXmsXm-3'; or 5'-VPXmsXfsXmXmXmXfXmXfXfXmXmXfXmXfXmXfXmXmXmsXmsXm-3'.

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

[0055] In some embodiments, the sense strand and antisense strand are in the following combination:

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

[0057] Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'

[0058] Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXfXmXmXmXfXmXmXfXmXmXmXmXmXmsXm-3′;

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

[0060] Justice Chain: 5'-XmsXmsXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'

[0061] Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXfXmXmXmXfXmXmXfXmXmXmXmsXm-3′;

[0062] or M-2: (21 / 23)

[0063] Justice chain: 5' -XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm -Cholesteryl -3'

[0064] Antisense strand: 5′-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmXmsXm-3′;

[0065] or M-2' (19 / 21)

[0066] Justice Chain: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'

[0067] Antisense strand: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXm-3'; or M3: (21 / 23)

[0068] Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'

[0069] Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXmXmXmXfXmXmXmXfXmXmXmXmXmXmsXm-3′;

[0070] or M3' (19 / 21)

[0071] Justice chain: 5'-XmsXmsXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'

[0072] Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXmXmXmXmXfXmXmXfXmXmXmXmsXm-3′;

[0073] or M4: (21 / 23)

[0074] Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'

[0075] Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXmXmXmXmXfXmXmXmXfXmXmXmXmXmXmsXm-3′;

[0076] or M4' (19 / 21)

[0077] Justice chain: 5'-XmsXmsXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'

[0078] Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXmXmXmXmXfXmXmXfXmXmXmXmsXm-3′;

[0079] or M5: (21 / 23)

[0080] Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'

[0081] Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXfXmXfXmXfXmXfXmXfXmXmXmXmXmXmsXm-3′;

[0082] or M5' (19 / 21)

[0083] Justice chain: 5'-XmsXmsXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3'

[0084] Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXfXmXfXmXfXmXfXmXfXmXmXmXmsXm-3′.

[0085] In some embodiments, the siRNA conjugate comprises a sense strand and an antisense strand, wherein the sense strand comprises the sense strand of any one of the siRNA conjugates shown in Tables 2 to 6 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate.

[0086] 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.

[0087] In a third aspect, the present invention provides a composition comprising any one of the above-mentioned siRNAs or siRNA conjugates.

[0088] In some embodiments, the composition is a pharmaceutical composition, and further comprises a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier or excipient involved in the present invention includes, but is 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, distearoylphosphatidylcholine, 1,2-dimyristyl glyceride, and dimethyl adipate.

[0089] In a fourth aspect, the present invention provides use of any of the above siRNAs, siRNA conjugates or pharmaceutical compositions in the preparation of a drug for preventing and / or treating a disease associated with MSTN expression.

[0090] In a fifth aspect, the present invention provides a method for preventing or treating a disease associated with MSTN expression, the method comprising administering the siRNA, siRNA conjugate or pharmaceutical composition of the present invention to a subject.

[0091] In some embodiments, the diseases associated with elevated MSTN levels include obesity and obesity-related diseases, muscle diseases such as skeletal muscle diseases, and neuromuscular diseases.

[0092] In some embodiments, the diseases associated with increased MSTN levels include obesity and obesity-related diseases, muscle diseases, and the obesity-related diseases include but are not limited to dysglycemia, dyslipidemia, hypertension, metabolic-related fatty liver disease, obstructive sleep apnea syndrome, polycystic ovary syndrome, cardiovascular disease, etc.; the muscle diseases include, for example, myeloid muscular atrophy, metabolic myopathy, Duchenne muscular dystrophy, mitochondrial myopathy, polymyositis, Guillain-Barre syndrome, myasthenia gravis, etc.

[0093] The pharmaceutical composition of the present invention can be used alone to treat obesity, skeletal muscle and neuromuscular diseases, or in combination with standard oral weight loss, skeletal muscle and neuromuscular disease drugs, providing experimental support for the implementation of diversified treatment plans for the above patients.

[0094] The usual suitable dosage range of the siRNA, siRNA conjugate or pharmaceutical composition for inhibiting MSTN gene expression of the present invention will be about 0.1 mg / kg to about 30.0 mg / kg, preferably about 5 mg / kg to about 15.0 mg / kg, based on the amount of siRNA contained therein.

[0095] The administration routes involved in the present 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 buccal administration and sublingual administration).

[0096] The siRNA and its conjugates and pharmaceutical compositions of the present invention can specifically target muscles, complementarily pair with muscle MSTN mRNA sequences, induce MSTN mRNA degradation, and thus inhibit the synthesis of MSTN genes in muscles, leading to a lasting reduction in MSTN protein, exerting a lasting muscle-building effect, while avoiding off-target effects, having low cytotoxicity, and being able to fundamentally prevent or treat MSTN-related obesity, skeletal muscle diseases, neuromuscular diseases and other related diseases.

[0097] In addition, compared with traditional small molecule drugs and antibody drugs, small nucleic acid drugs can directly regulate upstream gene expression 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 dosing is low (the drug can be given once every six months), and patient compliance is good.

[0098] Beneficial technical effects

[0099] The siRNA and its conjugates and pharmaceutical compositions provided by the present invention have strong inhibitory ability on the MSTN gene, can significantly reduce the expression of MSTN mRNA, and have low drug toxicity. Therefore, the siRNA and its conjugates and pharmaceutical compositions of the present invention can effectively prevent and / or treat diseases associated with MSTN expression, provide patients with more effective, safe and convenient therapeutic drugs, and have good drug development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 The siRNA conjugate cytotoxicity test results in Example 4 of the present invention;

[0101] Figure 2 The results of the siRNA conjugate in Example 5 of the present invention inhibiting the expression of MSTN gene in vivo (D7 and D14);

[0102] Figure 3 The results of the siRNA conjugate in Example 5 of the present invention inhibiting the expression of MSTN gene in vivo (D7, D14 and D28);

[0103] Figure 4 The results of the siRNA conjugate in Example 6 of the present invention inhibiting the expression of MSTN protein in vivo;

[0104] Figure 5 The results of the siRNA conjugate in Example 7 of the present invention inhibiting the expression of MSTN protein in vivo;

[0105] Figure 6 The results of the siRNA conjugate in Example 8 of the present invention inhibiting the expression of MSTN protein in vivo;

[0106] Figure 7 The results of the siRNA conjugate in Example 9 of the present invention inhibiting the expression of MSTN protein in vivo;

[0107] Figure 8 This is the result of the siRNA conjugate in Example 10 of the present invention inhibiting the expression of MSTN protein in vivo. DETAILED DESCRIPTION

[0108] definition

[0109] In the above and below, unless otherwise specified, capital letters C, G, U, A represent cytosine, guanine, uracil, and adenine nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorinated-modified nucleotide; lowercase letter d represents that the nucleotide adjacent to the left of the letter d is a deoxynucleotide; lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by thiophosphate subunits; the letter combination VP represents that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinylphosphonate (5'-(E)-vinylphosphonate, E-VP); Cholesteryl has the structure of formula (I) and is connected to the 3' end of the sense chain through a phosphate bond.

[0110] In the above and below, the "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and the "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorine group or deoxygenated. "Nucleotide analog" refers to a group that can replace nucleotides in nucleic acids but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide. Such as isonucleotides, bridged nucleic acids (BNA) or acyclic nucleotides. The "methoxy-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group is replaced by a methoxy group.

[0111] In the context of this article, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are each paired with the bases on the other strand in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one strand is always paired with thymine (or uracil) on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" means in the art that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary form.

[0112] As used herein, "including" is intended to be used interchangeably with the phrase "including but not limited to." The term "or" is used herein to mean and is interchangeable with the term "and / or" unless the context clearly indicates otherwise.

[0113] In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; "completely reverse complementary" means that there is no base mismatch between the two nucleotide sequences. In the above and below, especially when describing the preparation method of the siRNA, pharmaceutical composition or siRNA conjugate of the present invention, unless otherwise specified, the nucleoside monomer (nucleoside monomer) refers to the modified or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites) used in phosphoramidite solid phase synthesis according to the type and order of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid phase synthesis is a method used in siRNA synthesis known to those skilled in the art. The nucleoside monomers used in the present invention are all commercially available.

[0114] The term "corresponding siRNA" used in the context of the present invention refers to the same siRNA mentioned above, for example, when it is stated that "the sense strand comprises the sense strand of any siRNA shown in Table 1 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA", it means that the sense strand and antisense strand contained 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 siRNA conjugate shown in Table 2 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate", it means that the sense strand and antisense strand contained are from the same siRNA conjugate shown in Table 2 herein. In addition, in these contexts, "comprising" includes the case where it consists of these sequences.

[0115] Example

[0116] 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 examples (although representing specific embodiments of the present invention) are given for illustrative purposes only, because after reading the detailed description, various changes and modifications made within the spirit and scope of the present invention will become apparent to those skilled in the art.

[0117] The experimental techniques and methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0118] Example 1. siRNA design and synthesis

[0119] 1.1 siRNA design

[0120] OligoWalk was used to design a set of siRNAs targeting the human MSTN gene (human MSTN gene: NCBIrefseqID NM_005259.3; NCBI Gene ID: 2660). The human NM_005259.3 REFSEQ mRNA has a length of 2819 bases. At the same time, in order to avoid any sequence toxicity, sequences similar to human genes should be excluded.

[0121] Human NM_005259.3 REFSEQ mRNA (NCBI refseqID NM_005259.3):

[0122]

[0123] 1.2 siRNA sequence synthesis

[0124] siRNAs, including negative control siRNA (siCtrl), were synthesized according to standard oligonucleotide solid-phase synthesis protocols.

[0125] Oligonucleotide solid phase synthesis scheme: Use commercially available 5'DMT2'TBDMSrU phosphoramidite monomer, 5'DMT2'TBDMSrA (Bz) phosphoramidite monomer, 5'DMT2'TBDMSrC (Ac) phosphoramidite monomer, 5'DMT2'TBDMSrG (iBu) phosphoramidite monomer. Synthesize RNA at a scale of 500nM. Prepare the phosphoramidite solution at a concentration of 50mM, and use 0.3M benzylthiotetrazolium (BTT) acetonitrile solution as an activator. During the synthesis process, 0.1M oxidizing agent (pyridine: THF: water = 20: 78: 2) is used to convert trivalent phosphorus into a pentavalent phosphorus-stabilized phosphate backbone. After the synthesis is completed, the sequence is aminolyzed from the solid phase support and precipitated. Remove the 2'O tert-butyldimethylsilyl protecting group of 2' with triethylamine trihydrofluoric acid.

[0126] For the RNA sequence that has been synthesized, use ammonia water: methylamine = 1:1 aminolysis solution for 40 minutes at 55°C. After the aminolysis is completed, remove the solid phase carrier CPG powder and take the supernatant to dry. Add a protective group removal agent, react at 60°C for 2 hours, add n-butanol at a ratio of 1:5, stand at 20°C for 30 minutes, and centrifuge to obtain a precipitate. Add RNase-free water to dissolve and purify with reverse phase chromatography (0.1M triethylamine acetic acid (TEAA) and acetonitrile). The purified sample is ultrafiltered and desalted with PBS. Annealing is performed to obtain siRNA, and the obtained siRNA is verified. The results show that the target siRNA was successfully prepared.

[0127] 1.3 siRNA sequence modification and conjugate synthesis

[0128] The modified siRNA is synthesized according to the oligonucleotide solid phase synthesis scheme. The modified nucleotide group can be introduced into the siRNA of the present invention by using a nucleoside monomer with a corresponding modification. The method for preparing a nucleoside monomer with a corresponding modification is also well known to those skilled in the art. Cholesterol is conjugated to siRNA to synthesize siRNA conjugates with reference to the synthesis method disclosed in WO2019217459A1.

[0129] The structure of the conjugated group is shown below:

[0130]

[0131] Formula (I).

[0132] Annealing of oligoribonucleotides to produce siRNA conjugates: Prepare the RNA oligomers to be annealed with sterile RNase Free (RNA hydrolase-free) water to a 200 μM solution. Set up the annealing reaction system as follows: place the above solution (duplex concentration is 10 nM) with a total volume of 100 μL in a 95°C water bath for 10 minutes (≥100 nM requires high temperature for 20 minutes) → quickly place in a 60°C water bath to cool naturally → store the annealed solution at 4°C. Mix the complementary chains by combining equimolar RNA solutions. After identification, the siRNA molecule was constructed correctly. Prepare the siRNA solution into dry powder for use.

[0133] The sequences of the synthesized siRNA molecules are shown in Table 1 below:

[0134] Table 1. List of siRNA sequences targeting MSTN

[0135]

[0136]

[0137]

[0138] Table 2. Sequence list of siRNA conjugates targeting MSTN (M1 or M1' modification pattern)

[0139]

[0140]

[0141]

[0142] Note 1: YG-96M1 and YG-97M1 in the table are M1' modification patterns, and the remaining sequences are M1 modification patterns

[0143] Note 2: siCtrlM1 sense chain: XmsXmsXmXmXmXmXfXmXfXfXmXmXmXmXmXmXmXmdTdT-Cholesteryl; antisense chain: VPXmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXmXmXmXmsdTdT.

[0144] Table 3. Sequence list of siRNA conjugates targeting MSTN (M2 or M2' modification pattern)

[0145]

[0146]

[0147]

[0148] Note 1: YG-96M2 and YG-97M2 in the table are M2' modification patterns, and the remaining sequences are M2 modification patterns

[0149] Note 2: siCtrlM2 sense chain: XmsXmsXmXmXmXmXfXmXfXmXmXmXmXmXmXmXmXmdTdT-Cholesteryl; antisense chain: VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmdTdT.

[0150] Table 4 Sequence list of siRNA conjugates targeting MSTN (M3 or M3' modification mode)

[0151]

[0152]

[0153]

[0154] Note 1: YG-96M3 and YG-97M3 in the table are M3' modification patterns, and the remaining sequences are M3 modification patterns

[0155] Note 2: siCtrlM3 sense chain: XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmdTdT-Cholesteryl; antisense chain: VPXmsXfsXmXmXmXfXmXmXmXfXmXmXmXfXmXmXmXmdTdT.

[0156] Table 5 Sequence list of siRNA conjugates targeting MSTN (M4 or M4' modification mode)

[0157]

[0158]

[0159]

[0160] Note 1: YG-96M4 and YG-97M4 in the table are M4' modification patterns, and the remaining sequences are M4 modification patterns

[0161] Note 2: siCtrlM4 sense chain: XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmdTdT-Cholesteryl; antisense chain: VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXmXfXmXmXmXmdTdT.

[0162] Table 6 Sequence list of siRNA conjugates targeting MSTN (M5 or M5' modification mode)

[0163]

[0164]

[0165]

[0166] Note 1: In the table, YG-96M5 and YG-97M5 are M5' modification patterns, and the remaining sequences are M5 modification patterns

[0167] Note 2: siCtrlM4 sense chain: XmsXmsXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmdTdT-Cholesteryl; antisense chain: VPXmsXfsXmXmXmXfXmXfXfXmXfXmXfXmXfXmXmXmsXmdTdT.

[0168] Among them, capital letters C, G, U, A, and T represent cytosine, guanine, uracil, adenine nucleotides, and thymine deoxynucleotides; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by thiophosphate subunits; Cholesteryl has the structure of formula (I) and is connected to the 3' end of the sense chain through a phosphate bond; dT represents 2'-deoxythymine nucleotide.

[0169] Example 2. In vitro activity screening of siRNA RH30 cell line

[0170] 2.1 Experimental procedures

[0171] 2.1.1 Cell culture

[0172] RH30 cells (China Center for Type Culture Collection Cell Bank, GDC0611) were cultured in IMDM medium (Eallbio, supplemented with 10% FBS, 1% PS) at 37°C and 5% CO2. When the fusion rate reached 80%-90%, the cells were digested with trypsin, centrifuged, resuspended in DMEM high-glucose medium (Eallbio, supplemented with 10% FBS), counted, and transfected.

[0173] 2.1.2 Preparation of siRNA dilution solution

[0174] (1) The dry powder of the siRNA to be tested is centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM siRNA stock solution.

[0175] (2) Prepare 200 nM siRNA diluent Y:

[0176] a) taking 50 μl of the 100 μM siRNA stock solution prepared in step (1) above, adding 50 μl of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 50 μM;

[0177] b) taking 2 μl of the 50 μM siRNA dilution prepared in step a), adding 18 μl of ultrapure distilled water to obtain a siRNA stock solution X with a final concentration of 5 μM;

[0178] c) Take 2 μl of the prepared siRNA stock solution X and add 48 μl of Opti-medium to obtain 200 nM siRNA dilution solution Y.

[0179] 2.1.3 RH30 cell transfection

[0180] Take 0.6μl of Lipo®RNAiMAX (ThermoFisher, 13778150) transfection reagent, add 10μl Opti-medium to obtain Lipo®RNAiMAX transfection reagent diluent; mix the Lipo®RNAiMAX transfection reagent diluent and the 200nM siRNA diluent Y prepared in step 2.1.2 in a volume ratio of 1:1 to prepare a transfection mixture, let it stand for 5 minutes, take 10μl of the transfection mixture and add it to a 96-well plate, and add 90μl of the RH30 cells cultured in step 2.1.1 (final volume 100μl / well, the number of cells is 20,000 / well, and the concentration of siRNA in this system is 10nM); culture for 24 hours after the above transfection.

[0181] 2.1.4 RNA extraction

[0182] According to the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit product manual, total RNA from RH30 cells obtained in step 2.1.3 was extracted.

[0183] 2.1.5 Fluorescence quantitative PCR

[0184] The extracted total RNA was reverse transcribed and analyzed by real-time PCR using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit.

[0185] 2.1.6 Result Analysis

[0186] (1) The Ct value was automatically calculated using the software of the 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher);

[0187] (2) The relative expression of genes was calculated using the following formula:

[0188] ΔCt1=Ct(MSTN group)–Ct(ACTIN of MSTN group)

[0189] ΔCt2=Ct(siCtrl group)–Ct(ACTIN of siCtrl group)

[0190] ΔΔCt=ΔCt1 (MSTN group)-ΔCt2 (siCtrl group), where siCtrl group was the negative control group;

[0191] mRNA expression relative to siCtrl group = 2 -ΔΔCt

[0192] Inhibition rate (%) = (1 relative to the mRNA expression of the siCtrl group) × 100%.

[0193] 2.2 Experimental Results

[0194] The inhibitory effects of the siRNA of the present invention are shown in Table 7 below.

[0195] Table 7 Results of in vitro screening of siRNA in RH30 cell line

[0196]

[0197]

[0198]

[0199]

[0200] The results showed that some of the siRNAs of the present invention could significantly inhibit the expression of the MSTN gene at 10 nM.

[0201] Example 3. siRNA RH30 cell line IC50 determination

[0202] 3.1 Experimental procedures

[0203] Cell culture and transfection were performed in a manner similar to 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. The IC50 of siRNA in inhibiting MSTN gene expression was then determined.

[0204] 3.2 Results Analysis

[0205] (1) The Ct value was automatically calculated using the software of the 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher);

[0206] (2) Calculate the relative expression of the gene using the following formula:

[0207] ΔCt1=Ct(MSTN group)–Ct(ACTIN of MSTN group)

[0208] ΔCt2=Ct(siCtrl group)–Ct(ACTIN of siCtrl group)

[0209] ΔΔCt = ΔCt1-ΔCt2, where the siCtrl group was the negative control group;

[0210] mRNA expression relative to siCtrl group = 2 -ΔΔCt

[0211] Inhibition rate (%) = (1 relative to the mRNA expression of the siCtrl group) × 100%.

[0212] The log value of siRNA concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis. The "log (inhibitor) vs. normalized response --Variable slope" function module of the analysis software GraphPadPrism 8 was used to fit the dose-effect curve to obtain the IC50 value of each siRNA.

[0213] The fitting formula is: Y = 100 / (1 + 10^((LogIC50X) × HillSlope))

[0214] Where: HillSlope represents the slope of the percentage inhibition curve.

[0215] 3.3 Experimental Results

[0216] The IC50 determination results of the siRNA of the present invention are shown in Table 8 below:

[0217] Table 8. siRNA RH30 cell line IC50 determination results

[0218]

[0219]

[0220] The results of IC50 determination using RH30 cell line showed that some siRNAs of the present invention can significantly inhibit the expression of MSTN gene, and these sequences have good in vitro activity.

[0221] Example 4. Cytotoxicity of siRNA conjugates

[0222] 4.1 Experimental procedures

[0223] 4.1.1 Cell culture

[0224] RH30 cells were cultured in IMDM medium (Eallbio, supplemented with 10% FBS, 1% PS) at 37°C and 5% CO2. When the fusion rate reached 80%-90%, the cells were digested with trypsin, centrifuged, resuspended in DMEM high-glucose medium (Eallbio, supplemented with 10% FBS), counted, and transfected.

[0225] 4.1.2 RH30 cell transfection

[0226] Based on the above results of activity in RH30 cells, 17 siRNA conjugates were selected for toxicity determination. RH30 cells were transfected in a similar manner as in Example 2, and 15,000 RH30 cells were seeded per well. After 72 hours of culture, the cytotoxicity of each siRNA conjugate was measured by determining the ratio of cell viability / toxicity in each sample, where the concentrations of the transfected siRNA conjugates were 50 nM and 5 nM, respectively. Cell viability was measured by determining the intracellular ATP content using CellTiter-Glo (Promega, catalog number G7570) according to the manufacturer's protocol. ToxiLight TM (Lonza, catalog number LT07217) to measure cytotoxicity in the supernatant.

[0227] 4.2 Experimental Results

[0228] The cytotoxicity of siRNA conjugates is shown in Figure 1 .

[0229] The results show that some of the siRNA conjugates of the present invention have low cytotoxicity and good cell compatibility.

[0230] Example 5. Screening of siRNA conjugates for inhibiting target gene activity in muscle in vivo

[0231] 5.1 siRNA sequence modification

[0232] According to the in vitro screening results of Examples 2-4 above, some siRNA sequences with good in vitro activity in Table 2 were selected, and their conjugates were verified for in vivo activity.

[0233] 5.2 Experimental procedures

[0234] Wild-type male mice (C57BL / 6J) aged 6-8 weeks were purchased from Sibefor (Beijing) Biotechnology Co., Ltd., with 6 mice in each group. They were injected with 15 mg / kg of siRNA conjugates on day 0, and mice in the negative control group were given an equal volume of siCtrlM1. Quadriceps muscle samples were collected on day 7 (D7) and day 14 (D14) after administration, and the total RNA obtained was subjected to qRT-PCR analysis. According to the qPCR results, combined with the results of cytotoxicity experiments and cellular immunogenicity tests, comprehensive analysis was performed, and quadriceps muscle samples were collected on day 28 (D28) for the better siRNA conjugates, and qRT-PCR analysis was performed on the total RNA obtained from these mice.

[0235] 5.3 Experimental Results

[0236] The results of in vivo activity screening of siRNA conjugates are shown in Figure 2 .

[0237] Depend on Figure 2 The results showed that within 14 days of injection of siRNA conjugate drugs, YG-54M1, YG-95M1, YG-96M1, YG-97M1, YG-105M1, and YG-106M1 had a good knockdown effect in mice. This group of mice was selected for further observation until 28 days, and the relative expression level of MSTN gene in the quadriceps of mice was detected. The results are as follows Figure 3 As shown. Figure 3 The results showed that the knockdown effects of YG-95M1, YG-96M1, YG-97M1, YG-105M1, and YG-106M1 in mice were still very good on the 28th day.

[0238] Example 6. siRNA conjugates with modification pattern M1 or M1′ inhibit hMSTN expression level in serum in vivo

[0239] According to the in vivo experimental screening results of Example 5, siRNA conjugate sequences with better in vivo activity were selected to detect the target protein hMSTN content in serum.

[0240] 6.1 Experimental procedures

[0241] 6-8 week old male mice, 6 mice in each group, were subcutaneously administered with 15 mg / kg of the conjugate. 14 days (D14) and 28 days (D28) after administration, blood was collected from the orbital venous plexus of the mice, serum was separated, and the expression level of hMSTN in the serum was detected using an ELISA detection kit (R&D Systems, DGDF80).

[0242] 6.2 Experimental Results

[0243] The results of siRNA conjugate inhibiting hMSTN expression in serum are shown in Figure 4 .

[0244] 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.

[0245] Example 7. siRNA conjugates with modification pattern M2 or M2' inhibited hMSTN expression level in serum in vivo

[0246] 7.1 Experimental procedures

[0247] According to the experimental steps of Example 6, some siRNA conjugate sequences in Table 3 were tested for in vivo activity.

[0248] 7.2 Experimental Results

[0249] The results of the inhibition of hMSTN expression levels in serum by siRNA conjugates are shown in 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 effects were better than those of the corresponding siRNA with M1 modification pattern.

[0251] Example 8. siRNA conjugates with modification pattern M3 or M3' inhibit hMSTN expression level in serum in vivo

[0252] 8.1 Experimental Procedure

[0253] According to the experimental steps of Example 6, some siRNA conjugate sequences in Table 4 were tested for in vivo activity.

[0254] 8.2 Experimental Results

[0255] The results of the inhibition of hMSTN expression levels in serum by siRNA conjugates are shown in Figure 6 .

[0256] 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 effects were better than those of the corresponding siRNA with M1 modification pattern.

[0257] Example 9. siRNA conjugates with modification pattern M4 or M4' inhibit hMSTN expression level in serum in vivo

[0258] 9.1 Experimental Procedure

[0259] According to the experimental steps of Example 6, some siRNA conjugate sequences in Table 5 were tested for in vivo activity.

[0260] 9.2 Experimental Results

[0261] The results of the inhibition of hMSTN expression levels in serum by siRNA conjugates are shown in Figure 7 .

[0262] 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 effects were better than those of the corresponding siRNA with M1 modification pattern.

[0263] Example 10. siRNA conjugates with modification patterns M5 or M5' inhibit hMSTN expression levels in serum in vivo

[0264] 10.1 Experimental Procedure

[0265] According to the experimental steps of Example 6, some siRNA conjugate sequences in Table 6 were tested for in vivo activity.

[0266] 10.2 Experimental Results

[0267] The results of the inhibition of hMSTN expression levels in serum by siRNA conjugates are shown in Figure 8 .

[0268] 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 effects were better than those of the corresponding siRNA with M1 modification pattern.

Claims

1. A siRNA conjugate for inhibiting the expression of MSTN gene, comprising siRNA and a conjugated group conjugated to the siRNA, wherein the siRNA is a combination of SEQ ID NO: 97 and SEQ ID: 205, SEQ ID NO: 98 and SEQ ID NO: 206 having M2', M3', M4' and M5' modification patterns; A combination of SEQ ID NO:96 and SEQ ID NO:204, SEQ ID NO:106 and SEQ ID NO:214, or SEQ ID NO:107 and SEQ ID NO:215 having a modification pattern of M2, M3, M4, and M5; The conjugation site of siRNA and the conjugate group is at the 3' end of the siRNA sense strand, and the structure of the conjugate group is as follows: Formula (I); The sense strand and antisense strand in the conjugate have the following combined modification patterns: M-2： Justice chain: 5' -XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm -Cholesteryl -3' Antisense strand: 5′-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmXmsXm-3′; or M-2' Justice Chain: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3' Antisense strand: 5′-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXm-3′; or M3: Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3' Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXmXmXmXfXmXmXmXfXmXmXmXmXmXmsXm-3′; or M3' Justice chain: 5'-XmsXmsXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3' Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXmXmXmXfXmXmXfXmXmXmXmXmsXm-3′; or M4: Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3' Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXmXmXmXmXfXmXmXmXfXmXmXmXmXmXmsXm-3′; or M4' Justice chain: 5'-XmsXmsXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3' Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXmXmXmXmXfXmXmXfXmXmXmXmsXm-3′; or M5: Justice chain: 5'-XmsXmsXmXmXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3' Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXfXmXfXmXfXmXfXmXfXmXmXmXmXmXmsXm-3′; or M5' Justice chain: 5'-XmsXmsXmXmXmXfXmXfXdXfXmXmXmXmXmXmXmXmXmXmXm-Cholesteryl-3' Antisense strand: 5′-VPXmsXfsXmXmXmXfXmXfXmXfXmXfXmXfXmXfXmXmXmXmsXm-3′; in, Xm represents any nucleotide modified with 2'-methoxy, Xf represents any nucleotide modified with 2'-fluoro, Xd represents any nucleotide modified with 2'-deoxy, lowercase letter s represents that the two nucleotides adjacent to the letter s are connected by phosphorothioate subunits, VP represents 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.

2. A composition comprising the siRNA conjugate according to claim 1.

3. Use of the siRNA conjugate according to claim 1 or the composition according to claim 2 in the preparation of a medicament for preventing and / or treating a disease associated with MSTN gene expression.

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