Sirna for inhibiting PLN gene expression and conjugate thereof, and use thereof

Abstract

Provided are an siRNA for inhibiting PLN gene expression and a conjugate. 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 segment, and the first nucleotide sequence segment is a nucleotide sequence having a length of at least 15 nucleotides in an mRNA expressed by the PLN gene. The siRNA, the conjugate thereof, and a pharmaceutical composition thereof can effectively prevent and / or treat diseases associated with PLN expression, such as heart failure, cardiomyopathy and arrhythmia.

Description

siRNA for inhibiting PLN gene expression, conjugates thereof and application TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to siRNA for inhibiting PLN gene expression, conjugates thereof and application. BACKGROUND

[0002] Heart failure (hereinafter referred to as heart failure) is a global epidemic that is becoming increasingly serious, with a global patient population of 6430 million, of which the number of heart failure patients in China reached 1300 million as of 2023. Although the research on heart failure is continuously deepening, and the clinical treatment means are continuously increasing (such as drug intervention of angiotensin II or implantation of a ventricular assist device, etc.), the morbidity and mortality of heart failure patients are still high, and the long-term prognosis is poor, with a mortality rate of 50% within 5 years, so it is urgent to find a new treatment method.

[0003] Dilated cardiomyopathy refers to the presence of left or biventricular dilation or systolic dysfunction in the absence of abnormal loading conditions or severe coronary artery disease sufficient to cause ventricular remodeling. The initial symptoms of dilated cardiomyopathy are atypical and may manifest as decreased exercise tolerance, dyspnea, edema in the lower extremities and abdomen, chest tightness and palpitations, etc. Some patients with dilated cardiomyopathy may not notice any symptoms at first. However, patients with dilated cardiomyopathy can develop heart failure and severe arrhythmias, which can be life-threatening. The causes of dilated cardiomyopathy are diverse, including familial genetic factors.

[0004] Heart failure is characterized by a decline in cardiac contractile performance, and a common feature is impaired calcium sequestration in the sarcoplasmic reticulum (SR). The key factor is the reduced activity of the Ca 2+ -ATPase (SERCA2a) that mediates SR calcium uptake, leading to abnormal calcium uptake with increased unphosphorylated phospholamban (PLN). PLN is a major regulator of SERCA2a activity and is the only SERCA2a-related protein directly involved in the development of heart disease, including heart failure, to date. Dephosphorylated PLN is an inhibitor of SERCA, and phosphorylation of PLN can alleviate this inhibition. In addition, PLN is also a key mediator of beta-adrenergic stimulation, and changes in its phosphorylation are associated with functional changes in cardiac SR and contractility, making it a key regulator of cardiac contractility. Therefore, PLN has become a highly potential nucleic acid drug target for the treatment of heart failure.

[0005] PLN is a reversibly phosphorylated SR protein that can modify overall cardiac function. In many cases, ablation of PLN can successfully rescue the observed decline in cardiac function and Ca 2+ absorption in various animal models of heart failure. Therefore, it is of great significance to find a new method for treating heart failure by targeting PLN.2+ Abnormal uptake. Elevated PLN levels are detrimental in heart failure or cardiomyopathy. Upregulation of PLN inhibits SERCA2a activity and reduces calcium levels. 2+ In addition, the human R14del-PLN mutation leads to the development of cardiomyopathy, exhibiting clinical features of arrhythmogenic cardiomyopathy and dilated cardiomyopathy, potentially progressing to heart failure and premature death. Similar results have been observed in mice, where the R14del-PLN mutation causes dilated cardiomyopathy, resulting in PLN protein aggregation and cardiac dysfunction; reducing PLN expression has become a strategy for treating heart failure. However, evidence suggests that humans cannot completely lack PLN, otherwise it will cause severe dilated cardiomyopathy, and the safety and tolerability of PLN reduction is only 50%. Therefore, there is a need to develop more precise, safer, and more effective nucleic acid drugs with lower dosing frequency. Summary of the Invention

[0006] The purpose of this invention is to provide an siRNA molecule that can inhibit PLN gene expression, in order to provide a new treatment for PLN-related diseases, including heart failure, cardiomyopathy, and arrhythmia.

[0007] In a first aspect, this 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 nucleotide sequence I and nucleotide sequence II is a modified or unmodified nucleotide; nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; nucleotide sequence I is substantially identical to a first nucleotide sequence, the first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length in the mRNA expressing the PLN gene, preferably, the first nucleotide sequence being a nucleotide sequence of 15 to 25 nucleotides in length in the mRNA expressing the PLN gene, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides;

[0008] Preferably, the first nucleotide sequence is a nucleotide sequence of at least 15 nucleotides in length from the highly active region of the PLN gene-expressed mRNA, preferably a nucleotide sequence of 15 to 25 nucleotides. The highly active region is positions 292-357, 444-626, and 1127-1149 of the PLN gene-expressed mRNA, preferably positions 292-314, 315-337, 318-340, 327-349, 335-357, 444-466, 476-498, 604-626, and 1127-1149.

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

[0010] The high activity interval refers to the interval in which the designed siRNA and siRNA conjugate can effectively reduce the level of PLN mRNA. In FIG. 1, the interval is divided according to whether the maximum inhibition rate of siRNA and siRNA conjugate observed on PLN mRNA falls within 40-60%, 60%-80% or more than 80%.

[0011] In some embodiments, in the above-mentioned siRNA, the nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the first nucleotide sequence.

[0012] In some embodiments, in any of the above-mentioned siRNA, the nucleotide sequence II is substantially, substantially or completely reverse complementary to the first nucleotide sequence.

[0013] In some embodiments, in any of the above-mentioned siRNA, the sense strand and the antisense strand are the same or different in length, 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 and the antisense strand of the siRNA is 16 / 21, 19 / 21, 21 / 23, 19 / 24.

[0014] In some embodiments, in any of the above-mentioned siRNA, the nucleotide sequence I comprises at least 15 consecutive nucleotides, such as at least 15, 16, 17, 18, 19, 20 or 21 nucleotides, as shown in any of SEQ ID NO: 2-100.

[0015] In some embodiments, the nucleotide sequence of the sense strand is as shown in nucleotide sequence I, which differs from any of SEQ ID NO: 2-100 by 1, 2 or 3 nucleotides.

[0016] In some embodiments, the nucleotide sequence I is as shown in any of SEQ ID NO: 2-100.

[0017] In some embodiments, in any of the above-mentioned siRNA, the nucleotide sequence II comprises at least 15 consecutive nucleotides, such as at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides, as shown in any of SEQ ID NO: 101-199. In some embodiments, in any of the above-mentioned siRNA, the nucleotide sequence II comprises at least 15 consecutive nucleotides, such as at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides, as shown in any of SEQ ID NO: 101-199.

[0018] In some embodiments, the nucleotide sequence of the antisense strand is set forth in Nucleotide Sequence II, which differs from any one of SEQ ID NOs: 101-199 by 1, 2, or 3 nucleotides.

[0019] In some embodiments, the nucleotide sequence II is set forth in any one of SEQ ID NOs: 101-199.

[0020] In some embodiments, any one of the siRNAs described above, the siRNA is an siRNA molecule selected from the group consisting of YGND11-1 to YGND11-99 as set forth in Table 1.

[0021] In some embodiments, any one of the siRNAs described above, each of the nucleotides in the nucleotide sequence I and the nucleotide sequence II is a modified nucleotide.

[0022] In some embodiments, the modified nucleotide is a fluorine-modified nucleotide or a non-fluorine-modified nucleotide.

[0023] In some embodiments, the fluorine-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribosyl group of the nucleotide is replaced by fluorine, which has the structure shown in Formula (1); and the non-fluorine-modified nucleotide refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2'-position of the ribosyl group of the nucleotide is replaced by a non-fluorine group. In some embodiments, each non-fluorine-modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2'-position of the ribosyl group 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 ribosyl group is replaced by a non-fluorine group are well known to those skilled in the art, and can be selected from one of a 2'-alkoxy-modified nucleotide or a nucleotide analog, a 2'-substituted alkoxy-modified nucleotide or a nucleotide analog, a 2'-alkyl-modified nucleotide or a nucleotide analog, a 2'-substituted alkyl-modified nucleotide or a nucleotide analog, a 2'-amino-modified nucleotide or a nucleotide analog, a 2'-substituted amino-modified nucleotide or a nucleotide analog, and a 2'-deoxynucleotide. 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 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).

[0024] A nucleotide analogue refers to a group that can replace a nucleotide in a nucleic acid, but is structurally different from an adenine ribonucleoside, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine ribonucleotide. In some embodiments, a nucleotide analogue can be a heteronucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0025] A bridged nucleotide refers to a constrained or inaccessible nucleotide. A bridged nucleotide can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a fixed C3-endo sugar puckering bridged structure. In some embodiments, a bridged nucleotide can be an LNA, an ENA, a cET BNA, etc.; wherein the LNA is as shown in formula (6), the ENA is as shown in formula (7), and the cET BNA is as shown in formula (8).

[0026] An acyclic nucleotide is a type of nucleotide in which the sugar ring of the nucleotide is opened. In some embodiments, an acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycol nucleic acid (GNA); wherein the UNA is as shown in formula (9) and the GNA is as shown in formula (10).

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

[0028] A heteronucleotide refers to a compound formed by changing the position of a base on the ribose ring in a nucleotide. In some embodiments, a heteronucleotide can be a compound formed by moving the base from the 1'-position to the 2'-position or the 3'-position on the ribose ring. As shown in formula (11) or formula (12).

[0029] In formula (11) and formula (12), R is selected from H, OH, F, or a non-fluorine group as described above.

[0030] In formula (1) to formula (12), Base represents a base.

[0031] In some embodiments, in any of the above-described siRNAs, one or more of the nucleotides at positions 7, 9, 10, and 11 of the nucleotide sequence I, in the direction from the 5'-end to the 3'-end, are fluorine-modified nucleotides; and one or more of the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II, in the direction from the 5'-end to the 3'-end, are fluorine-modified nucleotides.

[0032] In some embodiments, in the nucleotide sequences I and II, in addition to fluorine modification, the other nucleotides are methoxy-modified.

[0033] In some embodiments, at least one of the phosphates in the phosphodiester backbone of at least one of the sense strand and the antisense strand of the siRNA is replaced with a phosphate having a modified group. In some embodiments, the phosphate having a modified group is a phosphorothioate group in which at least one of the oxygen atoms of the phosphodiester bond is replaced with a sulfur atom. In some embodiments, the phosphate having a modified group is a phosphorothioate group having the structure of Formula (13):

[0034] In some embodiments, in any of the siRNAs described above, the phosphorothioate group is linked at 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 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination thereof.

[0035] In some embodiments, in any of the siRNAs described above, the 5 '-terminal nucleotide of the antisense strand of the siRNA is a 5 '-phosphate nucleotide or a 5 '-phosphate analog modified nucleotide, such as Formula (14), Formula (15), and Formula (16):

[0036] 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 can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. In some embodiments, the conjugation site of the siRNA to the conjugate group can be at the 3 '-end or 5 '-end of the sense strand, at the 3 '-end of the antisense strand, or in the internal sequence of the siRNA.

[0037] In some embodiments, in the siRNA conjugate described above, the conjugate group is L96, which has the structure as follows:

[0038] In some embodiments, the siRNA conjugate is siRNA conjugate YGND11-1M to YGND11-99M shown in Table 2.

[0039] In a third aspect, the present disclosure provides a composition comprising any of the siRNAs or siRNA conjugates described above.

[0040] In some embodiments, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier or excipient involved in the present disclosure 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-dimyristin, dimethyl adipate.

[0041] In a fourth aspect, the present disclosure provides use of any of the above-mentioned siRNA, siRNA conjugate or composition in the manufacture of a medicament for preventing and / or treating a disease associated with PLN expression.

[0042] In a fifth aspect, the present disclosure provides a method for preventing and / or treating a disease associated with PLN expression, comprising administering to a subject a therapeutically effective amount or a prophylactically effective amount of the siRNA, siRNA conjugate or composition of the present disclosure.

[0043] In some embodiments, the disease associated with PLN expression comprises one or more of heart failure, cardiomyopathy, arrhythmia;

[0044] Preferably, the heart failure comprises one or more of acute heart failure, chronic heart failure, systolic heart failure, diastolic heart failure, left-sided heart failure, right-sided heart failure, global heart failure;

[0045] Preferably, the cardiomyopathy is dilated cardiomyopathy, comprising one or more of PLN, TTN, LMNA, RBM20, SCN5A, MYH7, TNNT2 and / or TPM1 mutation-induced dilated cardiomyopathy; more preferably, the dilated cardiomyopathy is genetic cardiomyopathy, comprising one or more of PLN p.Arg14del, Arg9Cys (R9C) and / or Arg25Cys (R25C) gene mutation-induced cardiomyopathy;

[0046] Preferably, the arrhythmia comprises ventricular tachycardia and / or ventricular fibrillation.

[0047] In a sixth aspect, the present disclosure provides the siRNA, siRNA conjugate or composition of the present disclosure for use in therapy.

[0048] The composition involved in the present disclosure can be used alone for the treatment of a disease associated with PLN expression, and can also be used in combination with standard oral drugs, thereby providing experimental support for diversified treatment options for clinical patients with the above-mentioned diseases.

[0049] The siRNA, siRNA conjugate or composition of the present disclosure for inhibiting the expression of PLN gene is generally suitable for a dosage 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, in terms of the amount of siRNA contained therein.

[0050] The administration route of the siRNA, siRNA conjugate or composition of the present disclosure includes 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.

[0051] The siRNA, siRNA conjugate and composition of the present disclosure can specifically target the heart, complementarily pair with the PLN mRNA sequence of the heart, induce the degradation of PLN mRNA, thereby inhibiting the protein synthesis and accumulation of PLN in the heart, improving the normal uptake of Ca 2+ by sarcoplasmic reticulum and the contractility of the heart, while avoiding off-target effects.

[0052] In addition, compared with traditional small molecule drugs and antibody drugs, small nucleic acid drugs can directly regulate the expression of upstream genes, and are less likely to produce drug resistance; moreover, small nucleic acid drugs have a long half-life in vivo, and thus have a low administration frequency (can be administered once every half year), and good patient compliance.

[0053] In summary, the siRNA, siRNA conjugate and composition of the present disclosure have a strong inhibitory effect on PLN gene, can significantly reduce the expression amount 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 heart failure, cardiomyopathy, arrhythmia and other diseases associated with PLN expression, provide more effective, safe and convenient therapeutic drugs for patients, and have a good drug development prospect. BRIEF DESCRIPTION OF DRAWINGS

[0054] FIG. 1 is a schematic diagram of the high-efficiency active region of PLN mRNA.

[0055] FIG. 2 is the result of a cytotoxicity experiment of the siRNA conjugate.

[0056] FIG. 3 is the result of an in vivo study of the siRNA conjugate for inhibiting the expression of PLN.

[0057] FIG. 4 is the result of off-target analysis of the siRNA conjugate.

[0058] FIG. 5 is the result of field potential of the siRNA conjugate in organoid research.

[0059] FIG. 6 is the result of the number of heartbeats per minute of the siRNA conjugate in organoid research.

[0060] Figure 7 is a result of siRNA conjugate organoid field potential duration. DETAILED DESCRIPTION

[0061] DEFINITIONS

[0062] In the present context, the words "comprising", "having", "including" or "containing" mean inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0063] In the present context, "sequence identity" means that the percentage of bases that are identical in the comparison of two sequences when aligned. Such alignment can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology edited by Ausubel et al. (2007). Preferably, the alignment is performed using default parameters. One alignment program using default parameters is BLAST. In particular, the program BLASTN using the following default parameters: genetic code = standard; filter = no; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = high score; databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR.

[0064] In the present context, the term siRNA means a small interfering ribonucleic acid RNAi molecule. It is a class of double-stranded RNA molecules, also referred to in the art as short interfering RNA or silencing RNA. It is also referred to in the present context as double-stranded ribonucleic acid. The siRNA typically comprises a sense strand and an antisense strand, wherein the antisense strand is complementary (such as at least 70% complementary, such as fully complementary) to a target sequence (suitably a mature mRNA sequence, such as a human PLN mRNA) and the sense strand is complementary to the antisense strand such that the sense strand and the antisense strand form a duplex or duplex region.

[0065] In some embodiments, the target sequence is a nucleotide sequence consisting of no less than 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 80, 100, 200, 300, or 400 consecutively linked nucleosides.

[0066] In the present context, the term "antisense strand" refers to the nucleic acid strand of a double-stranded ribonucleic acid which is substantially complementary to a region of the target sequence. The antisense strand is sometimes not completely reverse complementary to the target sequence, the mismatches are usually in one or more regions at the end, e.g. within 4, 3, 2 or 1 nucleotides of the 5'- and / or 3'-end.

[0067] In the present context, the term "sense strand" refers to the nucleic acid strand of a double-stranded ribonucleic acid which contains a region which is substantially complementary to a region of the antisense strand.

[0068] The sense and antisense strands of the siRNA can form a blunt end duplex, or can form a duplex comprising a 3'-overhang, which can be, for example, 1, 2 or 3 nucleotides in length, similar to the product of Dicer, which can form the RISC substrate in vivo. In some embodiments, only the sense strand has a 3'-overhang of 2 nucleotides in length. In some embodiments, only the antisense strand has a 3'-overhang of 2 nucleotides in length. In some embodiments, both the sense and antisense strands have a 3'-overhang of 2 nucleotides in length.

[0069] In the present context, siRNA includes unmodified siRNA and modified siRNA. In the present context, "unmodified siRNA" refers to an siRNA duplex in which each nucleotide in the double-stranded ribonucleic acid is an unmodified nucleotide, such as the siRNAs shown in Table 1 herein. In the present context, "modified siRNA" refers to an siRNA molecule in which one or more nucleotides in the siRNA molecule are modified to improve the stability or efficacy of the siRNA molecule.

[0070] In the present context, "siRNA conjugate" refers to a conjugate resulting from linking a conjugate group to an unmodified siRNA or a modified siRNA, such as the siRNA conjugates shown in Table 2 herein.

[0071] In the present context, when referring to any nucleotide position of any strand of an siRNA or siRNA conjugate, the 5' to 3' direction is meant, unless otherwise specified.

[0072] In the foregoing and hereinafter, unless otherwise specifically indicated, capital letters C, G, U, A, T represent cytosine, guanine, uracil, adenine and thymine deoxynucleotides; lower case letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy modified nucleotide; lower case letter f represents that the nucleotide adjacent to the left of the letter f is a fluoro modified nucleotide; lower case letter s represents that the two nucleotides adjacent to the left and right of the letter s are linked by phosphorothioate subunit; letter combination VP represents 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) linked to the 3 '-end of the sense strand by a phosphonate linkage.

[0073] In the foregoing and hereinafter, the term "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose sugar is replaced with a fluorine atom, and the term "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose sugar is replaced with a group other than fluorine. The term "nucleotide analog" refers to a group that can substitute for a nucleotide in a nucleic acid, but differs in structure from an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine deoxyribonucleotide, such as an iso-nucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide. The term "methoxy-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose sugar is replaced with a methoxy group. In the context of this document, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e., 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, in RNA, uracil (U)); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair comprises one purine and one pyrimidine. When the adenine on one strand always pairs with the thymine (or uracil) on the other strand, and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" in the art means that the bases at the corresponding positions in a double-stranded nucleic acid do not pair in a complementary manner. In the foregoing and hereinafter, "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 are no more than 1 base mismatch between the two nucleotide sequences; and "completely reverse complementary" means that there are no base mismatches between the two nucleotide sequences. For example, an siRNA molecule of the disclosure comprises one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is completely complementary to the shorter oligonucleotide, such a case can be referred to as "completely complementary" for the purposes described herein. Thus, "completely complementary" as used herein means that all of the bases in a contiguous sequence of a first polynucleotide will hybridize to the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence can comprise all or a portion of the first or second nucleotide sequence.

[0074] The term "complementary" as used herein can refer to base pairing between the sense strand and the antisense strand of an siRNA, base pairing between the antisense strand of an siRNA and the sequence of a target PLN mRNA.

[0075] In the foregoing and hereinafter, particularly in describing the preparation of siRNA, pharmaceutical composition or siRNA conjugate of the present disclosure, the nucleoside monomer refers to, according to the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared, modified or unmodified nucleoside phosphoramidite monomers (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also referred to as Nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis. The phosphoramidite solid-phase synthesis is a method used in siRNA synthesis well known to those skilled in the art. The nucleoside monomers used in the present disclosure are all commercially available.

[0076] In Tables 1 and 2, if the 5'-terminal nucleotide of the sense strand and the modified sense strand to which the conjugating group is linked is not marked with VP, it represents that the 5'-terminal nucleotide is not linked with a 5' phosphate group or a 5' phosphate derivative group, and its structure is shown as formula (II):

[0077] wherein Base represents a base, for example, A, U, G, C or T; R is a hydroxyl group or substituted with 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.

[0078] In Tables 1 and 2, if the 5'-terminal nucleotide of the antisense strand and the modified antisense strand is not marked with VP, it represents that the 5'-terminal nucleotide is not linked with a 5' phosphate group or a 5' phosphate derivative group, and its structure is also shown as formula (II).

[0079] The 3' position of the 3'-terminal nucleotide of the sense strand of Table 1 is a hydroxyl group, and the 3' position of the 3'-terminal nucleotide of the antisense strand of Table 1 and the modified antisense strand of Table 2 is a hydroxyl group.

[0080] In this context, the term "PLN" is a well-known gene and polypeptide. The PLN gene, PLN mRNA sequence is readily available, for example, using GenBank, UniProt, Online Mendelian Inheritance in Man (OMIM), etc.

[0081] The term "PLN gene" refers to a PLN gene from any species, such as a mouse, rat, monkey, canine, feline, or human PLN gene, and can include both wild-type PLN genes as well as variants of PLN genes. In some embodiments, the "PLN mRNA" is a human PLN mRNA, such as the mRNA of the sequence set forth in NCBI refseq ID NM_002667.5, and the PLN gene refers to the gene that transcribes the above-mentioned PLN mRNA.

[0082] "Inhibiting PLN gene expression" includes inhibition of a PLN gene at any level, such as inhibiting at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0083] The term "preventing" means that, prior to the onset of a disease, a subject is contacted (e.g., administered) with a siRNA, siRNA conjugate, or composition of the disclosure, which can reduce the risk of developing the disease, delay the progression, or reduce adverse outcomes, and does not necessarily mean complete inhibition of the disease.

[0084] The term "treating" means that, after the onset of a disease, a subject is contacted (e.g., administered) with a siRNA, siRNA conjugate, or composition of the disclosure, which can reduce the symptoms of the disease as compared to not being contacted, and does not necessarily mean complete inhibition of the symptoms of the disease. Onset of a disease means that the body has developed symptoms of the disease.

[0085] The term "effective amount" means the amount of a siRNA, siRNA conjugate, or composition of the disclosure that, when administered to a patient at one or more doses, produces the intended effect in the patient being treated or prevented. The effective amount can be readily determined by the attending physician, as one skilled in the art, by the consideration of numerous factors that affect the dosage and administration of a therapeutic compound, such as the species, age, and general health of the mammal, the severity of the disease, the mode of administration, and the use of any concomitant therapy, among others.

[0086] In the present context, the term "mode of administration" means any mode of administration which is medically acceptable, meaning any mode which produces an effective prophylactic or therapeutic level of the PLN-associated disease or condition without causing clinically unacceptable adverse side effects. For example, oral, enteral, mucosal, subcutaneous and / or parenteral administration. The term "parenteral" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal and intrasternal injection or infusion techniques. Other routes include, but are not limited to, nasal (e.g., via a nasogastric tube), transdermal, vaginal, rectal, sublingual and inhalation.

[0087] In the present context, "subject" or "individual" or "patient" means any subject, particularly a mammalian subject, in need of prophylaxis or treatment. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, etc.

[0088] In the present context, "disease associated with PLN expression" is intended to include any disease associated with the PLN gene or protein. Such a disease can be caused, for example, by an overproduction of the PLN protein. Exemplary diseases associated with PLN expression include one or more of heart failure, cardiomyopathy, arrhythmia.

[0089] Unless otherwise defined or indicated by context, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0090] siRNA

[0091] The present disclosure provides siRNAs for inhibiting the expression of the PLN gene. The siRNAs inhibit the expression of the PLN gene by assembling into an RNA-induced silencing complex (RISC) complex, cleaving the target mRNA (i.e., the PLN mRNA).

[0092] The siRNAs of the present disclosure contain nucleotide groups as the basic structural unit, which contain a phosphate group, a ribose group and a base, the structure of which is well known to those skilled in the art.

[0093] The siRNAs of the present disclosure comprise a sense strand and an antisense strand, and are thus double-stranded ribonucleic acids.

[0094] In the present disclosure, the nucleotide sequence of the antisense strand can be fully complementary or substantially complementary to the target sequence. When the nucleotide sequence of the antisense strand is substantially complementary to the target sequence, there are no more than 3 mismatched bases in the nucleotide sequence of the antisense strand to the target sequence. For example, the mismatched bases are 1, 2, or 3. When the nucleotide sequence of the antisense strand is fully complementary to the target sequence, there are no mismatched bases in the nucleotide sequence of the antisense strand to the target sequence.

[0095] Further, the antisense strand consists of at least 15 nucleotides. As a preference, the antisense strand consists of 19-26 nucleotides, more preferably 19-23 nucleotides, most preferably 19, 21, or 23 nucleotides.

[0096] In some alternative embodiments, the antisense strand is identical to the reverse complement of a sequence consisting of at least 15 consecutive nucleotides on the target sequence.

[0097] In some embodiments, the sense strand comprises a sequence that differs from at least 15 consecutive nucleotides in the target sequence by no more than 3 nucleotides. The sense strand comprises a region that is complementary to the antisense strand, and the nucleotide sequence of the sense strand is fully identical or substantially fully identical to the sequence of the binding region on the target sequence of the antisense strand. Thus, the nucleotide sequence of the sense strand is at least 15 consecutive nucleotides in the target sequence that binds the antisense strand; or, the nucleotide sequence of the sense strand differs from at least 15 consecutive nucleotides in the target sequence that binds the antisense strand by 1, 2, or 3 base-different nucleotides.

[0098] Further, the sense strand consists of at least 15 nucleotides. As a preference, the sense strand consists of 16-23 nucleotides, more preferably 19-23 nucleotides, most preferably 19, 21, or 23 nucleotides.

[0099] In the present disclosure, the length of the sense strand can be the same as or different from the length of the antisense strand. In some embodiments, the length ratio of the siRNA sense strand and antisense strand of the present disclosure can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26. In some preferred embodiments, the length ratio of the sense strand / antisense strand is 19 / 21, 20 / 22, or 21 / 23.

[0100] In some embodiments, after the sense strand is complementary to the antisense strand to form a double-stranded region, the sense strand, the antisense strand, or a combination thereof has overhanging nucleotides extending out of the double-stranded region. The number of overhanging nucleotides can be one or more, for example, two. In addition, the overhanging nucleotides can be located at the 5' end, the 3' end, or both ends of any of the antisense strand or the sense strand, and each of the overhanging nucleotides can be any type of nucleotide. In some embodiments, the sense strand is complementary to the antisense strand to form the double-stranded region, and the 3' end of the antisense strand has two overhanging nucleotides extending out of the double-stranded region, and the 3' end of the sense strand has a blunt end.

[0101] In some embodiments, each nucleotide of the sense strand is independently a modified nucleotide or an unmodified nucleotide. In some embodiments, each nucleotide of the antisense strand is independently a modified nucleotide or an unmodified nucleotide.

[0102] In some embodiments, the siRNA is an siRNA modifier. The siRNA modifier can improve the stability of the siRNA while maintaining a high PLN mRNA inhibitory activity.

[0103] The nucleic acids in certain embodiments of the present disclosure can be synthesized and / or modified by methods known in the art. Modifications that can be present in certain embodiments of the siRNAs of the present disclosure include, but are not limited to, the following:

[0104] 1) Modification of ribose: This is one of the most important modification ways of nucleic acids, which is the modification and replacement of groups at specific positions of the ribose ring, including but not limited to 2'-position modification (such as 2'-OMe modification, 2'-F modification), 4'-position modification, 5'-position modification, isomerization modification, etc.

[0105] 2) Modification of bases: mainly divided into three forms of purine modification, pyrimidine modification and base replacement, those skilled in the art will recognize that guanine, cytosine, adenine and uracil can be replaced by other moieties without significantly changing the base pairing properties of an oligonucleotide comprising a nucleotide bearing such a replacement moiety;

[0106] 3) Modification of phosphate backbone: including but not limited to modification of phosphorothioate, replacement of phosphate groups between nucleosides with groups that do not contain phosphorus atoms, such as replacement of P atoms with C atoms, S atoms and N atoms, etc.

[0107] 4) End modification: including but not limited to covalently linking specific groups at the 5'-end and / or 3'-end of the sense strand, 5'-end phosphorylation modification of the antisense strand.

[0108] In some embodiments, the siRNA modification comprises at least one modification of a nucleotide. The modification of the nucleotide does not result in a substantial reduction or loss of function of the siRNA to inhibit gene expression.

[0109] In some embodiments, each of the nucleotides in the sense strand and the antisense strand is a modified nucleotide, which is a fluoro-modified nucleotide or a non-fluoro-modified nucleotide. In some embodiments, the non-fluoro-modified nucleotide refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2'-position of the ribosyl group of the nucleotide is replaced by a non-fluoro group. In some embodiments, the non-fluoro group-replaced nucleotide or nucleotide analog is a 2'-alkoxy-modified nucleotide or nucleotide analog, a 2'-substituted alkoxy-modified nucleotide or nucleotide analog, a 2'-alkyl-modified nucleotide or nucleotide analog, a 2'-substituted alkyl-modified nucleotide or nucleotide analog, a 2'-amino-modified nucleotide or nucleotide analog, a 2'-substituted amino-modified nucleotide or nucleotide analog, or a 2'-deoxynucleotide.

[0110] In some embodiments, one or more of the nucleotides at positions 7, 9, 10, 11 of the sense strand, in the 5'-end to 3'-end direction, is a fluoro-modified nucleotide; and one or more of the nucleotides at positions 2, 6, 8, 9, 14, 16 of the antisense strand, in the 5'-end to 3'-end direction, is a fluoro-modified nucleotide. In some embodiments, the nucleotides at positions 7, 9, 10, 11 of the sense strand, in the 5'-end to 3'-end direction, are fluoro-modified nucleotides; and the nucleotides at positions 2, 6, 8, 9, 14, 16 of the antisense strand, in the 5'-end to 3'-end direction, are fluoro-modified nucleotides. In some embodiments, the nucleotides at positions 7, 9, 10, 11 of the sense strand, in the 5'-end to 3'-end direction, are fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides; and the nucleotides at positions 2, 6, 8, 9, 14, 16 of the antisense strand, in the 5'-end to 3'-end direction, are fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides.

[0111] In some embodiments, at least a portion of the phosphate groups in the phosphodiester backbone of at least one of the single strands of the siRNA is a phosphate group having a modification, which is a phosphorothioate group formed by replacing at least one of the oxygen atoms in the phosphodiester bond of the phosphate group with a sulfur atom. In some embodiments, the phosphorothioate group is linked at 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 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination thereof.

[0112] In some embodiments, the 5 '-terminal nucleotide of the antisense strand is a 5 '-phosphate nucleotide or a 5 '-phosphate analog modified nucleotide. In some embodiments, the 5 '-terminal nucleotide of the antisense strand is a 5 '-(E)-vinylphosphonate (E-VP) modified nucleotide.

[0113] siRNA conjugate

[0114] The present disclosure also provides siRNA conjugates, in which the siRNA linked to the conjugating group can be unmodified siRNA or siRNA modifications. In the context of the present disclosure, unless otherwise specified, "conjugation" refers to the linkage between two or more chemical moieties each having a specific function in a manner of covalent linkage; accordingly, "conjugate" refers to the compound formed by the covalent linkage between the respective chemical moieties. The siRNA molecules modified by the conjugating group have better tissue, organ targeting and the ability to promote endocytosis, while maintaining high inhibitory activity and stability, which can reduce the impact on other tissues or organs and reduce the amount of siRNA molecules used, so as to achieve the purposes of reducing toxicity and reducing costs.

[0115] The conjugation site of the siRNA to the conjugating group can be at the 3' end or the 5' end of the sense strand of the siRNA, at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugating group is at the 3' end of the sense strand of the siRNA.

[0116] In the present disclosure, the 3' end of the sense strand of the siRNA has a blunt end, the 3' end of the antisense strand has 2 overhanging nucleotides extending out of the double-stranded region, and the conjugating group is conjugated to the 3' end of the sense strand having a blunt end to form an siRNA conjugate.

[0117] In some embodiments, the conjugate group can be attached to the 2'-position hydroxyl group, the base, or the phosphate group of the nucleotide. When the conjugate group is attached to the internal sequence of the siRNA, the conjugate group is usually attached to the ribose sugar ring or the base, and when the conjugate group is attached to the end of the siRNA strand, the conjugate group is usually attached to the phosphate group of the nucleotide. In the present disclosure, the conjugate group can be a ligand commonly used in the field of siRNA administration. In some embodiments, the conjugate group can be selected from one or more of the following ligands formed by targeting molecules or derivatives thereof: saccharides, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folate; receptor ligands expressed by liver parenchymal cells, such as asialoglycoprotein, asialo sugar residues, lipoproteins (such as high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin; lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots, etc.

[0118] In some specific embodiments, the conjugate group is GalNAc.

[0119] In some specific embodiments, the GalNAc is conjugated to the 3' end of the sense strand via a phosphodiester bond.

[0120] Cells, subjects, and controls

[0121] As used herein, the terms cell, tissue, organ, and / or subject can be used in conjunction when referring to administration or dosing. In some aspects of the present application, the subject is a human or a vertebrate mammal, and thus the present application can be used to treat PLN-related diseases or disorders in both human and non-human subjects. In some embodiments of the present application, the subject is a human and the methods of the present application can be used in human prophylactic and therapeutic regimens.

[0122] Cells to which the methods of the present application can be applied include in vivo, in vitro, ex vivo cells, can be primary culture cells of tissues, or can be specific cell lines immortalized cells. The cells of the methods of the present application can be cardiac tissue cells including cardiomyocytes, cardiac myofibroblasts, cardiac vascular endothelial cells; can be skeletal muscle cells, or can be smooth muscle cells; can be liver cells including liver parenchymal cells, liver non-parenchymal cells (liver stellate cells, liver sinusoidal endothelial cells, liver Kupffer cells, intrahepatic bile duct epithelial cells); or can be kidney tissue cells including renal tubular cells, glomerular endothelial cells, renal interstitial fibroblasts, renal vascular endothelial cells.

[0123] Compositions

[0124] The siRNA or siRNA conjugate of the present disclosure can be prepared into a composition with a pharmaceutically acceptable carrier or excipient to reduce the expression of the PLN gene in cells, and can be used to treat PLN-related diseases or disorders.

[0125] The term "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient used for administration of a therapeutic agent. Such carriers or excipients include, but are not limited to, water for injection, sodium hydroxide, sodium phosphate monohydrate, sodium 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 glycerol, dimethyl adipate. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonates, sodium and calcium phosphates and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents can include starch and gelatin, while the lubricating agent (if present) is typically magnesium stearate, stearic acid or talc. If desired, tablets can be coated with material such as glycerol monostearate or glycerol distearate, to delay absorption in the gastrointestinal tract.

[0126] The inventors of the present disclosure found that the siRNA and siRNA conjugate of the present disclosure exhibit high PLN mRNA silencing activity, low cytotoxicity, low off-target effect, and can improve myocardial recovery capacity, improve electrophysiological function and have essentially no effect on the beating frequency of myocardial tissue, with high drug safety.

[0127] Dosing

[0128] The siRNA molecules (including unmodified siRNA, siRNA modifiers), siRNA conjugates, or compositions disclosed herein are used at a dose sufficient to inhibit PLN gene expression. In some embodiments of this disclosure, the dose of the siRNA, siRNA conjugate, or composition of this disclosure, based on the amount of siRNA contained therein, is 0.01 to 100.0 mg per kilogram of recipient body weight per dose, generally 1 to 50 mg / kg body weight, 5 to 40 mg / kg body weight, 10 to 30 mg / kg body weight, 1 to 20 mg / kg body weight, 1 to 10 mg / kg body weight, 5 to 15 mg / kg body weight, including extreme values. For example, based on the amount of siRNA contained therein, each single dose of the siRNA, siRNA conjugate, or composition of this disclosure can be in doses ranging from about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, etc. kg, 1.9mg / kg, 2mg / kg, 2.1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / kg, 4mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5mg / kg, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6mg / kg, 6.1mg / kg, 6.2 mg / kg, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7mg / kg, 7.1mg / kg, 7.2mg / kg, 7. 3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7.9mg / kg, 8mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4 mg / kg, 8.5 mg / kg, 8.6 mg / kg, 8.7 mg / kg, 8.8 mg / kg, 8.9 mg / kg, 9 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg body weight.

[0129] The amount of the siRNA, siRNA conjugate, or composition of the present disclosure to be used can be determined according to the weight, age, gender, severity of disease, and the like of the patient. The frequency of administration can be 1, 2, 3, 4, or more times per day, per week, per two weeks, per three weeks, per 1 month, per 2 months, per 3 months, per 4 months, per 5 months, per 6 months, per 7 months, per 8 months, per 9 months, per 10 months, per 11 months, or per year. The total number of times the siRNA, siRNA conjugate, or composition of the present disclosure is administered can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0130] Treatment

[0131] In some embodiments, the siRNAs or siRNA conjugates of the disclosure are administered alone. In other embodiments, they are administered in combination with one or more other therapeutic regimens for treating a PLN-associated disease or disorder. When administered in combination, the other therapeutic regimen can be administered prior to, concurrently with, and / or after administration of the PLN siRNA of the disclosure. The other therapeutic regimen can be other PLN siRNA or siRNA conjugate combinations or non-siRNA therapeutic agents. Non-limiting examples of non-siRNA therapeutic agents include: ACEI / ARB class drugs, beta-blockers, non-dihydropyridine calcium antagonists, diuretics, rivaroxaban, amiodarone, milrinone, spironolactone, soluble guanylate cyclase (sGC) stimulators, digitalis drugs, drugs that improve myocardial energy metabolism such as trimetazidine, coenzyme Q10, levocarnitine, phosphocreatine, potassium ion binders, and the like.

[0132] Medical uses

[0133] The siRNAs, siRNA conjugates, and compositions of the disclosure can be used to prevent and / or treat a disease associated with PLN expression, including one or more of heart failure, cardiomyopathy, arrhythmia.

[0134] In some embodiments, the heart failure includes one or more of acute heart failure, chronic heart failure, systolic heart failure, diastolic heart failure, left-sided heart failure, right-sided heart failure, and global heart failure.

[0135] In some embodiments, the cardiomyopathy is dilated cardiomyopathy, including one or more of dilated cardiomyopathy caused by PLN, TTN, LMNA, RBM20, SCN5A, MYH7, TNNT2, and / or TPM1 mutations; in some embodiments, the dilated cardiomyopathy is genetic cardiomyopathy, including one or more of cardiomyopathy caused by PLN p.Arg14del, Arg9Cys (R9C), and / or Arg25Cys (R25C) gene mutations.

[0136] In some embodiments, the arrhythmia includes ventricular tachycardia and / or ventricular fibrillation.

[0137] In some embodiments, the disclosure provides a method of inhibiting expression of a PLN gene in a cell, comprising contacting the cell with the siRNAs, siRNA conjugates, and compositions of the disclosure.

[0138] In some embodiments, the cell is an in vivo cell or an in vitro cell. In some particular embodiments, the cell is in a subject.

[0139] In some embodiments, the present disclosure provides methods of preventing and / or treating a disease associated with PLN expression, comprising administering to a subject or patient the siRNAs, siRNA conjugates, and compositions of the present disclosure.

[0140] In the present disclosure, a "subject" includes or is either a human or a non-human animal, preferably a vertebrate, and more preferably a mammal. The subject can include a transgenic organism. Most preferably, the subject is a human. Further, the subject has at least one of the following characteristics:

[0141] (1) abnormal expression of a PLN gene in vivo, more specifically, abnormally high expression of a PLN gene;

[0142] (2) has a disease associated with abnormal expression of a PLN gene;

[0143] (3) has a disease that would benefit from a reduction in PLN gene expression. For example, a human who has or is predisposed to a disease associated with abnormal expression of a PLN gene.

[0144] Kits

[0145] In some embodiments, the siRNA molecules (including unmodified siRNAs, siRNA modifications), siRNA conjugates, or compositions of the present disclosure and optionally other therapeutic agents can be packaged in a kit, in which the siRNA molecules, siRNA conjugates, pharmaceutically acceptable carriers, and optionally other therapeutic agents can be provided in liquid form or in dry form. The kits of the present disclosure can include a carrier that is compartmentalized to

[0146] In some embodiments, the kits include instructions for mixing the siRNA molecules (including unmodified siRNAs, siRNA modifications) or siRNA conjugates with the pharmaceutically acceptable carriers or other ingredients.

[0147] Examples

[0148] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description, but it is to be understood that both the detailed description and the specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

[0149] The experimental techniques and experimental methods used in this example are conventional techniques and methods, and are not specifically described unless otherwise specified. For example, the experimental methods not specifically described in the following examples are generally performed according to the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0150] Example 1. siRNA design and synthesis

[0151] 1.1 siRNA design

[0152] A set of siRNAs targeting the human PLN gene (human PLN gene: NCBI refseq ID NM_002667.5; NCBI Gene ID: 5350) was designed using OligoWalk online. The human NM_002667.5 REFSEQ mRNA has a length of 2989 bases. At the same time, to avoid any sequence toxicity, sequences similar to the human gene were also excluded.

[0153] Human NM_002667.5 REFSEQ mRNA (NCBI refseq ID NM_002667.5):

[0154] 1.2 siRNA sequence synthesis

[0155] The siRNAs, including the negative control siRNA (siCtrl), were synthesized according to the standard oligonucleotide solid-phase synthesis protocol.

[0156] Oligonucleotide solid-phase synthesis protocol: RNA was synthesized at a scale of 500 nmol using commercially available 5'-DMT-2'-TBDMS-rU phosphoramidite monomers, 5'-DMT-2'-TBDMS-rA(Bz) phosphoramidite monomers, 5'-DMT-2'-TBDMS-rC(Ac) phosphoramidite monomers, and 5'-DMT-2'-TBDMS-rG(iBu) phosphoramidite monomers. The phosphoramidite solution was prepared at a concentration of 50 mM, and 0.3 M benzylthio tetrazole (BTT) acetonitrile solution was used as the activator. During the synthesis, 0.1 M oxidizing reagent (pyridine:THF:water = 20:78:2) was used to convert the trivalent phosphorus to pentavalent phosphorus to stabilize the phosphate backbone. After the synthesis was completed, the sequence was aminolysed from the solid-phase support and precipitated. The 2' 2'-O-tert-butyldimethylsilyl protecting group was removed with triethylamine trihydrofluoride.

[0157] For the RNA sequence of the end of the synthesis, under the condition of 55°C, with ammonia solution: methylamine = 1:1 ammonia solution, ammonia for 40 minutes, after the end of the ammonia, remove the solid phase carrier, take the supernatant and dry. Add triethylamine trihydrofluoric acid: triethylamine: NMP = 6:4:3 protecting group remover, react at 60°C for 2 hours, add n-butanol at a ratio of 1:5, stand at -20°C for 30 minutes, centrifuge and take the precipitate. Add RNase-free water to dissolve, purify by reverse phase chromatography (0.1M triethylamine acetic acid (TEAA) and acetonitrile). The purified sample is desalted by ultrafiltration with PBS. And annealing, get siRNA, verify the siRNA obtained, and the results show that the target siRNA is successfully prepared.

[0158] 1.3 siRNA sequence modification and conjugate synthesis

[0159] Synthesize modified siRNA according to the oligonucleotide solid-phase synthesis scheme. The method for preparing nucleoside monomers with corresponding modifications by introducing modified nucleotide groups into the siRNA of the present disclosure using nucleoside monomers with corresponding modifications is also well known to those skilled in the art. Refer to the synthesis method disclosed in WO2014025805A1 or WO2017015109A1 to conjugate L96 to siRNA to synthesize siRNA conjugates.

[0160] The structure of the conjugate group L96 is as follows:

[0161] Annealing of oligoribonucleotides to produce siRNA conjugates: The RNA oligomer to be annealed is prepared into a 200 μΜ solution with sterile RNase Free H2O (RNAase-free water). The annealing reaction system is set as follows: place the above solution with a total volume of 100 μL (double-stranded concentration of 10 nmol) in a 95°C water bath for 10 minutes (≥100 nmol requires high temperature for 20 minutes) → quickly place in a 60°C water bath and naturally cool down → the solution after annealing is stored at 4°C. Mix the complementary strands by combining equal molar RNA solutions. It is identified that the siRNA conjugate is correctly constructed.

[0162] Prepare siRNA and siRNA conjugate solutions into dry powder for standby.

[0163] The sequence of the synthesized siRNA is shown in Table 1 below, and the schematic diagram of the high-efficiency active interval of PLN mRNA is shown in Figure 1.

[0164] Table 1. siRNA targeting PLN

[0165] wherein A, U, G, C represent adenine, uracil, guanine, cytosine nucleotides, respectively.

[0166] The synthetic siRNA conjugate sequences are shown in Table 2 below.

[0167] Table 2. siRNA conjugates targeting PLN

[0168] wherein the lower case letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lower case letter f represents that the nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide; the lower case letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate subunit; VP represents that the nucleotide adjacent to the right of the letter combination VP is a (5'-(E)-vinylphosphonate, E-VP) modified nucleotide; L96 represents a L96 conjugate group connected to the siRNA.

[0169] Example 2. In vitro activity screening of siRNA 293T cell line

[0170] 2.1 Experimental procedure

[0171] 2.1.1 Cell culture

[0172] 293T cells (China Typical Culture Collection Center (CCTCC), item number: CVCL_0063) were cultured at 37°C in a 5% CO2 environment using DMEM complete medium (Eallbio, added with 10% FBS), and when the confluence rate reached 80%-90%, the cells were trypsinized, counted and transfected.

[0173] 2.1.2 Preparation of siRNA diluent

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

[0175] (2) Preparation of 200 nM siRNA diluent Y

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

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

[0178] c) Take 2 μl of the prepared siRNA stock solution X, add 48 μl of Opti-medium (Opti-MEM I Medium, Gibco, Cat. No. 31985070) to obtain a 200 nM siRNA dilution Y.

[0179] 2.1.3 Transfection of 293T cells

[0180] Take transfection reagent (Thermo Fisher, Cat. No. 13778150) 0.6 μl, add 10 μl of Opti-medium to obtain transfection reagent dilution; mix transfection reagent dilution with 200 nM siRNA dilution Y prepared in 2.1.2 at a volume ratio of 1:1 to prepare a transfection mixture, stand for 5 minutes, take 10 μl of the transfection mixture and add to 90 μl of 293T cells cultured in 2.1.1 (final volume 100 μl / well, cell number 20000 / well, siRNA concentration in the system is 10 nM); incubate for 24 hours after transfection.

[0181] 2.1.4 RNA extraction

[0182] According to the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit kit (Nanjing Novozyme Biotech Co., Ltd., Cat. No. CL132-01) product manual, extract the total RNA of 293T cells obtained in 2.1.3.

[0183] 2.1.5 Fluorescence quantitative PCR

[0184] Use FlysisAmp Cells-to-CT 1-Step SYBR Green Kit kit to perform reverse transcription and real-time PCR analysis on the extracted total RNA.

[0185] 2.1.6 Result analysis

[0186] (1) Use the software of 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher) to automatically calculate the Ct value;

[0187] (2) Calculate the relative expression amount of the gene using the following formula:

[0188] ACt1 = Ct (PLN group) - Ct (ACTIN of PLN group)

[0189] ACt2 = Ct (siCtrl group) - Ct (ACTIN of siCtrl group)

[0190] ACt = ACt1 - ACt2, wherein the siCtrl group is the negative control group;

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

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

[0193] 2.2 Experimental results

[0194] The inhibition effect of the siRNA of the present disclosure is shown in Table 3 below:

[0195] Table 3. siRNA 293T cell line in vitro screening results

[0196] As can be seen from Table 3, some siRNAs of the present disclosure can significantly inhibit the expression of the PLN gene at 10 nM.

[0197] Example 3. Cytotoxicity experiment

[0198] 3.1 Experimental procedure

[0199] 3.1.1 Cell culture

[0200] The 293T cells were cultured at 37°C in an environment with 5% CO2 using DMEM complete medium (Eallbio, item number: 03.1002C; add 10% FBS), and when the fusion rate reached 80%-90%, the cells were trypsinized, counted and transfected.

[0201] 3.1.2 Transfection of 293T cells

[0202] Based on the results of Example 2, siRNAs with better in vitro activity were screened for toxicity determination. 293T cells were transfected according to a method similar to Example 2, and after 72 hours of culture, the cytotoxicity of each siRNA conjugate was measured by determining the ratio of cell viability / toxicity in each sample, wherein the concentration of the transfected siRNA conjugate was 50 nM, 5 nM, respectively. According to the manufacturer's protocol, cell viability was measured by determining intracellular ATP content using CellTiter-Glo assay. According to the manufacturer's protocol, ToxiLight was used to measure cytotoxicity in the supernatant. The results of cell toxicity are shown in Figure 2.

[0203] 3.2 Experimental results

[0204] The results show that some siRNA conjugates of the present disclosure have low cytotoxicity and good cell compatibility.

[0205] Based on the in vitro screening results of Example 2 and Example 3, the high-efficiency active interval for inhibiting PLN mRNA was determined, which is 292-357, 444-626 and 1127-1149 according to NCBI refseq ID NM_002667.5.

[0206] Example 4. In vivo activity screening of siRNA conjugates

[0207] According to the in vitro experimental screening results of Example 2 and Example 3, some siRNA sequences with better in vitro activity were selected to verify the in vivo activity of the siRNA conjugates.

[0208] 4.1 Experimental steps

[0209] 6-8 week old male mice (C57BL / 6) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. Each mouse was injected intravenously with 1 x 10 11Recombinant adeno-associated virus 8 (AAV8) vector with one genome copy. The recombinant AAV8 vector was packaged by AAV8 capsid protein expression plasmid and transfer plasmid carrying AAV8-hPLN, in which the human PLN sequence (NM_002667.5) of 354-2989 was controlled by TBG promoter (AAV8-TBG-hPLN), purchased from Yunnan Biotech (Guangzhou) Co., Ltd. AAV8-hPLN transgenic mouse model was constructed 14 days after injection. Then, each mouse was given a single subcutaneous dose of 3 mg / kg of the conjugate. The mice were sacrificed on day 7 (D7), day 14 (D14), and day 19 (D19) after administration, respectively, and 6 mice in each group, and the liver tissue was taken for PLN mRNA expression detection. Total RNA was extracted by Trizol method, mRNA reverse transcription used HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, Catalog No: R323-01), and real-time fluorescent quantitative PCR used ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Catalog No: Q711-03). The in vivo activity screening results of siRNA conjugates are shown in Tables 4 and 5.

[0210] Table 4. In vivo screening results of siRNA conjugates

[0211] Table 5. In vivo screening results of siRNA conjugates

[0212] 4.2 Experimental results

[0213] The chemically modified partial siRNA sequences have good activity in mice, and 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, and YGND11-13M, YGND11-21M, YGND11-31M, YGND11-41M and YGND11-73M have high efficiency of PLN gene expression knockdown.

[0214] Example 5. In vivo activity screening of siRNA conjugates with multiple doses

[0215] According to the in vivo experimental screening results of Example 4 above, some siRNA conjugates were selected for in vivo activity verification.

[0216] 5.1 Experimental steps

[0217] AAV8-hPLN transgenic mouse model was constructed using the method described in Example 4. The mice were administered with 0.3 mg / kg, 1 mg / kg, 3 mg / kg of the conjugate by single subcutaneous administration. The mice were sacrificed on the 19th day after administration, and the liver tissues were taken for PLN mRNA expression detection. The detection method and reagents were the same as in Example 4. The results of the multi-dose in vivo activity screening of the siRNA conjugate are shown in Table 6 and Figure 3.

[0218] Table 6. Results of multi-dose in vivo screening of siRNA conjugate

[0219] Note: N / A means data not measured

[0220] 5.2 Experimental results

[0221] The results show that the siRNA conjugates of the present disclosure all show good dose-dependent effect, and the inhibition rate is higher than 55% when the administration dose is 3 mg / kg. Among them, YGND11-21M has the highest inhibition efficiency on PLN gene expression, and the inhibition rate is as high as 71.57% when the administration dose is 3 mg / kg. In addition, YGND11-26M has an inhibition rate as high as 65.27% when the administration dose is 3 mg / kg.

[0222] Example 6. Off-target analysis of siRNA conjugate

[0223] 6.1 Experimental steps

[0224] Referring to the experimental step 2.1 in Reference Example 2, the 293T cell culture and transfection were completed. After 24 hours of culture after transfection, the total RNA of the 293T cells was extracted according to the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit product instruction. The total RNA was sent to a third-party company for transcriptome detection, and the differentially expressed genes were analyzed. In the differential expression gene analysis, the genes with padj≤0.05 and |log2FoldChange|>0.5145 were defined as differential genes, wherein padj≤0.05 represents the significance level after multiple test correction, ensuring that the identification result of the differential genes has a confidence level of more than 95%; and |log2FoldChange|>0.5145 corresponds to at least 1.43 times (2^0.5145) up-regulation or 70% (2^-0.5145) of the control group down-regulation of the gene expression, thereby excluding minor fluctuations and focusing on genes with significant changes in expression. The specific results are shown in Figure 4.

[0225] 6.2 Experimental results

[0226] The off-target analysis results show that the partial siRNA conjugates of the present disclosure cause less gene difference in cells, have higher safety, and can effectively prevent off-target effects.

[0227] Example 7. siRNA conjugate organoid electrophysiology results

[0228] 7.1 Experimental procedure

[0229] 7.1.1 Cell preparation

[0230] Human embryonic stem cells (hESCs) were selected, resuscitated, and inoculated in a 6-well plate pre-coated with 0.1% gelatin at a seeding density of 5x10 4 cells per well, and added with STEMPRO hESC SFM medium (Thermo Fisher, item number: A1000701) and placed in a CO2 incubator for culture. When the cell confluence reached 70%-80%, the cells were passaged or subjected to differentiation induction.

[0231] 7.1.2 Cardiomyocyte directional differentiation

[0232] (1) Mesoderm induction

[0233] The old medium in the 6-well plate was discarded, and the cells were gently washed twice with preheated PBS buffer for 5 minutes each time, 2 mL of mesoderm induction medium (STEMdiff TM , manufacturer: STEMCELL, item number: 05120) was added to each well, and the plate was shaken and placed in a CO2 incubator. The cell morphology was observed daily, and when the cells were spindle-shaped or spindle-shaped on the third day, the next stage was entered.

[0234] (2) Cardiac progenitor cell induction

[0235] The mesoderm induction medium was discarded, and the cells were washed twice with PBS buffer for 5 minutes each time, and 2 mL of cardiac progenitor cell induction medium (STEMdiff TM , manufacturer: STEMCELL, item number: 05230) was added to each well for continuous culture. From the fifth day, half of the medium was replaced daily, and on the seventh day, the cells gathered into small groups, which were cardiac progenitor cells.

[0236] (3) Cardiomyocyte induction

[0237] The cardiac progenitor cell induction medium was discarded, and the cells were washed twice with PBS buffer, and 2 mL of cardiomyocyte induction medium (STEMdiff TM , manufacturer: STEMCELL, item number: 05240) was added to each well for culture. From the tenth day, the medium was replaced daily, and the cells gradually showed spontaneous beating, and on the fourteenth day, typical cardiomyocyte morphology was formed, and the beating frequency was stable.

[0238] (4) Synchronous differentiation of cardiomyocytes

[0239] On day 4 of cardiomyocyte induction (cardiomyocyte progenitor cell induction phase), hESCs with 70%-80% confluence were digested into a single-cell suspension and cultured at 2×10⁻⁶ cells / cells. 4 Cells were seeded at a density of 100 cells / well in 6-well plates coated with 0.1% gelatin and cultured in Fibroblast Differentiation Medium (STEMCELL, catalog number 05250). Half the volume of medium was replaced daily. By day 14, the cells had differentiated into long spindle-shaped fibroblasts with prominent cytoplasmic processes.

[0240] 7.1.3 Construction of engineered organoids

[0241] Mix cardiomyocytes:cardiomyocytes:endothelial cells (Thermo Fisher, catalog number: C0035C) in a volume ratio of 2:1:1, gently pipetting to avoid air bubbles. Add an equal volume of pre-chilled extracellular matrix (Matrigel Matrix, Corning, catalog number: 354234) to the suspension on ice, and gently mix with a pipette. Slowly inject the mixture into the wells of a 3D-printed hemispherical PDMS mold (100 μL / well), ensuring uniform filling without voids or air bubbles.

[0242] 7.1.4 Organoid Spheroid Culture

[0243] Place the filling mold in a 24-well ultra-low adsorption plate (1 mold / well), and slowly add 1 mL of the corresponding culture medium (Organoid Maintenance Medium, manufacturer: STEMCELL, catalog number: 05810) along the well wall. Place in a CO2 incubator, and change half the volume of the medium daily for days 1-3, followed by a full volume replacement thereafter. Examine the culture periodically under a microscope and record changes in the morphology of the organoid spheroids. Maturation usually occurs after 15 days.

[0244] 7.1.5 siRNA conjugate transfection

[0245] Take 0.6 μl The transfection reagent (Thermo Fisher, catalog number: L3000015) was diluted with 10 μL of Opti-medium. This was then mixed with the siRNA dilution buffer and allowed to stand for 5 minutes. 10 μL of the transfection mixture was then added to the heart organoids cultured in section 7.1.4 (final siRNA concentration 62.5 nM) and incubated for 6 hours.

[0246] 7.1.6 Electrophysiological Recording

[0247] The multi-electrode microarray (MEA) device was used to record the field potential of the organoids, and the data of frequency, field potential duration, etc. were analyzed.

[0248] 7.2 Experimental results

[0249] 7.2.1 Field potential

[0250] The 50% (50% PW) of the pulse width after adding the siRNA conjugate was recorded, in seconds. The shorter the 50% PW, the shorter the calcium recovery, indicating that the SERCA2a recovery ability is strong, the inhibition effect of the PLN gene is weakened, and the siRNA conjugate is effective. The results of Figure 5 show that the siRNA conjugate reduces the 50% PW to varying degrees, indicating that the siRNA conjugate can effectively improve the myocardial recovery ability.

[0251] 7.2.2 Beats per minute (BPM)

[0252] In the electrocardio function detection, the beats per minute (BPM) is a commonly used index to measure the heart rate, which can reflect the beating frequency of the cardiac organoids. In the study of cardiac organoids, BPM is an important index to evaluate the cardiac function, rhythm and whether the organoids can simulate the beating of the real heart. The results of Figure 6 show that the siRNA conjugate has little effect on the beating frequency of the myocardial tissue, and the drug safety is high.

[0253] 7.2.3 Field potential duration (FPD)

[0254] In the study of cardiac organoids, the field potential duration (FPD) is an electrophysiological index to describe the duration of the electrical signal of the organoids in the electrophysiological experiment, which refers to the time interval between the two peaks of depolarization and repolarization, similar to the definition of the clinical electrocardiogram QT interval (the QT interval reflects the total time of ventricular depolarization and repolarization). The normal heart rate accelerates, and the QT interval shortens, and vice versa. The results of Figure 7 show that the siRNA conjugate shortens the FPD to varying degrees, indicating that it can effectively improve the electrophysiological function.

Claims

1. A siRNA for inhibiting PLN gene expression, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising nucleotide sequence I, and the antisense strand comprising nucleotide sequence II; each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified or unmodified nucleotide; nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; nucleotide sequence I is substantially identical to a first nucleotide sequence, the first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length in the mRNA expressing the PLN gene, preferably, the first nucleotide sequence being a nucleotide sequence of 15 to 25 nucleotides in length in the mRNA expressing the PLN gene, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; Preferably, the first nucleotide sequence is a nucleotide sequence of at least 15 nucleotides in length from the highly active region of the PLN gene-expressed mRNA, preferably a nucleotide sequence of 15 to 25 nucleotides. The highly active region is positions 292-357, 444-626, and 1127-1149 of the PLN gene-expressed mRNA, preferably positions 292-314, 315-337, 318-340, 327-349, 335-357, 444-466, 476-498, 604-626, and 1127-1149. Preferably, the mRNA expressing the PLN gene is as shown in NCBI refseqID NM_002667.5; specifically, the mRNA sequence is as shown in SEQ ID NO:

1.

2. The siRNA of claim 1, wherein, The nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with the first nucleotide sequence.

3. The siRNA of claim 1 or 2, wherein, The nucleotide sequence I comprises at least 15 consecutive nucleotides as shown in any of the sequences in SEQ ID NO:2-100, such as at least 15, 16, 17, 18, 19, 20, or 21 nucleotides; or, the nucleotide sequence of the positive strand is as shown in nucleotide sequence I, which differs from any of the sequences in SEQ ID NO:2-100 by 1, 2, or 3 nucleotides; preferably, the nucleotide sequence I is as shown in any of the sequences in SEQ ID NO:2-100. The nucleotide sequence II comprises at least 15 contiguous nucleotides, such as at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides, as set forth in any one of SEQ ID NOs: 101-199; or the nucleotide sequence of the antisense strand is as set forth in nucleotide sequence II, which differs from any one of SEQ ID NOs: 101-199 by 1, 2, or 3 nucleotides; preferably, the nucleotide sequence II is as set forth in any one of SEQ ID NOs: 101-199.

4. The siRNA according to any one of claims 1-3, which is selected from the siRNA molecules shown in Table 1.

5. The siRNA according to any one of claims 1 to 4, wherein, Each of the nucleotides in the nucleotide sequence I and the nucleotide sequence II is a modified nucleotide, which is a fluoro-modified nucleotide or a non-fluoro-modified nucleotide; Preferably, the fluorine-modified nucleotide refers to a nucleotide with the hydroxyl group at the 2'-position of the ribosyl group of the nucleotide being replaced by fluorine, having the structure shown in the following formula (1); the non-fluorine-modified nucleotide refers to a nucleotide or nucleotide analogue with the hydroxyl group at the 2'-position of the ribosyl group of the nucleotide being replaced by a non-fluorine group; preferably, the nucleotide or nucleotide analogue with the hydroxyl group at the 2'-position of the ribosyl group of the nucleotide being replaced by a non-fluorine group is a 2'-alkoxy-modified nucleotide or nucleotide analogue, a 2'-substituted alkoxy-modified nucleotide or nucleotide analogue, a 2'-alkyl-modified nucleotide or nucleotide analogue, a 2'-substituted alkyl-modified nucleotide or nucleotide analogue, a 2'-amino-modified nucleotide or nucleotide analogue, a 2'-substituted amino-modified nucleotide or nucleotide analogue, a 2'-deoxynucleotide; more preferably, the non-fluorine-modified nucleotide has the structure shown in any one of the following formulae (2)-(12): In formulae (1)-(12), Base represents a base; In formulae (9) and (10), R is selected from H, OH, or alkoxy (O-alkyl); In formulae (11) and (12), R is selected from H, OH, F, or the non-fluoro group.

6. The siRNA of claim 5, wherein, One or more of the nucleotides at positions 7, 9, 10, 11 of the nucleotide sequence I, in the 5'-end to 3'-end direction, is a fluoro-modified nucleotide; and one or more of the nucleotides at positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II, in the 5'-end to 3'-end direction, is a fluoro-modified nucleotide.

7. The siRNA of claim 5 or 6, wherein, At least a portion of the phosphate groups in the phosphodiester backbone of at least one of the single strands of the siRNA is a phosphate group having a modification, which is a phosphorothioate group formed by replacing at least one of the oxygen atoms in the phosphodiester bond of the phosphate group with a sulfur atom; Preferably, the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (13): Preferably, the phosphorothioate group is linked at at least one of the following positions 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 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination thereof; Preferably, the 5'-terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, such as shown in formula (14), formula (15) and formula (16):

8. An siRNA conjugate comprising an siRNA according to any one of claims 5 to 7 and a conjugate group conjugated to the siRNA, wherein, The conjugating group is L96, which has the structure shown below: Preferably, the siRNA conjugate is selected from the siRNA conjugates shown in Table 2.

9. A composition comprising the siRNA according to any one of claims 1-7 or the siRNA conjugate according to claim 8. Preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier or excipient. Preferably, the carrier or excipient includes, but is not limited to, water for injection, sodium hydroxide, sodium phosphate monobasic monohydrate, sodium phosphate monobasic dihydrate, phosphoric acid, sodium chloride, potassium chloride, hydrochloric acid, anhydrous potassium phosphate monobasic, anhydrous sodium phosphate dibasic, PEG2000, PEG6000, cholesterol, distearoylphosphatidylcholine, 1,2-dimyristylglycerol, dimethyl adipate.

10. Use of the siRNA according to any one of claims 1 to 7 or the siRNA conjugate according to claim 8 or the composition according to claim 9 for the manufacture of a medicament for the prevention and / or treatment of a disease associated with PLN expression.

11. Use according to claim 10, wherein, The disease associated with PLN expression comprises one or more of heart failure, cardiomyopathy, arrhythmia; Preferably, the heart failure comprises one or more of acute heart failure, chronic heart failure, systolic heart failure, diastolic heart failure, left-sided heart failure, right-sided heart failure, global heart failure; Preferably, the cardiomyopathy is dilated cardiomyopathy, comprising one or more of PLN, TTN, LMNA, RBM20, SCN5A, MYH7, TNNT2 and / or TPM1 mutation-induced dilated cardiomyopathy; more preferably, the dilated cardiomyopathy is genetic cardiomyopathy, comprising one or more of PLN p.Arg14del, Arg9Cys (R9C) and / or Arg25Cys (R25C) gene mutation-induced cardiomyopathy; Preferably, the arrhythmia comprises ventricular tachycardia and / or ventricular fibrillation.

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