siRNA Inhibiting PLN Gene Expression, Its Conjugates and Applications

Abstract

The present disclosure belongs to the field of biomedicine and relates to an siRNA and a conjugate for inhibiting PLN gene expression. The siRNA comprises a sense strand and an antisense strand. 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 complementary to form a double-stranded region. The nucleotide sequence I is substantially identical to a first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence with a length of at least 19 nucleotides in the mRNA of PLN gene expression. The siRNA, its conjugate and pharmaceutical composition of the present disclosure can effectively prevent and / or treat diseases associated with PLN expression, such as heart failure, cardiomyopathy, arrhythmia, etc., and have good prospects for drug development.

Description

Technical Field

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

[0002] Heart failure (HF) is a growing global epidemic, affecting 64.3 million people worldwide, including 13 million in my country by 2023. Despite ongoing research and a growing number of treatment options (such as angiotensin II and other medications, as well as implantable ventricular assist devices), HF patients still suffer from high rates of disability and mortality, poor long-term prognosis, and a 50% mortality rate within five years. Therefore, new treatments are urgently needed.

[0003] Dilated cardiomyopathy (DC) refers to left ventricular or biventricular dilation or systolic dysfunction in the absence of abnormal stress conditions or severe coronary artery disease sufficient to cause ventricular remodeling. The initial symptoms of DCM are atypical and may include decreased exercise tolerance, dyspnea, lower limb and abdominal edema, chest tightness, and palpitations. Some patients with DCM may not notice any symptoms initially. However, patients with DCM may develop heart failure and severe arrhythmias, which can be life-threatening. The causes of DCM vary, including familial genetic factors.

[0004] Heart failure is characterized by decreased cardiac contractility, and a common feature is impaired calcium chelation in the sarcoplasmic reticulum (SR). The key factor is the Ca2+ chelation that mediates SR calcium absorption. 2+ -ATPase (SERCA2a) activity is reduced, leading to Ca 2+ Abnormal absorption is accompanied by an increase in unphosphorylated phospholamban (PLN). PLN is a major regulator of SERCA2a activity and, to date, the only SERCA2a-related protein directly involved in the development of cardiac diseases, including heart failure. Dephosphorylated PLN is an inhibitor of SERCA, and phosphorylation of PLN alleviates this inhibition. Furthermore, PLN is a key mediator of β-adrenergic stimulation. Changes in its phosphorylation are associated with functional alterations in cardiac SR and contractility, making it a key regulator of cardiac contractility. Therefore, PLN has become a highly promising target for nucleic acid-based therapeutics for the treatment of heart failure.

[0005] PLN is a reversibly phosphorylated SR protein that modifies global cardiac function. In many cases, ablation of PLN can successfully rescue the decreased cardiac function and Ca2+ observed in various similar animal models of heart failure. 2+Abnormal uptake. In heart failure or cardiomyopathy, elevated PLN is harmful. Upregulation of PLN inhibits SERCA2a activity and reduces Ca 2+ uptake. Additionally, the human R14del-PLN mutation causes the development of cardiomyopathy, with clinical features of arrhythmogenic cardiomyopathy and dilated cardiomyopathy, and may progress to heart failure and premature death. Similar results are seen in mice, where the R14del-PLN mutation causes dilated cardiomyopathy, resulting in PLN protein aggregation, cardiac dysfunction, and reducing PLN expression has become a strategy for treating heart failure. However, there is evidence that humans cannot completely lack PLN, otherwise it will cause severe dilated cardiomyopathy, and the safe tolerance level of reduced PLN is 50%. Therefore, there is a need to develop more precise, safer and more effective nucleic acid drugs with lower dosing frequencies. Summary of the Invention

[0006] The object of the present invention is to provide an siRNA molecule capable of inhibiting PLN gene expression, with a view to providing a new treatment modality for PLN-related diseases including heart failure, cardiomyopathy, and arrhythmia, etc.

[0007] In a first aspect, the present disclosure provides an siRNA for inhibiting PLN 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 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 complementary to form a double-stranded region; the nucleotide sequence I is substantially identical to a first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence of at least 19 nucleotides in length in the mRNA of PLN gene expression, preferably, the first nucleotide sequence is a nucleotide sequence of 19-25 nucleotides in length in the mRNA of PLN gene expression, such as 19, 20, 21, 22, 23, 24, or 25 nucleotides;

[0008] Preferably, the first nucleotide sequence is a nucleotide sequence of at least 19 nucleotides in length in the highly active region of the mRNA of PLN gene expression, preferably a nucleotide sequence of 19-25 nucleotides in length, and the highly active region is positions 292-357, positions 444-626, and positions 1127-1149 of the mRNA of PLN gene expression, preferably positions 292-314, positions 315-337, positions 318-340, positions 327-349, positions 335-357, positions 444-466, positions 476-498, positions 604-626, and positions 1127-1149;

[0009] Preferably, the mRNA expressed by the PLN gene is as shown by NCBI refseqID NM_002667.5; specifically, the mRNA sequence is as shown by SEQ ID NO:1;

[0010] The high-activity interval refers to the interval in which both the designed siRNA and siRNA conjugate within this interval can effectively reduce the PLN mRNA level. In Figure 1 it, interval division was performed based on whether the maximum inhibition rate of the observed siRNA and siRNA conjugate on PLN mRNA fell into 40 - 60%, 60% - 80%, or greater than 80%.

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

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

[0013] In some embodiments, among any of the above siRNAs, the nucleotide sequence I contains at least 15 consecutive nucleotides shown by any sequence in SEQ ID NO:2 - 100, such as at least 15, 16, 17, 18, 19, 20, or 21 nucleotides; preferably, the nucleotide sequence I is as shown by any sequence in SEQ ID NO:2 - 100.

[0014] In some embodiments, among any of the above siRNAs, the nucleotide sequence II contains at least 15 consecutive nucleotides shown by any sequence in SEQ ID NO:101 - 199, such as at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides; preferably, the nucleotide sequence II is as shown by any sequence in SEQ ID NO:101 - 199.

[0015] In some embodiments, among any of the above siRNAs, the siRNA contains a sense strand and an antisense strand, which are:

[0016] 1) The sequence of the sense strand is as shown by SEQ ID NO:2, and the sequence of the antisense strand is as shown by SEQ ID NO:101;

[0017] 2) The sequence of the sense strand is as shown in SEQ ID NO:3, and the sequence of the antisense strand is as shown in SEQ ID NO:102;

[0018] 3) The sequence of the sense strand is as shown in SEQ ID NO:4, and the sequence of the antisense strand is as shown in SEQ ID NO:103;

[0019] 4) The sequence of the sense strand is as shown in SEQ ID NO:5, and the sequence of the antisense strand is as shown in SEQ ID NO:104;

[0020] 5) The sequence of the sense strand is as shown in SEQ ID NO:6, and the sequence of the antisense strand is as shown in SEQ ID NO:105;

[0021] 6) The sequence of the sense strand is as shown in SEQ ID NO:7, and the sequence of the antisense strand is as shown in SEQ ID NO:106;

[0022] 7) The sequence of the sense strand is as shown in SEQ ID NO:8, and the sequence of the antisense strand is as shown in SEQ ID NO:107;

[0023] 8) The sequence of the sense strand is as shown in SEQ ID NO:9, and the sequence of the antisense strand is as shown in SEQ ID NO:108;

[0024] 9) The sequence of the sense strand is as shown in SEQ ID NO:10, and the sequence of the antisense strand is as shown in SEQ ID NO:109;

[0025] 10) The sequence of the sense strand is as shown in SEQ ID NO:11, and the sequence of the antisense strand is as shown in SEQ ID NO:110;

[0026] 11) The sequence of the sense strand is as shown in SEQ ID NO:12, and the sequence of the antisense strand is as shown in SEQ ID NO:111;

[0027] 12) The sequence of the sense strand is as shown in SEQ ID NO:13, and the sequence of the antisense strand is as shown in SEQ ID NO:112;

[0028] 13) The sequence of the sense strand is as shown in SEQ ID NO:14, and the sequence of the antisense strand is as shown in SEQ ID NO:113;

[0029] 14) The sequence of the sense strand is as shown in SEQ ID NO:15, and the sequence of the antisense strand is as shown in SEQ ID NO:114;

[0030] 15) The sequence of the sense strand is as shown in SEQ ID NO: 16, and the sequence of the antisense strand is as shown in SEQ ID NO: 115;

[0031] 16) The sequence of the sense strand is as shown in SEQ ID NO: 17, and the sequence of the antisense strand is as shown in SEQ ID NO: 116;

[0032] 17) The sequence of the sense strand is as shown in SEQ ID NO: 18, and the sequence of the antisense strand is as shown in SEQ ID NO: 117;

[0033] 18) The sequence of the sense strand is as shown in SEQ ID NO: 19, and the sequence of the antisense strand is as shown in SEQ ID NO: 118;

[0034] 19) The sequence of the sense strand is as shown in SEQ ID NO: 20, and the sequence of the antisense strand is as shown in SEQ ID NO: 119;

[0035] 20) The sequence of the sense strand is as shown in SEQ ID NO: 21, and the sequence of the antisense strand is as shown in SEQ ID NO: 120;

[0036] 21) The sequence of the sense strand is as shown in SEQ ID NO: 22, and the sequence of the antisense strand is as shown in SEQ ID NO: 121;

[0037] 22) The sequence of the sense strand is as shown in SEQ ID NO: 23, and the sequence of the antisense strand is as shown in SEQ ID NO: 122;

[0038] 23) The sequence of the sense strand is as shown in SEQ ID NO: 24, and the sequence of the antisense strand is as shown in SEQ ID NO: 123;

[0039] 24) The sequence of the sense strand is as shown in SEQ ID NO: 25, and the sequence of the antisense strand is as shown in SEQ ID NO: 124;

[0040] 25) The sequence of the sense strand is as shown in SEQ ID NO: 26, and the sequence of the antisense strand is as shown in SEQ ID NO: 125;

[0041] 26) The sequence of the sense strand is as shown in SEQ ID NO: 27, and the sequence of the antisense strand is as shown in SEQ ID NO: 126;

[0042] 27) The sequence of the sense strand is as shown in SEQ ID NO: 28, and the sequence of the antisense strand is as shown in SEQ ID NO: 127;

[0043] 28) The sequence of the sense strand is as shown in SEQ ID NO:29, and the sequence of the antisense strand is as shown in SEQ ID NO:128;

[0044] 29) The sequence of the sense strand is as shown in SEQ ID NO:30, and the sequence of the antisense strand is as shown in SEQ ID NO:129;

[0045] 30) The sequence of the sense strand is as shown in SEQ ID NO:31, and the sequence of the antisense strand is as shown in SEQ ID NO:130;

[0046] 31) The sequence of the sense strand is as shown in SEQ ID NO:32, and the sequence of the antisense strand is as shown in SEQ ID NO:131;

[0047] 32) The sequence of the sense strand is as shown in SEQ ID NO:33, and the sequence of the antisense strand is as shown in SEQ ID NO:132;

[0048] 33) The sequence of the sense strand is as shown in SEQ ID NO:34, and the sequence of the antisense strand is as shown in SEQ ID NO:133;

[0049] 34) The sequence of the sense strand is as shown in SEQ ID NO:35, and the sequence of the antisense strand is as shown in SEQ ID NO:134;

[0050] 35) The sequence of the sense strand is as shown in SEQ ID NO:36, and the sequence of the antisense strand is as shown in SEQ ID NO:135;

[0051] 36) The sequence of the sense strand is as shown in SEQ ID NO:37, and the sequence of the antisense strand is as shown in SEQ ID NO:136;

[0052] 37) The sequence of the sense strand is as shown in SEQ ID NO:38, and the sequence of the antisense strand is as shown in SEQ ID NO:137;

[0053] 38) The sequence of the sense strand is as shown in SEQ ID NO:39, and the sequence of the antisense strand is as shown in SEQ ID NO:138;

[0054] 39) The sequence of the sense strand is as shown in SEQ ID NO:40, and the sequence of the antisense strand is as shown in SEQ ID NO:139;

[0055] 40) The sequence of the sense strand is as shown in SEQ ID NO:41, and the sequence of the antisense strand is as shown in SEQ ID NO:140;

[0056] 41) The sequence of the sense strand is as shown in SEQ ID NO: 42, and the sequence of the antisense strand is as shown in SEQ ID NO: 141;

[0057] 42) The sequence of the sense strand is as shown in SEQ ID NO: 43, and the sequence of the antisense strand is as shown in SEQ ID NO: 142;

[0058] 43) The sequence of the sense strand is as shown in SEQ ID NO: 44, and the sequence of the antisense strand is as shown in SEQ ID NO: 143;

[0059] 44) The sequence of the sense strand is as shown in SEQ ID NO: 45, and the sequence of the antisense strand is as shown in SEQ ID NO: 144;

[0060] 45) The sequence of the sense strand is as shown in SEQ ID NO: 46, and the sequence of the antisense strand is as shown in SEQ ID NO: 145;

[0061] 46) The sequence of the sense strand is as shown in SEQ ID NO: 47, and the sequence of the antisense strand is as shown in SEQ ID NO: 146;

[0062] 47) The sequence of the sense strand is as shown in SEQ ID NO: 48, and the sequence of the antisense strand is as shown in SEQ ID NO: 147;

[0063] 48) The sequence of the sense strand is as shown in SEQ ID NO: 49, and the sequence of the antisense strand is as shown in SEQ ID NO: 148;

[0064] 49) The sequence of the sense strand is as shown in SEQ ID NO: 50, and the sequence of the antisense strand is as shown in SEQ ID NO: 149;

[0065] 50) The sequence of the sense strand is as shown in SEQ ID NO: 51, and the sequence of the antisense strand is as shown in SEQ ID NO: 150;

[0066] 51) The sequence of the sense strand is as shown in SEQ ID NO: 52, and the sequence of the antisense strand is as shown in SEQ ID NO: 151;

[0067] 52) The sequence of the sense strand is as shown in SEQ ID NO: 53, and the sequence of the antisense strand is as shown in SEQ ID NO: 152;

[0068] 53) The sequence of the sense strand is as shown in SEQ ID NO: 54, and the sequence of the antisense strand is as shown in SEQ ID NO: 153;

[0069] 54) The sequence of the sense strand is as shown in SEQ ID NO: 55, and the sequence of the antisense strand is as shown in SEQ ID NO: 154;

[0070] 55) The sequence of the sense strand is as shown in SEQ ID NO: 56, and the sequence of the antisense strand is as shown in SEQ ID NO: 155;

[0071] 56) The sequence of the sense strand is as shown in SEQ ID NO: 57, and the sequence of the antisense strand is as shown in SEQ ID NO: 156;

[0072] 57) The sequence of the sense strand is as shown in SEQ ID NO: 58, and the sequence of the antisense strand is as shown in SEQ ID NO: 157;

[0073] 58) The sequence of the sense strand is as shown in SEQ ID NO: 59, and the sequence of the antisense strand is as shown in SEQ ID NO: 158;

[0074] 59) The sequence of the sense strand is as shown in SEQ ID NO: 60, and the sequence of the antisense strand is as shown in SEQ ID NO: 159;

[0075] 60) The sequence of the sense strand is as shown in SEQ ID NO: 61, and the sequence of the antisense strand is as shown in SEQ ID NO: 160;

[0076] 61) The sequence of the sense strand is as shown in SEQ ID NO: 62, and the sequence of the antisense strand is as shown in SEQ ID NO: 161;

[0077] 62) The sequence of the sense strand is as shown in SEQ ID NO: 63, and the sequence of the antisense strand is as shown in SEQ ID NO: 162;

[0078] 63) The sequence of the sense strand is as shown in SEQ ID NO: 64, and the sequence of the antisense strand is as shown in SEQ ID NO: 163;

[0079] 64) The sequence of the sense strand is as shown in SEQ ID NO: 65, and the sequence of the antisense strand is as shown in SEQ ID NO: 164;

[0080] 65) The sequence of the sense strand is as shown in SEQ ID NO: 66, and the sequence of the antisense strand is as shown in SEQ ID NO: 165;

[0081] 66) The sequence of the sense strand is as shown in SEQ ID NO: 67, and the sequence of the antisense strand is as shown in SEQ ID NO: 166;

[0082] 67) The sequence of the sense strand is as shown in SEQ ID NO: 68, and the sequence of the antisense strand is as shown in SEQ ID NO: 167;

[0083] 68) The sequence of the sense strand is as shown in SEQ ID NO: 69, and the sequence of the antisense strand is as shown in SEQ ID NO: 168;

[0084] 69) The sequence of the sense strand is as shown in SEQ ID NO: 70, and the sequence of the antisense strand is as shown in SEQ ID NO: 169;

[0085] 70) The sequence of the sense strand is as shown in SEQ ID NO: 71, and the sequence of the antisense strand is as shown in SEQ ID NO: 170;

[0086] 71) The sequence of the sense strand is as shown in SEQ ID NO: 72, and the sequence of the antisense strand is as shown in SEQ ID NO: 171;

[0087] 72) The sequence of the sense strand is as shown in SEQ ID NO: 73, and the sequence of the antisense strand is as shown in SEQ ID NO: 172;

[0088] 73) The sequence of the sense strand is as shown in SEQ ID NO: 74, and the sequence of the antisense strand is as shown in SEQ ID NO: 173;

[0089] 74) The sequence of the sense strand is as shown in SEQ ID NO: 75, and the sequence of the antisense strand is as shown in SEQ ID NO: 174;

[0090] 75) The sequence of the sense strand is as shown in SEQ ID NO: 76, and the sequence of the antisense strand is as shown in SEQ ID NO: 175;

[0091] 76) The sequence of the sense strand is as shown in SEQ ID NO: 77, and the sequence of the antisense strand is as shown in SEQ ID NO: 176;

[0092] 77) The sequence of the sense strand is as shown in SEQ ID NO: 78, and the sequence of the antisense strand is as shown in SEQ ID NO: 177;

[0093] 78) The sequence of the sense strand is as shown in SEQ ID NO: 79, and the sequence of the antisense strand is as shown in SEQ ID NO: 178;

[0094] 79) The sequence of the sense strand is as shown in SEQ ID NO: 80, and the sequence of the antisense strand is as shown in SEQ ID NO: 179;

[0095] 80) The sequence of the sense strand is as shown in SEQ ID NO: 81, and the sequence of the antisense strand is as shown in SEQ ID NO: 180;

[0096] 81) The sequence of the sense strand is as shown in SEQ ID NO: 82, and the sequence of the antisense strand is as shown in SEQ ID NO: 181;

[0097] 82) The sequence of the sense strand is as shown in SEQ ID NO: 83, and the sequence of the antisense strand is as shown in SEQ ID NO: 182;

[0098] 83) The sequence of the sense strand is as shown in SEQ ID NO: 84, and the sequence of the antisense strand is as shown in SEQ ID NO: 183;

[0099] 84) The sequence of the sense strand is as shown in SEQ ID NO: 85, and the sequence of the antisense strand is as shown in SEQ ID NO: 184;

[0100] 85) The sequence of the sense strand is as shown in SEQ ID NO: 86, and the sequence of the antisense strand is as shown in SEQ ID NO: 185;

[0101] 86) The sequence of the sense strand is as shown in SEQ ID NO: 87, and the sequence of the antisense strand is as shown in SEQ ID NO: 186;

[0102] 87) The sequence of the sense strand is as shown in SEQ ID NO: 88, and the sequence of the antisense strand is as shown in SEQ ID NO: 187;

[0103] 88) The sequence of the sense strand is as shown in SEQ ID NO: 89, and the sequence of the antisense strand is as shown in SEQ ID NO: 188;

[0104] 89) The sequence of the sense strand is as shown in SEQ ID NO: 90, and the sequence of the antisense strand is as shown in SEQ ID NO: 189;

[0105] 90) The sequence of the sense strand is as shown in SEQ ID NO: 91, and the sequence of the antisense strand is as shown in SEQ ID NO: 190;

[0106] 91) The sequence of the sense strand is as shown in SEQ ID NO:92, and the sequence of the antisense strand is as shown in SEQ ID NO:191;

[0107] 92) The sequence of the sense strand is as shown in SEQ ID NO:93, and the sequence of the antisense strand is as shown in SEQ ID NO:192;

[0108] 93) The sequence of the sense strand is as shown in SEQ ID NO:94, and the sequence of the antisense strand is as shown in SEQ ID NO:193;

[0109] 94) The sequence of the sense strand is as shown in SEQ ID NO:95, and the sequence of the antisense strand is as shown in SEQ ID NO:194;

[0110] 95) The sequence of the sense strand is as shown in SEQ ID NO:96, and the sequence of the antisense strand is as shown in SEQ ID NO:195;

[0111] 96) The sequence of the sense strand is as shown in SEQ ID NO:97, and the sequence of the antisense strand is as shown in SEQ ID NO:196;

[0112] 97) The sequence of the sense strand is as shown in SEQ ID NO:98, and the sequence of the antisense strand is as shown in SEQ ID NO:197;

[0113] 98) The sequence of the sense strand is as shown in SEQ ID NO:99, and the sequence of the antisense strand is as shown in SEQ ID NO:198; or

[0114] 99) The sequence of the sense strand is as shown in SEQ ID NO:100, and the sequence of the antisense strand is as shown in SEQ ID NO:199.

[0115] 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.

[0116] In some embodiments, the fluorine-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose of the nucleotide is replaced by fluorine and has the structure shown in the following formula (1); the non-fluorine-modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2'-position of the ribose of the nucleotide is replaced by a non-fluorine group. In some embodiments, each non-fluorine-modified nucleotide is independently selected from a nucleotide or nucleotide analog in which the hydroxyl group at the 2'-position of the ribose of the nucleotide is replaced by a non-fluorine group. These nucleotides or nucleotide analogs in which the hydroxyl group at the 2'-position of the ribose is replaced by a non-fluorine 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 as shown in formula (4). In some embodiments, the 2'-deoxynucleotide (DNA) is as shown in formula (5).

[0117]

[0118] A nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenosine ribonucleoside, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide, or thymidine ribonucleotide. In some embodiments, the nucleotide analog may be a heteronucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0119] A bridged nucleotide refers to a constrained or inaccessible nucleotide. A bridged nucleotide may contain a bridged structure with a fixed C3'-endo sugar puckering having a five-membered ring, a six-membered ring, or a seven-membered ring. In some embodiments, the bridged nucleotide may be LNA, ENA, cET BNA, etc.; where LNA is as shown in formula (6), ENA is as shown in formula (7), and cET BNA is as shown in formula (8).

[0120]

[0121] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of nucleotides. In some embodiments, the 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).

[0122]

[0123] In the above formulas (9) and (10), R is selected from H, OH or alkoxy (O-alkyl).

[0124] An isonucleotide refers to a compound formed by changing the position of the base on the ribose ring in a nucleotide. In some embodiments, the isonucleotide can be a compound formed by moving the base from the 1'-position to the 2'-position or 3'-position of the ribose ring. As shown in formula (11) or formula (12).

[0125]

[0126] In formulas (11) and (12), R is selected from H, OH, F or the non-fluorine groups as described above.

[0127] In formulas (1) to (12), Base represents a base.

[0128] In some embodiments, in any of the above-mentioned siRNAs, in the direction from the 5'-end to the 3'-end, one or more nucleotides at positions 7, 9, 10, and 11 of the nucleotide sequence I are fluorine-modified nucleotides; and, in the direction from the 5'-end to the 3'-end, one or more nucleotides at positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluorine-modified nucleotides.

[0129] In some embodiments, at least a part of the phosphate ester groups in the phospho-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA are phosphate ester groups with a modifying group. In some embodiments, the phosphate ester group with a modifying group is a phosphorothioate 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 a modifying group is a phosphorothioate group having the structure shown in formula (13):

[0130]

[0131] In some embodiments, in any of the siRNAs described above, the phosphorothioate linkage is present at least at one position selected from the group consisting of: 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;

[0132] In some embodiments, in any of the siRNAs described above, the 5'-terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analogue, as shown in Formula (14), Formula (15) and Formula (16):

[0133]

[0134] In a second aspect, the present disclosure provides an siRNA conjugate comprising any of the siRNAs described above and a conjugate group conjugated to the siRNA. In some embodiments, the pharmaceutically acceptable conjugate group in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent or tetravalent. In some embodiments, the conjugation site of the siRNA and 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 also in the internal sequence of the siRNA.

[0135] In some embodiments, in the siRNA conjugate described above, the conjugate group is L96, and the structure is shown as follows:

[0136]

[0137] In some embodiments, the siRNA conjugate comprises a sense strand and an antisense strand, and is:

[0138] 1) The sequence of the sense strand is as shown in SEQ ID NO:200, and the sequence of the antisense strand is as shown in SEQ ID NO:299;

[0139] 2) The sequence of the sense strand is as shown in SEQ ID NO:201, and the sequence of the antisense strand is as shown in SEQ ID NO:300;

[0140] 3) The sequence of the sense strand is as shown in SEQ ID NO:202, and the sequence of the antisense strand is as shown in SEQ ID NO:301;

[0141] 4) The sequence of the sense strand is as shown in SEQ ID NO: 203, and the sequence of the antisense strand is as shown in SEQ ID NO: 302;

[0142] 5) The sequence of the sense strand is as shown in SEQ ID NO: 204, and the sequence of the antisense strand is as shown in SEQ ID NO: 303;

[0143] 6) The sequence of the sense strand is as shown in SEQ ID NO: 205, and the sequence of the antisense strand is as shown in SEQ ID NO: 304;

[0144] 7) The sequence of the sense strand is as shown in SEQ ID NO: 206, and the sequence of the antisense strand is as shown in SEQ ID NO: 305;

[0145] 8) The sequence of the sense strand is as shown in SEQ ID NO: 207, and the sequence of the antisense strand is as shown in SEQ ID NO: 306;

[0146] 9) The sequence of the sense strand is as shown in SEQ ID NO: 208, and the sequence of the antisense strand is as shown in SEQ ID NO: 307;

[0147] 10) The sequence of the sense strand is as shown in SEQ ID NO: 209, and the sequence of the antisense strand is as shown in SEQ ID NO: 308;

[0148] 11) The sequence of the sense strand is as shown in SEQ ID NO: 210, and the sequence of the antisense strand is as shown in SEQ ID NO: 309;

[0149] 12) The sequence of the sense strand is as shown in SEQ ID NO: 211, and the sequence of the antisense strand is as shown in SEQ ID NO: 310;

[0150] 13) The sequence of the sense strand is as shown in SEQ ID NO: 212, and the sequence of the antisense strand is as shown in SEQ ID NO: 311;

[0151] 14) The sequence of the sense strand is as shown in SEQ ID NO: 213, and the sequence of the antisense strand is as shown in SEQ ID NO: 312;

[0152] 15) The sequence of the sense strand is as shown in SEQ ID NO: 214, and the sequence of the antisense strand is as shown in SEQ ID NO: 313;

[0153] 16) The sequence of the sense strand is as shown in SEQ ID NO: 215, and the sequence of the antisense strand is as shown in SEQ ID NO: 314;

[0154] 17) The sequence of the sense strand is as shown in SEQ ID NO: 216, and the sequence of the antisense strand is as shown in SEQ ID NO: 315;

[0155] 18) The sequence of the sense strand is as shown in SEQ ID NO: 217, and the sequence of the antisense strand is as shown in SEQ ID NO: 316;

[0156] 19) The sequence of the sense strand is as shown in SEQ ID NO: 218, and the sequence of the antisense strand is as shown in SEQ ID NO: 317;

[0157] 20) The sequence of the sense strand is as shown in SEQ ID NO: 219, and the sequence of the antisense strand is as shown in SEQ ID NO: 318;

[0158] 21) The sequence of the sense strand is as shown in SEQ ID NO: 220, and the sequence of the antisense strand is as shown in SEQ ID NO: 319;

[0159] 22) The sequence of the sense strand is as shown in SEQ ID NO: 221, and the sequence of the antisense strand is as shown in SEQ ID NO: 320;

[0160] 23) The sequence of the sense strand is as shown in SEQ ID NO: 222, and the sequence of the antisense strand is as shown in SEQ ID NO: 321;

[0161] 24) The sequence of the sense strand is as shown in SEQ ID NO: 223, and the sequence of the antisense strand is as shown in SEQ ID NO: 322;

[0162] 25) The sequence of the sense strand is as shown in SEQ ID NO: 224, and the sequence of the antisense strand is as shown in SEQ ID NO: 323;

[0163] 26) The sequence of the sense strand is as shown in SEQ ID NO: 225, and the sequence of the antisense strand is as shown in SEQ ID NO: 324;

[0164] 27) The sequence of the sense strand is as shown in SEQ ID NO: 226, and the sequence of the antisense strand is as shown in SEQ ID NO: 325;

[0165] 28) The sequence of the sense strand is as shown in SEQ ID NO: 227, and the sequence of the antisense strand is as shown in SEQ ID NO: 326;

[0166] 29) The sequence of the sense strand is as shown in SEQ ID NO: 228, and the sequence of the antisense strand is as shown in SEQ ID NO: 327;

[0167] 30) The sequence of the sense strand is as shown in SEQ ID NO: 229, and the sequence of the antisense strand is as shown in SEQ ID NO: 328;

[0168] 31) The sequence of the sense strand is as shown in SEQ ID NO: 230, and the sequence of the antisense strand is as shown in SEQ ID NO: 329;

[0169] 32) The sequence of the sense strand is as shown in SEQ ID NO: 231, and the sequence of the antisense strand is as shown in SEQ ID NO: 330;

[0170] 33) The sequence of the sense strand is as shown in SEQ ID NO: 232, and the sequence of the antisense strand is as shown in SEQ ID NO: 331;

[0171] 34) The sequence of the sense strand is as shown in SEQ ID NO: 233, and the sequence of the antisense strand is as shown in SEQ ID NO: 332;

[0172] 35) The sequence of the sense strand is as shown in SEQ ID NO: 234, and the sequence of the antisense strand is as shown in SEQ ID NO: 333;

[0173] 36) The sequence of the sense strand is as shown in SEQ ID NO: 235, and the sequence of the antisense strand is as shown in SEQ ID NO: 334;

[0174] 37) The sequence of the sense strand is as shown in SEQ ID NO: 236, and the sequence of the antisense strand is as shown in SEQ ID NO: 335;

[0175] 38) The sequence of the sense strand is as shown in SEQ ID NO: 237, and the sequence of the antisense strand is as shown in SEQ ID NO: 336;

[0176] 39) The sequence of the sense strand is as shown in SEQ ID NO: 238, and the sequence of the antisense strand is as shown in SEQ ID NO: 337;

[0177] 40) The sequence of the sense strand is as shown in SEQ ID NO: 239, and the sequence of the antisense strand is as shown in SEQ ID NO: 338;

[0178] 41) The sequence of the sense strand is as shown in SEQ ID NO: 240, and the sequence of the antisense strand is as shown in SEQ ID NO: 339;

[0179] 42) The sequence of the sense strand is as shown in SEQ ID NO: 241, and the sequence of the antisense strand is as shown in SEQ ID NO: 340;

[0180] 43) The sequence of the sense strand is as shown in SEQ ID NO: 242, and the sequence of the antisense strand is as shown in SEQ ID NO: 341;

[0181] 44) The sequence of the sense strand is as shown in SEQ ID NO: 243, and the sequence of the antisense strand is as shown in SEQ ID NO: 342;

[0182] 45) The sequence of the sense strand is as shown in SEQ ID NO: 244, and the sequence of the antisense strand is as shown in SEQ ID NO: 343;

[0183] 46) The sequence of the sense strand is as shown in SEQ ID NO: 245, and the sequence of the antisense strand is as shown in SEQ ID NO: 344;

[0184] 47) The sequence of the sense strand is as shown in SEQ ID NO: 246, and the sequence of the antisense strand is as shown in SEQ ID NO: 345;

[0185] 48) The sequence of the sense strand is as shown in SEQ ID NO: 247, and the sequence of the antisense strand is as shown in SEQ ID NO: 346;

[0186] 49) The sequence of the sense strand is as shown in SEQ ID NO: 248, and the sequence of the antisense strand is as shown in SEQ ID NO: 347;

[0187] 50) The sequence of the sense strand is as shown in SEQ ID NO: 249, and the sequence of the antisense strand is as shown in SEQ ID NO: 348;

[0188] 51) The sequence of the sense strand is as shown in SEQ ID NO: 250, and the sequence of the antisense strand is as shown in SEQ ID NO: 349;

[0189] 52) The sequence of the sense strand is as shown in SEQ ID NO: 251, and the sequence of the antisense strand is as shown in SEQ ID NO: 350;

[0190] 53) The sequence of the sense strand is as shown in SEQ ID NO: 252, and the sequence of the antisense strand is as shown in SEQ ID NO: 351;

[0191] 54) The sequence of the sense strand is as shown in SEQ ID NO: 253, and the sequence of the antisense strand is as shown in SEQ ID NO: 352;

[0192] 55) The sequence of the sense strand is as shown in SEQ ID NO: 254, and the sequence of the antisense strand is as shown in SEQ ID NO: 353;

[0193] 56) The sequence of the sense strand is as shown in SEQ ID NO: 255, and the sequence of the antisense strand is as shown in SEQ ID NO: 354;

[0194] 57) The sequence of the sense strand is as shown in SEQ ID NO: 256, and the sequence of the antisense strand is as shown in SEQ ID NO: 355;

[0195] 58) The sequence of the sense strand is as shown in SEQ ID NO: 257, and the sequence of the antisense strand is as shown in SEQ ID NO: 356;

[0196] 59) The sequence of the sense strand is as shown in SEQ ID NO: 258, and the sequence of the antisense strand is as shown in SEQ ID NO: 357;

[0197] 60) The sequence of the sense strand is as shown in SEQ ID NO: 259, and the sequence of the antisense strand is as shown in SEQ ID NO: 358;

[0198] 61) The sequence of the sense strand is as shown in SEQ ID NO: 260, and the sequence of the antisense strand is as shown in SEQ ID NO: 359;

[0199] 62) The sequence of the sense strand is as shown in SEQ ID NO: 261, and the sequence of the antisense strand is as shown in SEQ ID NO: 360;

[0200] 63) The sequence of the sense strand is as shown in SEQ ID NO: 262, and the sequence of the antisense strand is as shown in SEQ ID NO: 361;

[0201] 64) The sequence of the sense strand is as shown in SEQ ID NO: 263, and the sequence of the antisense strand is as shown in SEQ ID NO: 362;

[0202] 65) The sequence of the sense strand is as shown in SEQ ID NO: 264, and the sequence of the antisense strand is as shown in SEQ ID NO: 363;

[0203] 66) The sequence of the sense strand is as shown in SEQ ID NO: 265, and the sequence of the antisense strand is as shown in SEQ ID NO: 364;

[0204] 67) The sequence of the sense strand is as shown in SEQ ID NO: 266, and the sequence of the antisense strand is as shown in SEQ ID NO: 365;

[0205] 68) The sequence of the sense strand is as shown in SEQ ID NO: 267, and the sequence of the antisense strand is as shown in SEQ ID NO: 366;

[0206] 69) The sequence of the sense strand is as shown in SEQ ID NO: 268, and the sequence of the antisense strand is as shown in SEQ ID NO: 367;

[0207] 70) The sequence of the sense strand is as shown in SEQ ID NO: 269, and the sequence of the antisense strand is as shown in SEQ ID NO: 368;

[0208] 71) The sequence of the sense strand is as shown in SEQ ID NO: 270, and the sequence of the antisense strand is as shown in SEQ ID NO: 369;

[0209] 72) The sequence of the sense strand is as shown in SEQ ID NO: 271, and the sequence of the antisense strand is as shown in SEQ ID NO: 370;

[0210] 73) The sequence of the sense strand is as shown in SEQ ID NO: 272, and the sequence of the antisense strand is as shown in SEQ ID NO: 371;

[0211] 74) The sequence of the sense strand is as shown in SEQ ID NO: 273, and the sequence of the antisense strand is as shown in SEQ ID NO: 372;

[0212] 75) The sequence of the sense strand is as shown in SEQ ID NO: 274, and the sequence of the antisense strand is as shown in SEQ ID NO: 373;

[0213] 76) The sequence of the sense strand is as shown in SEQ ID NO: 275, and the sequence of the antisense strand is as shown in SEQ ID NO: 374;

[0214] 77) The sequence of the sense strand is as shown in SEQ ID NO: 276, and the sequence of the antisense strand is as shown in SEQ ID NO: 375;

[0215] 78) The sequence of the sense strand is as shown in SEQ ID NO: 277, and the sequence of the antisense strand is as shown in SEQ ID NO: 376;

[0216] 79) The sequence of the sense strand is shown in SEQ ID NO: 278, and the sequence of the antisense strand is shown in SEQ ID NO: 377;

[0217] 80) The sequence of the sense strand is shown in SEQ ID NO: 279, and the sequence of the antisense strand is shown in SEQ ID NO: 378;

[0218] 81) The sequence of the sense strand is shown in SEQ ID NO: 280, and the sequence of the antisense strand is shown in SEQ ID NO: 379;

[0219] 82) The sequence of the sense strand is shown in SEQ ID NO: 281, and the sequence of the antisense strand is shown in SEQ ID NO: 380;

[0220] 83) The sequence of the sense strand is shown in SEQ ID NO: 282, and the sequence of the antisense strand is shown in SEQ ID NO: 381;

[0221] 84) The sequence of the sense strand is shown in SEQ ID NO: 283, and the sequence of the antisense strand is shown in SEQ ID NO: 382;

[0222] 85) The sequence of the sense strand is shown in SEQ ID NO: 284, and the sequence of the antisense strand is shown in SEQ ID NO: 383;

[0223] 86) The sequence of the sense strand is shown in SEQ ID NO: 285, and the sequence of the antisense strand is shown in SEQ ID NO: 384;

[0224] 87) The sequence of the sense strand is shown in SEQ ID NO: 286, and the sequence of the antisense strand is shown in SEQ ID NO: 385;

[0225] 88) The sequence of the sense strand is shown in SEQ ID NO: 287, and the sequence of the antisense strand is shown in SEQ ID NO: 386;

[0226] 89) The sequence of the sense strand is shown in SEQ ID NO: 288, and the sequence of the antisense strand is shown in SEQ ID NO: 387;

[0227] 90) The sequence of the sense strand is shown in SEQ ID NO: 289, and the sequence of the antisense strand is shown in SEQ ID NO: 388; [[ID='35']]

[0228] 91) The sequence of the sense strand is shown in SEQ ID NO: 290, and the sequence of the antisense strand is shown in SEQ ID NO: 389;

[0229] 92) The sequence of the sense strand is shown as SEQ ID NO: 291, and the sequence of the antisense strand is shown as SEQ ID NO: 390;

[0230] 93) The sequence of the sense strand is shown as SEQ ID NO: 292, and the sequence of the antisense strand is shown as SEQ ID NO: 391;

[0231] 94) The sequence of the sense strand is shown as SEQ ID NO: 293, and the sequence of the antisense strand is shown as SEQ ID NO: 392;

[0232] 95) The sequence of the sense strand is shown as SEQ ID NO: 294, and the sequence of the antisense strand is shown as SEQ ID NO: 393;

[0233] 96) The sequence of the sense strand is shown as SEQ ID NO: 295, and the sequence of the antisense strand is shown as SEQ ID NO: 394;

[0234] 97) The sequence of the sense strand is shown as SEQ ID NO: 296, and the sequence of the antisense strand is shown as SEQ ID NO: 395;

[0235] 98) The sequence of the sense strand is shown as SEQ ID NO: 297, and the sequence of the antisense strand is shown as SEQ ID NO: 396; or

[0236] 99) The sequence of the sense strand is shown as SEQ ID NO: 298, and the sequence of the antisense strand is shown as SEQ ID NO: 397.

[0237] In a third aspect, the present disclosure provides a composition comprising any one of the above-mentioned siRNAs or siRNA conjugates.

[0238] In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carriers or excipients involved in the present disclosure 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, distearoyl phosphatidylcholine, 1,2-dimyristoyl glycerol, dimethyl adipate.

[0239] In a fourth aspect, the present disclosure provides the use of any one of the above-mentioned siRNAs, siRNA conjugates or pharmaceutical compositions in the preparation of a drug for preventing and / or treating a disease associated with PLN expression.

[0240] In a fifth aspect, the present disclosure provides a method for preventing and / or treating a disease associated with PLN expression, the method comprising administering to a subject the siRNA, siRNA conjugate or pharmaceutical composition of the present disclosure.

[0241] In some embodiments, the disease associated with PLN expression includes one or more of heart failure, cardiomyopathy, and arrhythmia;

[0242] Preferably, the heart failure includes one or more of acute heart failure, chronic heart failure, systolic heart failure, diastolic heart failure, left heart failure, right heart failure, and global heart failure;

[0243] Preferably, the cardiomyopathy is a genetic cardiomyopathy, including one or more of cardiomyopathies caused by p.Arg14del, Arg9Cys (R9C), and / or Arg25Cys (R25C) gene mutations; more preferably, the cardiomyopathy is dilated cardiomyopathy, including one or more of dilated cardiomyopathies caused by TTN, LMNA, RBM20, SCN5A, MYH7, TNNT2, and / or TPM1 mutations;

[0244] Preferably, the arrhythmia includes ventricular tachycardia and / or ventricular fibrillation.

[0245] The pharmaceutical composition involved in the present disclosure can be used alone in the treatment of diseases associated with PLN expression, or in combination with standard oral drugs, providing experimental support for diverse treatment regimens for clinical patients with the above diseases.

[0246] Based on the amount of siRNA contained therein, the generally suitable dosage range of the siRNA, siRNA conjugate or pharmaceutical composition for inhibiting PLN gene expression involved in the present disclosure will be in the range of about 0.1 mg / kg to about 10.0 mg / kg, preferably about 0.3 mg / kg to about 3.0 mg / kg.

[0247] The administration routes involved in the present disclosure include intravenous administration, subcutaneous administration, intrathecal injection, intramuscular administration, transdermal administration, airway administration (aerosol), ocular administration, nasal administration, rectal administration, pulmonary administration, and topical administration (including buccal administration and sublingual administration), etc.

[0248] The siRNA, siRNA conjugate and pharmaceutical composition of the present disclosure can specifically target the heart, complementarily pair with the cardiac PLN mRNA sequence, induce the degradation of PLN mRNA, thereby inhibiting the protein synthesis and aggregation of PLN in the heart, improving the normal uptake of Ca 2+ by the sarcoplasmic reticulum and cardiac contractility, while avoiding off-target effects.

[0249] 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 vivo, so the dosing frequency is low (once every six months), and patient compliance is good.

[0250] In summary, the siRNA, siRNA conjugate, and pharmaceutical composition provided by the present disclosure have a strong inhibitory ability on the PLN gene, can significantly reduce the expression level of PLN mRNA, and have low drug toxicity. Therefore, the siRNA, siRNA conjugate, and pharmaceutical composition of the present disclosure can effectively prevent and / or treat diseases associated with PLN expression, such as heart failure, cardiomyopathy, and arrhythmia, providing more effective, safe, and convenient therapeutic drugs for patients and having good prospects for drug development. Brief Description of the Drawings

[0251] Figure 1 It is a schematic diagram of the high-efficiency active interval of PLN mRNA.

[0252] Figure 2 It is the result of the cytotoxicity experiment of the siRNA conjugate.

[0253] Figure 3 It is the in vivo research result of the siRNA conjugate inhibiting PLN expression. Detailed Description of the Embodiments

[0254] Definitions

[0255] In the above and below, unless otherwise specified, the capital letters C, G, U, and A represent cytosine, guanine, uracil, and adenine nucleotides; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide; the lowercase letter s indicates that there is a phosphorothioate subunit linkage between the two nucleotides adjacent to the left and right of the letter s; the letter combination VP indicates that the nucleotide adjacent to the right of the letter combination VP is a (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide; L96 has the structure of formula (I) and is linked to the 3'-end of the sense strand through a phosphodiester bond.

[0256] As used above and below, the term "fluorine-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose moiety of the nucleotide is replaced by fluorine, and the term "non-fluorine-modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2'-position of the ribose moiety of the nucleotide is replaced by a non-fluorine group. A "nucleotide analogue" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide or thymidine deoxyribonucleotide. Such as an isonucleotide, a bridged nucleic acid (BNA) or an acyclic nucleotide. The term "methoxy-modified nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2'-position of the ribose moiety with a methoxy group. In the context of this article, the expressions "complementary" or "reverse complementary" can be used interchangeably and have the meanings well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary manner. 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 a purine and a 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 to be complementary to each other, and the sequence of one strand can be deduced from the sequence of its complementary strand. Correspondingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at corresponding positions do not pair in a complementary form. As used 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; "essentially reverse complementary" means that there is no more than 1 base mismatch between the two nucleotide sequences; "fully reverse complementary" means that there are no base mismatches between the two nucleotide sequences. As used above and below, especially when describing the preparation methods of the siRNAs, pharmaceutical compositions or siRNA conjugates of the present disclosure, unless otherwise specified, the term "nucleoside monomer" refers to the modified or unmodified nucleoside phosphoramidite monomers (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites) used in the phosphoramidite solid-phase synthesis according to the types and sequences of nucleotides in the siRNA or siRNA conjugate to be prepared. The phosphoramidite solid-phase synthesis is a method well known to those skilled in the art for siRNA synthesis. The nucleoside monomers used in the present disclosure are all commercially available.

[0257] In Table 1 and Table 2, if there is no "VP" marked to the left of the 5'-terminal nucleotide of the sense strand and the modified sense strand conjugated with the linker group, it means that the 5'-terminal nucleotide is not linked to a 5'-phosphate group or a 5'-phosphate-derived group, and its structure is as shown in Formula (II):

[0258]

[0259] Wherein, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or is substituted by various groups known to those skilled in the art. For example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino, 2'-deoxynucleotide.

[0260] In Table 1 and Table 2, if there is no "VP" marked to the left of the 5'-terminal nucleotide of the antisense strand and the modified antisense strand, it means that the 5'-terminal nucleotide is not linked to a 5'-phosphate group or a 5'-phosphate-derived group, and its structure is also as shown in Formula (II).

[0261] In Table 1 and Table 2, the 3'-position of the 3'-terminal nucleotide of the sense strand, the antisense strand and the modified antisense strand is a hydroxyl group.

[0262] Examples

[0263] Other objects, features and advantages of the present disclosure 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 disclosure) are given for illustrative purposes only, because various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art after reading this detailed description.

[0264] The experimental techniques and methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods without specific conditions described in the following examples are usually carried out under conventional conditions such as those described by 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. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0265] Example 1. siRNA Design and Synthesis

[0266] 1.1 siRNA Design

[0267] Design a set of siRNAs targeting the human PLN gene (human PLN gene: NCBI refseq ID NM_002667.5; NCBI Gene ID: 5350) using OligoWalk online. The human NM_002667.5 REFSEQ mRNA has a length of 2989 bases. At the same time, to avoid toxicity caused by any sequence, sequences similar to human genes also need to be excluded.

[0268]

[0269] 1.2 siRNA Sequence Synthesis

[0270] Synthesize siRNA according to the standard solid-phase oligonucleotide synthesis protocol, including negative control siRNA (siCtrl).

[0271] Solid-phase oligonucleotide synthesis protocol: Use commercially available 5'-DMT-2'-TBDMS-rU phosphoramidite monomer, 5'-DMT-2'-TBDMS-rA(Bz) phosphoramidite monomer, 5'-DMT-2'-TBDMS-rC(Ac) phosphoramidite monomer, 5'-DMT-2'-TBDMS-rG(iBu) phosphoramidite monomer to synthesize RNA on a 500 nmol scale. Prepare the phosphoramidite solution at a concentration of 50 mM, and use a 0.3 M benzylthiotetrazole (BTT) acetonitrile solution as the activator. During the synthesis, use a 0.1 M oxidation reagent (pyridine:THF:water = 20:78:2) to convert trivalent phosphorus to pentavalent phosphorus to stabilize the phosphate backbone. After the synthesis is completed, ammonolyze the sequence from the solid-phase support and sediment. Remove the 2'O-tert-butyldimethylsilyl protecting group at the 2' position with triethylamine trihydrofluoride.

[0272] For the synthesized RNA sequence, under the condition of 55 °C, ammonolyze it with an ammonolysis solution of ammonia:methylamine = 1:1 for 40 minutes. After the ammonolysis is completed, remove the solid-phase support and take the supernatant to dryness. Add a protecting group removing agent of triethylamine trihydrofluoride:triethylamine:NMP = 6:4:3, react at 60 °C for 2 hours, add n-butanol in a ratio of 1:5, let it stand at -20 °C for 30 minutes, and centrifuge to obtain the precipitate. Dissolve it in RNase-free water and purify it by reverse-phase chromatography (0.1 M triethylamine acetate (TEAA) and acetonitrile). Ultrafilter and desalt the purified sample with PBS. And perform annealing to obtain siRNA, and verify the obtained siRNA, and it is found that the target siRNA is successfully prepared.

[0273] 1.3 Modification of siRNA Sequence and Synthesis of Conjugates

[0274] Synthesize modified siRNA according to the solid-phase oligonucleotide synthesis protocol. The modified nucleotide groups can be introduced into the siRNA of the present disclosure by using nucleoside monomers with corresponding modifications, and the methods for preparing nucleoside monomers with corresponding modifications are also well-known to those skilled in the art. Synthesize siRNA conjugates by conjugating L96 to siRNA with reference to the synthesis methods disclosed in WO2014025805A1 or WO2017015109A1.

[0275] The structure of the conjugate group L96 is shown as follows:

[0276]

[0277] Annealing of oligoribonucleotides to produce siRNA conjugates: The RNA oligomers to be annealed were prepared into a 200 μΜ solution with sterile RNase-Free H2O (RNase-free). Set up the annealing reaction system as follows: Place 100 μL of the above solution (double-stranded concentration is 10 nmol) in a 95 °C water bath for 10 minutes (20 minutes at high temperature is required for a demand of ≥100 nmol) → quickly place it in a 60 °C water bath and let it cool naturally → store the solution after annealing at 4 °C. Mix complementary strands by combining equimolar RNA solutions. After identification, the siRNA conjugate was correctly constructed.

[0278] Prepare the siRNA and siRNA conjugate solutions into dry powders for later use.

[0279] The sequences of the synthesized siRNAs are shown in Table 1 below, and a schematic diagram of the highly active region of PLN mRNA is as Figure 1 shown.

[0280] Table 1. siRNA sequence table targeting PLN

[0281]

[0282]

[0283]

[0284]

[0285] Among them, A, U, G, and C represent adenine, uracil, guanine, and cytosine nucleotides. The sequences of the synthesized siRNA conjugates are shown in Table 2 below:

[0286] Table 2: siRNA conjugates targeting PLN

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293] Among them, the lowercase letter m indicates that the nucleotide adjacent to the left of this letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of this letter f is a fluoro-modified nucleotide; the lowercase letter s indicates that there is a phosphorothioate subunit linkage between the two nucleotides adjacent to the left and right of this letter s; VP indicates that the nucleotide adjacent to the right of this letter combination VP is a vinylphosphonate (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide; L96 represents the L96 conjugate group linked to siRNA.

[0294] Example 2. In vitro activity screening of siRNA on 293T cell line

[0295] 2.1 Experimental procedure

[0296] 2.1.1 Cell culture

[0297] Culture 293T cells (China Center for Type Culture Collection (CCTCC), catalog number: CVCL_0063) in DMEM complete medium (Eallbio, supplemented with 10% FBS) at 37°C and 5% CO2. When the confluence rate reaches 80%-90%, digest the cells with trypsin, count them and perform transfection.

[0298] 2.1.2 Preparation of siRNA dilution

[0299] (1) Centrifuge the dry powder of the siRNA to be tested at low temperature and high speed, and then dissolve it with ultrapure distilled water to prepare a stock solution of 100 μM siRNA.

[0300] (2) Prepare a 200 nM siRNA dilution Y

[0301] a) Take 50 μl of the 100 μM siRNA stock solution prepared in the above step (1), add 50 μl of ultrapure distilled water to obtain a siRNA dilution with a final concentration of 50 μM.

[0302] b) Take 2 μl of the 50 μM siRNA dilution prepared in step a), add 18 μl of ultrapure distilled water to obtain a siRNA stock solution X with a final concentration of 5 μM.

[0303] c) Take 2 μl of the prepared siRNA stock solution X, add 48 μl of Opti-medium (Opti-MEM I Medium, Gibco, catalog number: 31985070) to obtain a 200 nM siRNA dilution Y.

[0304] 2.1.3 Transfection of 293T cells

[0305] Take 0.6 μl of RNAiMAX transfection reagent (Thermo Fisher, catalog number: 13778150) was added to 10 μl of Opti-medium to obtain RNAiMAX transfection reagent dilution; The RNAiMAX transfection reagent dilution was mixed with the 200 nM siRNA dilution Y prepared in 2.1.2 at a volume ratio of 1:1 to prepare a transfection mixture, which was allowed to stand for 5 minutes. Then, 10 μl of the transfection mixture was added to a 96-well plate, and 90 μl of the 293T cells cultured in 2.1.1 was added (final volume 100 μl / well, cell number 20,000 cells / well, and the concentration of siRNA in this system was 10 nM); After the above transfection, the cells were cultured for 24 hours.

[0306] 2.1.4 RNA Extraction

[0307] According to the product manual of the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Nanjing Novoprotein Scientific Co., Ltd., catalog number: CL132-01), the total RNA of the 293T cells obtained in 2.1.3 was extracted.

[0308] 2.1.5 Fluorescent Quantitative PCR

[0309] The extracted total RNA was subjected to reverse transcription and real-time PCR analysis using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit.

[0310] 2.1.6 Result Analysis

[0311] (1) Using the software of the 7500 Real-Time Fluorescent Quantitative PCR Instrument (Thermo Fisher), the Ct value was automatically calculated;

[0312] (2) The relative expression level of the gene was calculated using the following formula:

[0313] ΔCt1 = Ct (PLN group) – Ct (ACTIN of the PLN group)

[0314] ΔCt2 = Ct (siCtrl group) – Ct (ACTIN of the siCtrl group)

[0315] ΔΔCt = ΔCt1 - ΔCt2, where the siCtrl group represents the non-specific siRNA sequence and is the negative control group; the mRNA expression relative to the siCtrl group = 2 -ΔΔCt

[0316] Inhibition rate (%) = (1 - mRNA expression relative to the siCtrl group) × 100%

[0317] 2.2 Experimental results

[0318] The inhibitory effects of the siRNAs of the present disclosure are shown in Table 3 below:

[0319] Table 3. In vitro screening results of siRNA in 293T cell line

[0320]

[0321]

[0322]

[0323] As can be seen from Table 3, some of the siRNAs of the present disclosure can significantly inhibit the expression of the PLN gene at 10 nM.

[0324] Example 3. Cytotoxicity experiment

[0325] 3.1 Experimental procedures

[0326] 3.1.1 Cell culture

[0327] The 293T cells were cultured in DMEM complete medium (Eallbio, catalog number: 03.1002C; supplemented with 10% FBS) in an environment of 37 °C and 5% CO2. When the confluence rate reached 80%-90%, the cells were digested with trypsin, counted and transfected.

[0328] 3.1.2 Transfection of 293T cells

[0329] Based on the results of Example 2 above, some siRNAs with better in vitro activity were screened for toxicity determination. 293T cells were transfected in a method similar to that of Example 2. After culturing for 72 hours, 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. According to the manufacturer's protocol, the intracellular ATP content was determined by using CellTiter-Glo assay to measure cell viability. According to the manufacturer's protocol, the cytotoxicity in the supernatant was measured using ToxiLightTM. The cytotoxicity results are shown in Figure 2 .

[0330] 3.2 Experimental results

[0331] The results show that some of the siRNA conjugates of the present disclosure have low cytotoxicity and good cell compatibility.

[0332] Based on the in vitro screening results of Example 2 and Example 3, the highly efficient active intervals for inhibiting PLN mRNA were thus determined, which are positions 292 - 357, 444 - 626, and 1127 - 1149 calculated according to NCBI refseqID NM_002667.5.

[0333] Example 4. In Vivo Activity Screening of siRNA Conjugates

[0334] According to the in vitro experimental screening results of the above Example 2 and Example 3, some siRNA sequences with better in vitro activities were selected to verify the in vivo activities of the above siRNA conjugates.

[0335] 4.1 Experimental Procedures

[0336] Male mice (C57BL / 6) at 6 - 8 weeks of age, purchased from Beijing SparkJade Biotechnology Co., Ltd., were intravenously injected with 1×10 11 genomic copies of recombinant adeno - associated virus 8 (AAV8) vector. The recombinant AAV8 vector was packaged by the AAV8 capsid protein expression plasmid and the transfer plasmid carrying AAV8 - hPLN. The transfer plasmid carried positions 354 - 2989 of the human PLN sequence (NM_002667.5) and was controlled by the TBG promoter (AAV8 - TBG - hPLN), purchased from Vazyme Biotech Co., Ltd. (Guangzhou). The construction of the AAV8 - hPLN transgenic mouse model was completed 14 days after injection. Then, the conjugates were administered subcutaneously at a dose of 3 mg / kg per mouse. The mice were sacrificed on the 7th day (D7), 14th day (D14), and 19th day (D19) after administration, 6 mice in each group, and liver tissues were taken for PLN mRNA expression detection. Total RNA was extracted by the Trizol method, mRNA reverse transcription was performed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, product number: R323 - 01), and real - time fluorescence quantitative PCR was performed using the ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, product number: Q711 - 03). The in vivo activity screening results of the siRNA conjugates are shown in Tables 4 and 5.

[0337] Table 4. In Vivo Screening Results of siRNA Conjugates

[0338]

[0339]

[0340] Table 5. In Vivo Screening Results of siRNA Conjugates

[0341]

[0342] 4.2 Experimental Results

[0343] The chemically modified partial siRNA sequences have good activity in mice. YGND11-13M, YGND11-16M, YGND11-17M, YGND11-18M, YGND11-21M, YGND11-26M, YGND11-31M, YGND11-41M, and YGND11-73M can reduce the expression level of PLN in mice, among which YGND11-13M, YGND11-21M, YGND11-31M, YGND11-41M, and YGND11-73M have high knockdown efficiency on the expression of the PLN gene.

[0344] Example 5. Multi-dose In Vivo Activity Screening of siRNA Conjugates

[0345] According to the in vivo experiment screening results of Example 4 above, select some siRNA conjugates for in vivo activity verification.

[0346] 5.1 Experimental Procedures

[0347] Use the method described in Example 4 to construct an AAV8-hPLN transgenic mouse model. Subcutaneously administer the conjugate to each mouse at a dose of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Sacrifice the mice on the 19th day after administration, and take liver tissues for PLN mRNA expression detection. The detection methods and reagents are the same as those in Example 4. The multi-dose in vivo activity screening results of the siRNA conjugate are shown in Table 6 below and Figure 3 as shown.

[0348] Table 6. Multi-dose In Vivo Screening Results of siRNA Conjugates

[0349]

[0350]

[0351] Note: N / A indicates that the data was not measured

[0352] 5.2 Experimental Results

[0353] The results show that the siRNA conjugates of the present disclosure all show good dose-dependent effects. Among them, YGND11-21M has the highest inhibitory efficiency on the expression of the PLN gene, and the inhibitory efficiency is as high as 71.57% at a dosing dose of 3 mg / kg. In addition, the inhibitory efficiency of YGND11-26M is as high as 65.27% at a dosing dose of 3 mg / kg.

Claims

1. An siRNA for inhibiting PLN gene expression, the siRNA comprising a sense strand and an antisense strand, the sequence of the sense strand being as shown in SEQ ID NO:22, the sequence of the antisense strand being as shown in SEQ ID NO:121, and each nucleotide in the sense strand and the antisense strand being a modified or unmodified nucleotide.

2. The siRNA according to claim 1, wherein Each nucleotide in the sense strand and the antisense strand is a modified nucleotide, and the modified nucleotide is a fluorine-modified nucleotide or a methoxy-modified nucleotide; The fluorine-modified nucleotide refers to a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose of the nucleotide with fluorine, and it has the structure shown in the following formula (1); the methoxy-modified nucleotide refers to a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose with a methoxy group, and it has the structure shown in the following formula (2): In formula (1) to formula (2), Base represents a base; In the direction from the 5'-end to the 3'-end, the 7th, 9th, 10th, and 11th nucleotides of the sense strand are fluorine-modified nucleotides; and, in the direction from the 5'-end to the 3'-end, the 2nd, 6th, 8th, 9th, 14th, and 16th nucleotides of the antisense strand are fluorine-modified nucleotides; The phosphate ester group in the phospho-sugar backbone of the sense strand and the antisense strand has a phosphate ester group with a modifying group, and the phosphate ester group with a modifying group is a phosphorothioate group having the structure shown in formula (13): The connection of the phosphorothioate group is present at the following positions: between the 1st and 2nd nucleotides of the sense strand and the antisense strand; between the 2nd and 3rd nucleotides of the sense strand and the antisense strand; between the 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; The 5'-terminal nucleotide of the antisense strand is a nucleotide modified with a 5'-phosphate analogue, as shown in formula (15):

3. A siRNA conjugate comprising the siRNA according to claim 2 and a conjugate group conjugated to the siRNA, wherein, The conjugating group is L96, and the structure is as shown below: The siRNA conjugate comprises a sense strand and an antisense strand, the sequence of the sense strand being as shown in SEQ ID NO:22, and the sequence of the antisense strand being as shown in SEQ ID NO:

319.

4. A composition comprising the siRNA according to claim 1 or 2 or the siRNA conjugate according to claim 3.

5. The composition according to claim 4, wherein The composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier or excipient.

6. The composition according to claim 5, wherein, The carrier or excipient includes one or more of 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, distearoyl phosphatidylcholine, 1,2-dimyristoyl glycerol, dimethyl adipate.

7. Use of the siRNA according to claim 1 or 2, or the siRNA conjugate according to claim 3, or the composition according to any one of claims 4 to 6 in the preparation of a medicament for preventing and / or treating a disease associated with PLN expression, wherein the disease associated with PLN expression is one or more of heart failure, cardiomyopathy, and arrhythmia.

8. The use according to claim 7, wherein The heart failure includes one or more of acute heart failure and chronic heart failure.

9. Use according to claim 7, wherein The heart failure includes one or more of systolic heart failure and diastolic heart failure.

10. The use according to claim 7, wherein The heart failure includes one or more of left heart failure, right heart failure, and whole heart failure.

11. The use according to claim 7, wherein, The cardiomyopathy is hereditary cardiomyopathy, including one or more of cardiomyopathy caused by p.Arg14del, Arg9Cys (R9C), and / or Arg25Cys (R25C) gene mutations.

12. The use according to claim 7, wherein, The cardiomyopathy is dilated cardiomyopathy, including one or more of dilated cardiomyopathy caused by mutations in TTN, LMNA, RBM20, SCN5A, MYH7, TNNT2, and / or TPM1.

13. The use according to claim 7, wherein, The arrhythmia includes ventricular tachycardia and / or ventricular fibrillation.

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