Adeno-associated virus capsid protein mutant and adeno-associated virus

Abstract

Provided are a modified adeno-associated virus capsid protein and the use thereof. The capsid protein comprises a substitution of about 5-8 amino acids as compared with a wild-type AAV capsid protein, and an AAV comprising the mutated capsid protein has increased infectivity to target tissue or target cells (such as heart or cardiomyocytes) than an AAV comprising the unmutated wild-type AAV capsid protein.

Description

A mutant of adeno-associated virus capsid protein and adeno-associated virus Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an adeno-associated virus capsid protein mutant and its uses. Background Technology

[0002] Vectors used in gene therapy can be divided into viral vectors and non-viral vectors. Among viral vectors, the most commonly used include adenovirus vectors, lentivirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors.

[0003] Adeno-associated virus (AAV) belongs to the family Parvoviridae and the genus Dependovirus. The virion consists of a 25 nm icosahedral capsid containing a 4.7 kb single-stranded DNA genome with two open reading frames: Rep and Cap. The non-structural Rep gene encodes four regulatory proteins essential for viral replication, while Cap encodes three structural proteins (VP1-3) that assemble into a 60-subunit capsid. This viral capsid mediates the AAV vector's ability to overcome many biological barriers to viral transduction, including cell surface receptor binding, endocytosis, intracellular transport, and unpacking in the nucleus.

[0004] AAV (anti-inflammatory vasculature) is widely used in gene transduction, gene therapy, vaccination, and oncolytic therapy. It possesses numerous advantages, including low pathogenicity, broad tissue infectivity, wide host cell range (it can infect and express in both proliferating and non-proliferating cells), low immunogenicity, long-term expression of exogenous genes in vivo, and no integration into the host cell genome. It is widely used in experimental and clinical research. In 2017, the US FDA approved the first AAV-mediated gene therapy for the treatment of a rare inherited eye disease. Patients received a single subretinal administration of a drug delivered via an rAAV vector, and the expression of the therapeutic drug was observed continuously for over four years.

[0005] Hypertrophic cardiomyopathy (HCM) caused by functional mutations is a hereditary heart disease characterized by abnormal thickening of the myocardium, which can lead to cardiac dysfunction. The MYBPC3 gene encodes cardiac myosin-binding protein C (MyBP-C), a protein located in the sarcomeres of the myocardium, crucial for the contraction and relaxation of the myocardium. MyBP-C belongs to the immunoglobulin superfamily, consists of 1273 amino acid residues, and has a molecular weight of 149 kDa. MyBP-C regulates myocardial contractility through its interaction with myosin and also participates in the regulation of calcium homeostasis. Mutations in the MYBPC3 gene are one of the common causes of hypertrophic cardiomyopathy (HCM). The most common mutation types are nonsense and frameshift mutations, with carriers accounting for approximately 30–40% of all HCM patients. In some regions, it is the leading pathogenic gene for HCM.

[0006] Heart failure (HF) is a severe syndrome that progresses to the end stage of various cardiovascular diseases. Its core pathological feature is impaired contractile and diastolic function of myocardial cells, leading to an inability of the heart to pump blood to meet the body's metabolic demands. It has become a major public health problem with high morbidity and mortality rates worldwide. Current clinical treatment mainly involves drug intervention and device therapy, which can alleviate symptoms and slow disease progression, but cannot fundamentally repair the function of damaged myocardial cells. SERCA2a (sarcoplasmic reticulum / endoplasmic reticulum Ca 2+ SERCA2a (-ATPase 2A) is a key transporter protein located on the sarcoplasmic reticulum membrane of cardiomyocytes and is a core regulatory protein for calcium homeostasis in cardiomyocytes. SERCA2a has a molecular weight of approximately 110 kDa and belongs to the PIIA-ATPase subfamily. Its structure mainly includes a transmembrane helical domain, a cytoplasmic domain, and an intraluminal loop. During cardiac diastole, SERCA2a provides energy by hydrolyzing ATP and transferring cytoplasmic calcium in a 2:1 stoichiometric ratio. 2+ Calcium is transported against its concentration gradient to the sarcoplasmic reticulum lumen, a process that accounts for more than 70% of diastolic calcium uptake in cardiomyocytes. During cardiac systole, membrane depolarization induces voltage-gated calcium uptake. 2+ Channels open, intracellular Ca 2+ Increased concentration activates the opening of Rynoldsin receptor 2 (RyR2), triggering local release of Ca from the sarcoplasmic reticulum (SR). 2+ This leads to myocardial contraction. Decreased SERCA2a levels and activity affect cytoplasmic calcium... 2+ The removal rate and amount of SERCA2α can inhibit myocardial relaxation to some extent. Therefore, restoring or enhancing the expression and activity of SERCA2α to correct myocardial calcium homeostasis has become a key target and research risk for treating heart failure at the cellular and molecular level and overcoming existing treatment bottlenecks.

[0007] In normal myocardium, inhibitor-1 (I-1c) maintains the phosphorylation level of phosphatase-1 (PLB) by inhibiting protein phosphatase 1 (PP1) activity, ensuring that SERCA2a efficiently pumps cytoplasmic calcium back to the sarcoplasmic reticulum and safeguarding the normal diastolic and systolic cycles of the myocardium. However, in heart failure, the expression level of I-1c in myocardial tissue is significantly reduced, leading to overactivation of PP1 and dephosphorylation of PLB. Dephosphorylated PLB binds to SERCA2a, inhibiting its calcium ion transport activity, resulting in impaired cytoplasmic calcium clearance. This leads to both decreased diastolic function and weakened myocardial contractility, while simultaneously accelerating cardiomyocyte apoptosis and ventricular remodeling. Inhibitor-1 (I-1c) binds to protein phosphatase-1 (PP1), leading to phosphorylation of PLB and thus increasing SERCA2a activity.

[0008] Adeno-associated virus (AAV) has shown potential as a gene therapy vector in treating cardiac diseases such as hepatocellular carcinoma (HCM), dilated cardiomyopathy, hereditary arrhythmias, hereditary hypertension, lipoprotein pancreatic enzyme deficiency, transthyretin amyloidosis, and heart failure. Studies have shown that AAV can be used to deliver novel genes or edit genes to compensate for loss-of-function mutations, making it suitable for treating recessive monogenic diseases. For example, delivery of miR-1 via the AAV9 vector can improve cardiac function in a mouse model of HCM, reducing myocardial hypertrophy and fibrosis. Delivery of the I-1c gene via the AAV9 vector restores calcium cycling by inhibiting protein phosphatase 1 (PP1). Currently, several AAV-based gene therapy drugs for cardiac diseases are in clinical trials, including those for hereditary cardiomyopathy, heart failure, and Fabry disease.

[0009] The tissue specificity (tropism) of AAV vectors is primarily determined by the type of their capsid protein. Different AAV serotypes exhibit varying affinities for cardiac tissue. AAV9 has been extensively studied due to its highly efficient cardiomyocyte transduction ability. Studies have shown that AAV9 vectors demonstrate higher transduction efficiency in cardiomyocytes and exhibit stronger fluorescence signals. Therefore, modifying or mutating the capsid protein is an effective strategy to enhance its infectivity. Summary of the Invention

[0010] This invention relates to an adeno-associated virus (AAV) capsid protein mutant and to a viral vector (such as an AAV viral vector) containing the capsid protein mutant, delivered to desired cells or tissues. Specifically, the invention comprises a mutated capsid protein having one or more modifications (such as substitution, insertion, or mutation) in the amino acid sequence relative to the wild-type AAV capsid protein, which, when present in an AAV viral vector, exhibits increased infectivity to target tissues or cells (e.g., cardiac cells, cardiomyocytes) compared to an AAV viral vector containing the unmutated wild-type AAV capsid protein.

[0011] This invention provides an adeno-associated virus (AAV) capsid protein mutant, wherein the capsid protein mutant comprises an insertion of a heterologous polypeptide containing approximately 5-8 amino acids relative to the wild-type AAV capsid protein. The heterologous polypeptide includes an amino acid sequence selected from SVLMKEYD (SEQ ID NO:1), YMLDK (SEQ ID NO:3), or ARGNDYAR (SEQ ID NO:4), and the insertion site is located between amino acid positions 588 and 589 of the wild-type AAV9 capsid protein or at the corresponding position in other serotype capsid proteins. Compared to AAV viruses containing the corresponding wild-type AAV capsid protein, AAV viruses containing the capsid protein mutant exhibit enhanced cardiomyocyte infectivity.

[0012] In some preferred embodiments, the heterologous polypeptide comprises an amino acid sequence selected from YMLDK (SEQ ID NO:3) or ARGNDYAR (SEQ ID NO:4).

[0013] In some preferred embodiments, the amino acid sequence of the heterologous polypeptide is ARGNDYAR (SEQ ID NO:4).

[0014] In some specific embodiments, the AAV described in this invention can be derived from any serotype of AAV, for example, the AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, and AAVrh10.

[0015] The capsid protein mutants of the present invention exhibit increased infection or transfection capacity in target cells or tissues (such as cardiomyocytes), for example, by increasing the DNA or RNA expression of the target gene by at least 2, 3, 4, 5, 10, 20, or more times. In other embodiments, the capsid protein mutants of the present invention exhibit increased expression levels of the target protein in target cells or tissues (such as cardiomyocytes), for example, by at least 2, 3, 4, 5, 10, or more times.

[0016] Another aspect of the present invention provides a recombinant adeno-associated virus (rAAV), the recombinant adeno-associated virus comprising: i. the adeno-associated virus capsid protein mutant provided in the first aspect of the present invention;

[0017] ii. Heterologous nucleic acids encoding gene products.

[0018] In some specific implementations, the gene product is selected from one or two gene products or combinations thereof from any of the following groups:

[0019] i.MYBPC3, KCNH2, TRPM4, DSG2, ATP2A2 proteins;

[0020] ii. CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A, SOS1, NPR1, ERBB4, VIP, MYH7 proteins; or

[0021] iii. SERCA2a, I-1c protein.

[0022] In some specific embodiments, the gene product is myosin-binding protein C (MYBPC3). In some embodiments, the amino acid sequence of MYBPC3 has at least 90%, 95%, 98%, 99%, or 100% homology with the polypeptide sequence of SEQ ID NO:5.

[0023] In some implementations, MYBPC3 is the full-length MYBPC3 or a functional variant thereof. In some implementations, MYBPC3 is a truncated MYBPC3.

[0024] In some embodiments, the heterologous nucleic acid encoding MYBPC3 has at least 90%, 95%, 98%, 99%, or 100% homology with SEQ ID NO:6.

[0025] In some embodiments, the heterologous nucleic acid encoding the gene product is operatively linked to a promoter. In some specific embodiments, the promoter is:

[0026] i. Muscle-specific promoters; and / or

[0027] ii. Cardiac cell-specific promoters; and / or

[0028] iii. Cardiac cell-specific promoters.

[0029] The heart-specific promoter is selected from myosin (Des), α-myosin heavy chain (a-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (TNNC1 or cTnC) promoters, and cardiac troponin T (TNNT2) promoters. In some preferred embodiments, the promoter is the human cardiac troponin T (hTNNT2) promoter. In some embodiments, the promoter has the same cell type specificity as the approximately 600 bp native cardiac troponin T promoter (SEQ ID NO:11). In some embodiments, the promoter is a truncated version of the native cardiac troponin T promoter.

[0030] In some specific embodiments, the truncated cardiac troponin T promoters are named hTNNT2-319, hTNNT2-400bp, hTNNT2-501bp, or hTNNT2-544bp, respectively. In some more specific embodiments, the truncated hTNNT2-319, hTNNT2-400bp, hTNNT2-501bp, and hTNNT2-544bp promoters have sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or sequences having at least 90%, 95%, 98%, 99%, or 100% homology with them.

[0031] In some preferred embodiments, the truncated cardiac troponin T promoter is hTNNT2-501bp or hTNNT2-544bp. The hTNNT2-501bp or hTNNT2-544bp cardiac troponin T promoter has a sequence of SEQ ID NO:9, SEQ ID NO:10, or at least 90%, 95%, 98%, 99%, or 100% homology with it, respectively.

[0032] The truncated version of the cardiac troponin T promoter described above in this invention has increased infection or transfection capacity in target cells or tissues (such as cardiomyocytes) compared to the original promoter, for example, by increasing the DNA or RNA expression of the target gene by at least 1, 1.5, 2, 3, 5, or more times. In other embodiments, the truncated version of the cardiac troponin T promoter described above in this invention exhibits increased expression levels of the target protein in target cells or tissues (such as cardiomyocytes) compared to the original promoter, for example, by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more.

[0033] In some specific embodiments, the recombinant adeno-associated virus comprises a polynucleotide expression cassette containing the heteronucleotide encoding MYBPC3 described above, the cardiac troponin T promoter, and other expression regulatory elements.

[0034] In some implementations, other expression regulatory elements are introns, which are located after the promoter. In some cases, an intron can refer to any sequence that can be transcribed but not translated. In some cases, an intron can refer to any sequence that has been transcribed and removed from the mature RNA transcript in the cell. The introns are selected from VH4 introns, SV40 introns, Chi introns, chicken β-action introns, U12 introns, RHD introns, etc.

[0035] In some embodiments, other expression regulatory elements may also include a polyadenylation signal (polyA). Polyadenylation protects mRNA from exonuclease attack and is crucial for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation comprises multiple consecutive adenosine monophosphates, typically containing an AAUAAA repeat sequence. The polyadenylation signal described in this invention is located downstream of the coding sequence encoding a VEGF antagonist. In some embodiments, the polyadenylation signal includes simian vacuolar virus 40 (SV40), human growth hormone (HGH), bovine growth hormone (BGH), or β-globin (RGB).

[0036] In some embodiments, the polynucleotide expression cassette of the present invention has a functional adenoviral inverted terminal repeat (ITR) side-joined at the 5' and 3' ends. A functional adenoviral inverted terminal repeat (ITR) refers to an ITR sequence used for integration, replication, and packaging AAV viral particles. The inverted terminal repeat is a serotype of adeno-associated virus ITR selected from AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAV9 ITR, AAV10 ITR, AAV11 ITR, and AAV12 ITR.

[0037] In some specific embodiments, the polynucleotide expression cassette of the present invention comprises, in sequence from 5'ITR to 3'ITR, a 5'ITR, an hTNNT2 promoter, a heteronucleotide encoding MYBPC3, a BGH polyadenylation signal, and a 3'ITR.

[0038] In some more specific embodiments, the polynucleotide expression cassette of the present invention comprises the nucleic acid sequence described in SEQ ID NO:12 from 5'ITR to 3'ITR.

[0039] Another aspect of the present invention provides a recombinant adeno-associated virus (rAAV) comprising a heterologous nucleic acid encoding myosin-binding protein C (MYBPC3) or a functional variant thereof, wherein the encoding gene is operatively linked to a promoter which is a truncated version of the cardiac troponin T promoter, preferably having a sequence having SEQ ID NO:9, SEQ ID NO:10 or a sequence having at least 90%, 95%, 98%, 99% or 100% homology thereto.

[0040] In some specific embodiments, the recombinant adeno-associated virus includes a polynucleotide expression cassette containing the heteronucleotide encoding MYBPC3 described above, the cardiac troponin T promoter, and other expression regulatory elements.

[0041] In some implementations, other expression regulatory elements are introns, which are located after the promoter. In some cases, an intron can refer to any sequence that can be transcribed but not translated. In some cases, an intron can refer to any sequence that has been transcribed and removed from the mature RNA transcript in the cell. The introns are selected from VH4 introns, SV40 introns, Chi introns, chicken β-action introns, U12 introns, RHD introns, etc.

[0042] In some embodiments, other expression regulatory elements may also include a polyadenylation signal (polyA). Polyadenylation protects mRNA from exonuclease attack and is crucial for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation comprises multiple consecutive adenosine monophosphates, typically containing an AAUAAA repeat sequence. The polyadenylation signal described in this invention is located downstream of the coding sequence encoding a VEGF antagonist. In some embodiments, the polyadenylation signal includes simian vacuolar virus 40 (SV40), human growth hormone (HGH), bovine growth hormone (BGH), or β-globin (RGB).

[0043] In some embodiments, the polynucleotide expression cassette of the present invention has a functional adenoviral inverted terminal repeat (ITR) side-joined at the 5' and 3' ends. A functional adenoviral inverted terminal repeat (ITR) refers to an ITR sequence used for integration, replication, and packaging AAV viral particles. The inverted terminal repeat is a serotype of adeno-associated virus ITR selected from AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAV9 ITR, AAV10 ITR, AAV11 ITR, and AAV12 ITR.

[0044] In some preferred embodiments, the polynucleotide expression cassette sequentially comprises, from 5'ITR to 3'ITR, a 5'ITR, an hTNNT2 promoter, a heteronucleotide encoding MYBPC3, a BGH polyadenylation signal, and a 3'ITR.

[0045] In some more specific embodiments, the polynucleotide expression cassette contains the nucleic acid sequence described in SEQ ID NO:12 from the 5'ITR to the 3'ITR.

[0046] In some embodiments, the gene product of the present invention is SERCA2a and / or I-1c protein or a functional variant thereof. In some preferred embodiments, the gene product of the present invention is SERCA2a and I-1c protein.

[0047] In some specific embodiments, SERCA2a has at least 90%, 95%, 98%, 99%, or 100% homology with the polypeptide sequence of SEQ ID NO:13.

[0048] In some specific embodiments, I-1c has at least 90%, 95%, 98%, 99%, or 100% homology with the polypeptide sequence of SEQ ID NO:14.

[0049] In some specific embodiments, the recombinant adeno-associated virus of the present invention comprises a polynucleotide expression cassette, wherein the polynucleotide expression cassette comprises, in sequence from 5'ITR to 3'ITR:

[0050] The polynucleotide expression cassette contains, in sequence from 5'ITR to 3'ITR:

[0051] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0052] (b) Polyadenylation signal;

[0053] (c) SERCA2a encoded sequence;

[0054] (d) miniCMV promoter;

[0055] (e) CMV promoter;

[0056] (f) PI introns;

[0057] (i) I-1c encoded sequence;

[0058] (j) Polyadenylation signal;

[0059] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0060] In some more specific embodiments, the polynucleotide expression cassette of the present invention comprises the nucleic acid sequence described in SEQ ID NO:15 from the 5'ITR to the 3'ITR.

[0061] In some more specific embodiments, the nucleic acid described in this invention further includes codon optimization or CpG removal. In some preferred embodiments, the polynucleotide expression cassette described in this invention comprises the nucleic acid sequence described in SEQ ID NO:16 from the 5' ITR to the 3' ITR.

[0062] In this case, the capsid protein of the recombinant adeno-associated virus (AAV) can be derived from any AAV serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. Any of these AAV serotypes can serve as a gene delivery vector. For example, the AAV capsid can be a wild-type capsid or a natural capsid. Wild-type AAV capsids of particular interest include AAV2, AAV5, AAV8, and AAV9. Similar to ITRs, the capsid is not necessarily wild-type; rather, as long as the capsid can transduce specific cells or tissues, the wild-type VP1, VP2, or VP3 sequences can be altered by nucleotide insertion, deletion, or substitution. In other words, the AAV capsid can be a variant AAV capsid, which includes one or more amino acid substitutions, deletions, or insertions relative to the parental capsid protein or the AAV capsid protein.

[0063] In some preferred embodiments, the capsid protein serotype is a heart-specific capsid protein, such as wild-type AAV1, AAV2, AAV6, AAV8, AAV9 or a mutant thereof, preferably wild-type AAV9 or a mutant thereof.

[0064] Another aspect of the present invention provides a pharmaceutical composition comprising:

[0065] a) The recombinant adeno-associated virus provided by the present invention;

[0066] b) Pharmaceutically acceptable excipients.

[0067] In some embodiments, pharmaceutically acceptable excipients in the pharmaceutical composition include buffers, stabilizers, and surfactants.

[0068] In some specific embodiments, the buffer is selected from citrate, phosphate, histidine, Tris or HEPES buffer, preferably citrate; the stabilizer is selected from sucrose, trehalose, mannitol, lactose, galactose, glucose, maltose, preferably sucrose; the surfactant is selected from poloxamer 188, polysorbate 20, polysorbate 80, polyethylene glycol hydroxystearate (HS15) or vitamin E polyethylene glycol succinate (TPGS), preferably poloxamer 188.

[0069] In some specific embodiments, the pharmaceutical composition further comprises a second salt selected from sodium chloride, magnesium chloride, or potassium chloride, preferably sodium chloride.

[0070] In some specific embodiments, the pharmaceutical composition further comprises an amino acid selected from aspartic acid, arginine, glycine, histidine, or proline, preferably aspartic acid.

[0071] In some embodiments, the pH of the pharmaceutical composition of the present invention is 5.5–8.0. In some preferred embodiments, the pH of the pharmaceutical composition of the present invention is 6.0–8.0. In some preferred embodiments, the pH of the pharmaceutical composition of the present invention is 5.5–7.5. In some preferred embodiments, the pH of the pharmaceutical composition of the present invention is 5.5–6.5. In some more preferred embodiments, the pH of the pharmaceutical composition of the present invention is 6.0 ± 0.2. The pharmaceutical composition can be adjusted to the desired endpoint pH using sodium hydroxide or hydrochloric acid as a pH adjuster.

[0072] In some specific embodiments, the pharmaceutical composition comprises:

[0073] 5mM to 100mM buffer;

[0074] 2.5% to 10 wt% stabilizer;

[0075] Surfactants of 0.001% to 0.05 wt%;

[0076] Amino acids ranging from 0 to 150 mM;

[0077] A second type of salt, 0–10 wt%.

[0078] In some specific embodiments, the pharmaceutical composition comprises:

[0079] Sodium citrate, 5mM to 100mM;

[0080] 2.5%–10 wt% sucrose;

[0081] 0.001% to 0.05 wt% of poloxamer 188;

[0082] 0–150 mM aspartic acid;

[0083] 0-10 wt% sodium chloride.

[0084] In some specific embodiments, the pharmaceutical composition comprises:

[0085] Sodium citrate, 5mM to 40mM;

[0086] 2.5%–10 wt% sucrose;

[0087] 0.001% to 0.05 wt% of poloxamer 188;

[0088] 5–40 mM aspartic acid;

[0089] 0-5 wt% sodium chloride.

[0090] In some specific embodiments, the pharmaceutical composition comprises:

[0091] 40mM sodium citrate;

[0092] 5 wt% sucrose;

[0093] 0.005 wt% poloxamer 188;

[0094] 40mM aspartic acid.

[0095] In some specific embodiments, the pharmaceutical composition comprises:

[0096] 40mM sodium citrate;

[0097] 5 wt% sucrose;

[0098] 0.005 wt% poloxamer 188;

[0099] 40mM of arginine.

[0100] In some specific embodiments, the pharmaceutical composition comprises:

[0101] 40mM sodium citrate;

[0102] 5 wt% sucrose;

[0103] 0.005 wt% poloxamer 188;

[0104] 40mM proline.

[0105] In some specific embodiments, the pharmaceutical composition comprises:

[0106] 20mM sodium citrate;

[0107] 5 wt% sucrose;

[0108] 0.05wt% poloxamer 188;

[0109] 20mM aspartic acid;

[0110] 100mM sodium chloride (approximately 0.5844wt%).

[0111] In some embodiments, the pharmaceutical composition of the present invention comprises a recombinant adeno-associated virus genome concentration of approximately 1*103 7 vg / ml up to 1*10 14vg / ml. In some preferred embodiments, the genomic concentration of the recombinant adeno-associated virus (AAV) of the present invention is approximately 1*10⁻⁶. 9 vg / ml up to 1*10 13 vg / ml; preferably 1*10 9 vg / ml up to 1*10 12 vg / ml; preferably 1*10 10 vg / ml up to 1*10 13 vg / ml; preferably 1*10 11 vg / ml up to 1*10 14 vg / ml.

[0112] In some preferred embodiments, the genome concentration of the recombinant adeno-associated virus (AAV) of the present invention is approximately 1 x 10⁻⁶. 7 1.5 x 10 7 2 x 10 7 2.5 x 10 7 3 x 10 7 3.5 x 10 7 4 x 10 7 4.5 x 10 7 5 x 10 7 5.5 x 10 7 6 x 10 7 6.5 x 10 7 7 x 10 7 7.5 x 10 7 8x10 7 8.5 x 10 7 9x10 7 9.5 x 10 7 1x10 8 1.5 x 10 8 2 x 10 8 2.5 x 10 8 3 x 10 8 3.5 x 10 8 4 x 10 8 4.5 x 10 8 5 x 10 8 5.5 x 10 8 6 x 10 8 6.5 x 10 8 7 x 10 8 7.5 x 10 8 8x10 8 8.5 x 10 8 9x10 8、9.5x10 8 ,1x 10 9 、1.5x 10 9 ,2x10 9 、2.5x 10 9 、3x 10 9 、3.5x 10 9 ,4x10 9 、4.5x 10 9 ,5x10 9 、5.5x10 9 ,6x10 9 、6.5x10 9 ,7x10 9 、7.5x10 9 ,8x10 9 、8.5x10 9 ,9x10 9 、9.5x10 9 ,1x 10 10 、1.5x 10 10 ,2x10 10 、2.5x 10 10 、3x 10 10 、3.5x 10 10 ,4x10 10 、4.5x 10 10 ,5x10 10 、5.5x10 10 ,6x10 10 、6.5x10 10 ,7x10 10 、7.5x10 10 、8x10 10 、8.5x10 10 ,9x10 10 、9.5x10 10 ,1x 10 11 、1.5x 10 11 ,2x10 11 、2.5x 10 11 、3x 10 11 、3.5x10 11 ,4x10 11 、4.5x 10 11 ,5x10 11 、5.5x10 11 ,6x10 11 、6.5x10 11 ,7x10 117.5 x 10 11 8x10 11 8.5 x 10 11 9x10 11 9.5 x 10 11 1x10 12 1.5 x 10 12 2 x 10 12 2.5 x 10 12 3 x 10 12 3.5 x 10 12 4 x 10 12 4.5 x 10 12 5 x 10 12 5.5 x 10 12 6 x 10 12 6.5 x 10 12 7 x 10 12 7.5 x 10 12 8x10 12 8.5 x 10 12 9x10 12 9.5 x 10 12 1x10 13 1.5 x 10 13 2 x 10 13 2.5 x 10 13 3 x 10 13 3.5 x 10 13 4 x 10 13 4.5 x 10 13 5 x 10 13 5.5 x 10 13 6 x 10 13 6.5 x 10 13 7 x 10 13 7.5 x 10 8 8x10 13 8.5 x 10 13 9x10 13 9.5 x 10 13 1x10 14 (All units are vg / ml)

[0113] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition of the present invention is administered via intravenous, subcutaneous, intramuscular, or intracardiac injection. In a preferred embodiment, the recombinant adeno-associated virus or pharmaceutical composition of the present invention is administered via intramuscular, intravenous, or intracardiac injection.

[0114] In some embodiments, the pharmaceutical composition of the present invention is a liquid composition or a pharmaceutical composition obtained by freeze-drying a liquid composition.

[0115] In some embodiments, the pharmaceutical composition of the present invention is stored in a unit-dose container. In some specific embodiments, the unit-dose container is a vial or a syringe. In some preferred embodiments, the vial is a glass vial; in other preferred embodiments, the syringe is a pre-filled syringe.

[0116] Another aspect of the present invention provides the use of the recombinant adeno-associated virus in the preparation of a medicament for the prevention or treatment of diseases or conditions caused by MYBPC3 mutations.

[0117] Another aspect of the present invention provides a method for preventing or treating diseases or conditions caused by MYBPC3 mutations, the method comprising administering an effective amount of the recombinant adeno-associated virus or pharmaceutical composition according to the present invention to an individual in need.

[0118] In some specific embodiments, the disease or condition caused by the MYBPC3 mutation described in this invention is cardiomyopathy. In some preferred embodiments, the disease or condition caused by the MYBPC3 mutation described in this invention is hypertrophic cardiomyopathy. In some preferred embodiments, the prevention or treatment of the disease or condition caused by the MYBPC3 mutation includes expressing the MYBPC3 protein in the heart of the subject and / or increasing MYBPC3 activity and / or increasing cardiac function.

[0119] Another aspect of the present invention provides the use of the recombinant adeno-associated virus in the preparation of a medicament for the prevention or treatment of heart failure.

[0120] Another aspect of the present invention provides a method for preventing or treating heart failure, the method comprising administering to an individual in need an effective amount of the recombinant adeno-associated virus or pharmaceutical composition according to the present invention.

[0121] In some specific embodiments, the heart failure described in this invention is caused by ischemia, arrhythmia, myocardial infarction, abnormal cardiac contractility, or abnormal cardiac function. 2+ Metabolic causes. In some preferred embodiments, the prevention or treatment of heart failure includes expressing the SERCA2a protein in the subject's heart and / or increasing SERCA2a activity. Attached Figure Description

[0122] Figure 1. Fluorescence image of mouse heart tissue section in Example 2.

[0123] Figure 2 shows DNA copies of the target gene in different tissue samples of mice in Example 2.

[0124] Figure 3 shows the protein expression levels of primary mouse cardiomyocytes and IPSC-CM cells in Example 3.

[0125] Figures 4A-4F show the evaluation of cardiac function in mice after intravenous injection of the drug in the eye in Example 6.

[0126] Figure 5 shows a schematic diagram of the polynucleotide expression cassette in Example 7.

[0127] Figures 6A-6B show the evaluation of cardiac function in mice after intravenous administration in Example 8.

[0128] definition

[0129] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0130] As used herein, the term "AAV" refers to naturally occurring adeno-associated virus and recombinant forms of adeno-associated virus (rAAV), and includes mutant forms of AAV. The term AAV further includes, but is not limited to, AAV 1, AAV 2, AAV 3, AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV 10, avian AAV, bovine AAV, canine AAV, equine AAV, sheep AAV, primate AAV, and non-primate AAV.

[0131] The rAAV of this invention comprises capsid proteins. “Capsid proteins” are structural proteins encoded by the cap gene of AAV. rAAV comprises three capsid proteins, designated VP1, VP2, and VP3, all of which are transcribed from a single cap gene via alternative splicing. The molecular weights of VP1, VP2, and VP3 are approximately 87 kDa, 72 kDa, and 62 kDa, respectively. Upon translation, the capsid proteins form a spherical 60-mer protein shell around the viral genome. The functions of the capsid proteins are to protect the viral genome, deliver the genome, and interact with the host.

[0132] The capsid protein described in this invention can be derived from any adeno-associated virus (AAV) serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. Any of these AAV serotypes can serve as a gene delivery vector. For example, the AAV capsid can be a wild-type capsid or a natural capsid. Wild-type AAV capsids of particular interest include AAV1, AAV2, AAV6, AAV8, and AAV9. Similar to ITRs, the capsid is not necessarily wild-type; rather, as long as the capsid can transduce specific cells or tissues, the wild-type VP1, VP2, or VP3 sequences can be altered through nucleotide insertion, deletion, or substitution. In other words, the AAV capsid can be a variant AAV capsid, which includes one or more amino acid substitutions, deletions, or insertions relative to the parental capsid protein or the AAV capsid protein.

[0133] The term "variant" refers to a mutant of a reference nucleotide or amino acid, for example, having at least one difference in sequence relative to the native nucleotide or amino acid (such as insertion, substitution, or deletion of an amino acid or nucleotide). For the mutant nucleotide or amino acid described in this invention, it may also be described as a modified amino acid or nucleotide.

[0134] The term "polynucleotide expression cassette" as used in this article refers to a cassette comprising two or more functional nucleotide sequences operatively linked together. These nucleotide sequences may include expression regulatory elements, translation initiation sequences, coding sequences, and termination sequences, and are typically composed of DNA.

[0135] The term "coding sequence" as used in this article refers to a nucleotide sequence that encodes a gene product in vivo or in vitro. A coding sequence or gene can encode a polypeptide or protein molecule.

[0136] The term "promoter" as used in this article refers to a DNA sequence that guides RNA polymerase binding and thereby promotes RNA synthesis. Promoters and their corresponding protein expression can be universal (meaning they have strong activity in cells, tissues, and species) or cell-specific, tissue-specific, or species-specific.

[0137] "Heart-specific promoter" refers to a promoter whose activity in cardiac cells is at least twice that in any other non-cardiac cell type. Preferably, the heart-specific promoter suitable for use in the vector of the present invention has an activity in cardiac cells that is at least 5, at least 10, at least 15, at least 20, at least 25, or at least 50 times that in non-cardiac cell types.

[0138] The term "intron" as used in this article refers to any sequence that can be transcribed but not translated. In some cases, an intron can refer to any sequence that has been transcribed and removed from the mature RNA transcript in the cell.

[0139] The term "operably linked" refers to the juxtaposition of genetic elements in a relationship that allows them to operate in the intended manner. For example, if a promoter helps initiate transcription of a coding sequence, it is operably linked to the coding region. Intermediate residues may exist between the promoter and the coding region as long as this function is maintained.

[0140] The term "inverted terminal repeat (ITR)" refers to the ITR sequence used for replication and packaging of AAV viruses.

[0141] The terms "include" or "contain" mean that, in addition to the elements required in the subject matter, elements not mentioned in the subject matter may also be included. For example, for a polynucleotide expression cassette that includes a promoter, other elements besides the promoter (such as ITRs, enhancers, introns, coding genes, polyadenylated nucleotide sequences, etc.) may be included.

[0142] The terms "polyadenylation sequence," "polyadenylation region," and "polyadenylation signal" refer to the recognition region required by endonucleases to cleave RNA transcripts, followed by the polyadenylation concordance sequence AATAAA. The polyadenylation sequence provides the PolyA site, a site on the RNA transcript where adenine residues are added via post-transcriptional polyadenylation.

[0143] The term "homology" refers to the degree of similarity between two or more nucleotide or amino acid sequences when they are compared, expressed as a percentage, such as 85%, 90%, 95%, 99%, or 100%.

[0144] The term "functional variant" refers to a protein that, despite differences in its amino acid sequence (such as mutations, truncations, or insertions), retains essentially the same function (such as activity and specificity) as the original protein.

[0145] The terms “transduced,” “infected,” “transfected,” or “converted” generally refer to methods used to administer, introduce, or insert foreign DNA (vectors) into cells. When DNA is introduced into cells via a virus or viral vector, the cells are transduced with foreign DNA. When DNA is introduced into cells via non-viral methods, the cells are transfected with foreign DNA. The terms “transduced” and “infected” are used interchangeably herein to refer to cells that have received foreign DNA or polynucleotides from a virus or viral vector.

[0146] "Pharmaceutical composition" means containing one or more recombinant adeno-associated viruses (rAAV) as described herein, as well as other components such as physiologically / pharmaceutically viable carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, to facilitate the absorption and / or expression of the active ingredient, and thereby to exert its biological activity. In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0147] Pharmaceutically acceptable buffer solutions are well known in the art and include, but are not limited to: inorganic acid salts, such as phosphates (sodium or potassium), bicarbonates, etc.; organic acid salts, such as citrates, acetates, succinates, etc.; acidic buffer solutions, such as acetic acid, phosphoric acid, hydrochloric acid, carbonic acid, succinic acid, citrate, histidine hydrochloride, malic acid, etc.; and basic buffers, such as sodium hydroxide, Tris, HEPES, etc.

[0148] "Citrate" buffer is a buffer solution that includes citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, magnesium citrate, etc. A preferred citrate buffer is sodium citrate.

[0149] Amino acids are well known in this field, including both natural and non-natural amino acids (synthetic amino acids). Examples include: alanine (Ala); valine (Val); leucine (Leu); isoleucine (Ile); proline (Pro); phenylalanine (Phe); tryptophan (Trp); methionine (Met); glycine (Gly); serine (Ser); threonine (Thr); cysteine ​​(Cys); tyrosine (Tyr); asparagine (Asn); glutamine (Gln); selenocysteine ​​(Sec); pyrrolidone (Pyl); lysine (Lys); arginine (Arg); histidine (His); aspartic acid (Asp); glutamic acid (Glu), etc.

[0150] It has been found that adding moderate levels (e.g., between about 1% and about 10%) of one or more sugars and / or sugar alcohols as stabilizers contributes to the stability of liquid formulations and / or lyophilized formulations. Any sugar can be used as a stabilizer in the pharmaceutical compositions of the present invention; non-limiting examples include monosaccharides, disaccharides, or polysaccharides, or water-soluble dextran, including, for example, fructose, glucose, mannose, sorbitol, xylose, maltose, lactose, sucrose, dextran, trehalose, amylopectin, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, and carboxymethyl cellulose. Sugar alcohols are defined as hydrocarbons having about 4 to about 8 carbon atoms and a hydroxyl group. Non-limiting examples of sugar alcohols that can be used in the pharmaceutical compositions provided by the present invention include mannitol, sorbitol, inositol, galactitol, euonymol, xylitol, and arabinitol.

[0151] The pharmaceutical compositions of the present invention can primarily use nonionic surfactants known in the pharmaceutical field, including but not limited to polysorbate 80 (Tween 80; PS80), polysorbate 20 (Tween 20; PS20) and various poloxamers or pranic derivatives, including pranic F-68 and BRIJ 35, or mixtures thereof.

[0152] "Lyophilized formulation" refers to a pharmaceutical composition or formulation obtained by a vacuum freeze-drying step after the liquid or solution form has been processed.

[0153] In representative embodiments, the pharmaceutical compositions of the present invention have physiologically compatible pH values. In representative aspects, the pH values ​​of the pharmaceutical compositions are about 5.0 to about 9.0, about 5.5 to about 8.0, about 6.0 to about 8.0, about 5.5 to about 7.5, about 6.0 to about 7.5, and about 6.5 to about 7.5. In some embodiments, the pH of the formulation is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In a representative aspect, the pH of the pharmaceutical composition is about 7.0 or about 7.5. In some embodiments, the pH of the pharmaceutical composition is about 6.0 ± 0.2. In some embodiments, the pH of the pharmaceutical composition is about 7.0 ± 0.2. In some embodiments, the pH of the pharmaceutical composition is about 8.0 ± 0.2.

[0154] As used herein, the term “about” indicates an approximate range of plus or minus 10% from a specified value. For example, the expression “about 20%” includes a range of 18-22%. As used herein, “about” also includes the exact amount. Therefore, “about 20%” means both “about 20%” and “20%”.

[0155] The formulations or pharmaceutical compositions of the present invention contain other pharmaceutically acceptable ingredients. In a representative aspect, the formulations or pharmaceutical compositions contain any one or a combination of the following: acidifiers, anticoagulants, antimicrobial preservatives, antioxidants, preservatives, alkalis, inorganic salts, etc.

[0156] As used herein, the term "effective dose" refers to the amount of rAAV virus or pharmaceutical composition that is effective at the dose and time required to achieve the desired preventive or therapeutic effect. Therapeutic effective doses of rAAV virus or pharmaceutical composition can vary depending on factors such as the disease state, age, sex, and weight of the subject being treated, and the ability of the rAAV virus or pharmaceutical composition to elicit the desired response in the subject. Dosing regimens can be adjusted to provide an optimal therapeutic response. Therapeutic effective doses are generally also the amount in which any toxic or harmful effects of the rAAV virus or pharmaceutical composition are outweighed by the beneficial effects of treatment. "Prophylactic effective dose" refers to the amount of rAAV virus or pharmaceutical composition that is effective at the dose and time required to achieve the desired preventive effect, such as the prevention or suppression of various conditions. Prophylactic doses can be used in subjects before or early in the course of disease, and in some cases, prophylactic effective doses may be greater than or less than therapeutic effective doses. Dosage is largely dependent on the condition and size of the subject being treated, as well as the treatment formulation, frequency of treatment, and route of administration. Regimens for continued treatment, including dosage, formulation, and frequency, can be guided by initial response and clinical judgment.

[0157] As used in this article, the term "cardiomyopathy" refers to a decline in heart function due to any cause. Subjects with cardiomyopathy are often at risk of arrhythmias or sudden cardiac death, or both.

[0158] As used in this article, the term "hypertrophic cardiomyopathy" refers to a heart and myocardial disease in which a portion of the myocardium is hypertrophic. Detailed Implementation

[0159] The present invention will be further described below with reference to embodiments, but these embodiments do not constitute any limitation on the present invention.

[0160] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0161] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0162] Example 1: Construction of Mutants

[0163] AAV9 mutants were constructed using site-directed mutagenesis. The mutants contained the amino acid sequences of the polypeptide sequences SVLMKEYD (SEQ ID NO:1), SVLLKYD (SEQ ID NO:2), YMLDK (SEQ ID NO:3), and ARGNDYAR (SEQ ID NO:4), which were inserted between amino acid positions 588 and 589 of the wild-type AAV9 capsid protein, as shown in Table 1.

[0164] Construction of AAV9 capsid mutant plasmid: Primers with homologous arms were designed to insert the polypeptide sequence for PCR amplification. The PCR product was subjected to DNA gel electrophoresis, and the target band was excised and recovered from the gel. The AAV9 backbone vector was digested with enzymes to obtain the digested vector. Digestion conditions: 37℃, >3 hours. The PCR fragment and the digested vector were ligated with homologous recombinase. Ligation conditions: 50℃, 10 minutes. 10 μL of the ligation product was added to stbl3 competent cells, incubated on ice for 20 minutes, heat-shocked at 42℃ for 60 seconds, and then quickly placed on ice for 2-3 minutes. 2XYT medium was added, and the cells were incubated at 37℃ and 220 rpm for 1 hour. The cells were then plated on Kana plates and incubated overnight at 37℃. Clones were picked the next day for sequencing. Successfully sequenced clones were subjected to plasmid extraction to obtain the target plasmid.

[0165] Table 1

[0166] The AAV9 mutant virus packaging was generated through cell co-transfection with three plasmids: a first plasmid (pAAV-CBA-EGFP) containing an expression cassette (EGFP reporter gene or Mybpc3) flanked by an ITR; a second plasmid (the mutant plasmid encoding the Rep / Cap gene constructed in Example 1); and a third plasmid containing an adenovirus helper gene. The target gene plasmid / capsid protein plasmid / helper plasmid were mixed at a 1:2:1 ratio, filtered, and added to the packaging reagent. After mixing and incubation at room temperature for 30 minutes, the mixture was added to suspended HEK293 cells. Cells were cultured at 120 rpm, 37°C, and 5% CO2 for 72 hours, then centrifuged. Cells were incubated overnight at -80°C, allowed to return to room temperature, and then lysed with acid. The pH was adjusted to neutral, filtered through a 0.22 μm filter, and purified using an AAVX purification column to obtain the virus. The viral titer was determined by PCR and used for further processing.

[0167] Example 2: Mouse tail vein injection transduction experiment

[0168] Male C57BL / 6 mice, aged 6–8 weeks, were purchased and acclimatized for 5 days. Each mouse was then injected intravenously with 1E12vg of various AAV mutants (constructed as in Example 1, carrying the EGFP reporter gene); three mice were placed in each group. Heart perfusion was performed 21 days later, and heart tissue sections and immunohistochemical staining were conducted. Fluorescence images were taken after DAPI staining. The results are shown in Figure 1.

[0169] Heart tissue was collected and lysed in 500 μL of DNA extraction buffer in a cell wall-breaking tube at a grinding frequency of 70 rpm for 60 s, repeated 4 times. After grinding, genomic DNA was extracted from the heart tissue using a DNA extraction kit (Qiagen, 69504). The copy number of the target gene was detected by qPCR. The qPCR system (20 μL) consisted of: 5 μL genomic DNA, 10 μL Tagman Fast Advanced Master Mix, 0.5 μL upstream primer, 0.5 μL downstream primer, 0.5 μL probe, and 3.5 μL water. The qPCR reaction program was: pre-denaturation, 95℃, 5 min; cycling reaction, 95℃, 30 s; 60℃, 30 s; 72℃, 60 s; for a total of 40 cycles. The DNA copy number of the target gene in different tissue samples was calculated. The results are shown in Figure 2, where the horizontal axis represents different capsid mutant groups in the horizontal and coronal planes, and the vertical axis represents the vector gene copy number per diploid genome.

[0170] Example 3: Infection experiment with primary mouse cardiomyocytes or IPSC-CM cells

[0171] 1. Infection of primary mouse cardiomyocytes

[0172] Primary mouse cardiomyocytes were digested and seeded at 2E5 cells / well in 24-well plates. The culture medium was DMEM / medium medium + 10% FBS + 1% penicillin antibody, and incubated overnight at 37°C with 5% CO2. Primary mouse cardiomyocytes were then infected with AAV9-MYBPC3 and AAV9-051-MYBPC3 (constructed as in Example 1), with MOIs of 4E3, 2E4, 1E5, 4E5, and 2E6, respectively. The virus was added to the cells after medium replacement, and the cells were incubated at 37°C with 5% CO2 for 6 hours. The culture medium was then replaced with fresh complete medium (DMEM / medium medium + 10% FBS + 1% penicillin antibody). After 72 hours, the cell supernatant was collected, centrifuged at 2000 rpm for 5 minutes, and the supernatant was used for ELISA detection.

[0173] 2. Infection of human IPSC-CM cells

[0174] After digestion, IPSC-CM cells were seeded at 2E5 cells / well in 24-well plates and cultured overnight at 37°C with 5% CO2 using IPSC maintenance medium (Rongchuang Biotechnology). AAV9-MYBPC3 and AAV9-051-MYBPC3 (constructed as in Example 1) were used to infect IPSC-CM cells with MOIs of 4E3, 2E4, 1E5, 4E5, and 2E6, respectively. The virus was added to the cells after medium replacement, and the cells were cultured at 37°C with 5% CO2 for 6 hours, followed by replacement with fresh IPSC maintenance medium. After 72 hours, the cell supernatant was collected, centrifuged at 2000 rpm for 5 minutes, and the supernatant was used for ELISA detection.

[0175] 3. MYBPC3 protein detection

[0176] Human MYBPC3 capture antibody was coated onto ELISA plates using 1X PBS at a concentration of 1 μg / mL (100 μL / well) and incubated overnight at 4°C. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 1X PBST (0.05%), 300 μL each time. The ELISA plate was blotted dry, and 300 μL / well of blocking buffer (5% skim milk) was added. Blocking was performed at 37°C for 1 hour. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 1X PBST (0.05%), 300 μL each time. The ELISA plate was blotted dry, and 100 μL / well of diluted sample was added. The sample was diluted with the coating buffer and incubated at 37°C for 1 hour. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 1X PBST (0.05%), 300 μL each time. The ELISA plate was then blotted dry. Human MYBPC3 detection antibody was diluted 1:3000 with blocking buffer, and 100 μL / well was added to the plate and incubated at 37°C for 1 hour. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 300 μL of 1XPBST (0.05%) each time. The ELISA plate was blotted dry. Anti-biotin detection antibody was diluted 1:4000 with blocking buffer, and 100 μL / well was added to the plate and incubated at 37°C for 1 hour. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 300 μL of 1XPBST (0.05%) each time. The ELISA plate was blotted dry. 100 μL / well of TMB chromogenic buffer was added, and the reaction was incubated at 37°C for 5-10 min. The reaction was stopped by adding 50 μL / well of 2N H2SO4, and the plate was read using an OD450 microplate reader. The MYBPC3 protein content was calculated, and the results are shown in Figure 3, where the horizontal axis represents the genomic titer, and the vertical axis represents the MYBPC3 expression level (ng / mg).

[0177] Example 4: IPSC-CM cell plasmid transfection experiment

[0178] A polynucleotide expression cassette containing regulatory elements and the MYBPC3 coding sequence was constructed using standard recombinant DNA cloning techniques or general molecular biology techniques. The cassette, from 5'ITR to 3'ITR, sequentially includes the 5'ITR, hTNNT2 promoter, target gene encoding MYBPC3, BGH polyadenylation signal, and 3'ITR. Specifically, plasmid construction included: designing homologous arm primers for different promoter sequences (hTNNT2-400, hTNNT2-319, hTNNT2-501, hTNNT2-544) for PCR amplification; performing DNA gel electrophoresis on the PCR products; and recovering the target band by gel excision. The target gene vector was digested with enzymes to obtain a digested vector (the nucleic acid sequence of pAAV-hTNNT2-501-MYBPC3 from the 5'ITR to 3'ITR ends is shown in SEQ ID NO:12). Digestion conditions: 37℃, >3 hours. The PCR fragment and the digested vector were ligated using a homologous recombinase at 50℃ for 10 minutes. Add 10 μL of the ligation product to stbl3 competent cells, incubate on ice for 20 minutes, heat shock at 42°C for 60 seconds, then quickly place on ice for 2-3 minutes. Add 2XYT medium, incubate at 37°C and 220 rpm for 1 hour, plate on Kana plates, and incubate overnight at 37°C. The next day, select clones for sequencing. Successfully sequenced clones are then subjected to plasmid extraction to obtain the target plasmid.

[0179] Human IPSC-CM cells were infected: After digestion, IPSC-CM cells were seeded at 2E5 cells / well in 24-well plates and cultured overnight at 37°C with 5% CO2 in IPSC maintenance medium (Rongchuang Biotechnology). AAV virus was then used to infect IPSC-CM cells with an MOI of 2E5. The virus was added to the cells after medium replacement and cultured at 37°C with 5% CO2 for 6 hours, followed by replacement with fresh IPSC maintenance medium. After 72 hours, cells were harvested, the supernatant was removed, and adherent cells were used for RNA extraction. RNA extraction from IPSC-CM cells was performed using the Vazyme Fastpure complex cell / Tissue Total RNA Isolation Kit (RC113-C1), and cDNA preparation was performed using the Vazyme Reverse Transcription Kit (R433-01). The prepared cDNA was then used for qPCR detection. The relative expression levels of mRNA were detected by qPCR. The qPCR system (20 μl) consisted of: 5 μl genomic DNA, 10 μl Tagman Fast Advanced Master Mix, 0.5 μl upstream primer, 0.5 μl downstream primer, 0.5 μl probe, and 3.5 μl water. The qPCR reaction program was: pre-denaturation, 95℃, 5 min; cycling reaction, 95℃, 30 s; 60℃, 30 s; 72℃, 60 s; for a total of 40 cycles. The relative expression levels of mRNA in different tissue samples were calculated. The relative expression levels of MYBPC3 RNA in plasmids containing different promoters are shown in Table 2.

[0180] Table 2

[0181] Example 5: AAV Infection Experiment in Primary Mouse Cardiac Cells

[0182] The AAV9 mutant virus packaging was generated through cell co-transfection with three plasmids: a first plasmid (such as the plasmid containing different promoter sequences constructed in Example 4) containing an expression cassette (Mybpc3) with the target gene flanked by an ITR; a mutant plasmid (AAV9-051, the second plasmid) encoding the Rep / Cap gene constructed in Example 1; and a third plasmid containing an adenovirus helper gene. The target gene plasmid / capsid protein plasmid / helper plasmid were mixed at a ratio of 1:2:1, filtered, and added to the packaging reagent. After mixing, the mixture was incubated at room temperature for 30 minutes and then added to suspended HEK293 cells. Cells were cultured at 120 rpm, 37°C, and 5% CO2 for 72 hours, then centrifuged. Cells were incubated at -80°C overnight, allowed to return to room temperature, and then lysed with acid. The pH was adjusted to neutral, filtered through a 0.22 μm filter, and purified using an AAVX purification column to obtain the virus. The viral titer was determined by PCR and used for later use.

[0183] Infection of primary mouse cardiomyocytes: After digestion, primary mouse cardiomyocytes were seeded at a rate of 2E5 cells / well in 24-well plates. The cells were cultured overnight at 37°C with 5% CO2 in DMEM / medium medium + 10% FBS + 1% penicillin antibody. AAV virus was then used to infect the primary mouse cardiomyocytes at MOIs of 4E3, 2E4, 1E5, 4E5, and 2E6. The virus was added to the cells after medium replacement and cultured at 37°C with 5% CO2 for 6 hours. The culture was then replaced with fresh complete medium (DMEM / medium medium + 10% FBS + 1% penicillin antibody). After 72 hours, the cell supernatant was collected, centrifuged at 2000 rpm for 5 minutes, and the supernatant was used for ELISA detection.

[0184] MYBPC3 protein detection: Human MYBPC3 capture antibody was coated onto ELISA plates using 1X PBS at a concentration of 1 μg / mL (100 μL / well) and incubated overnight at 4°C. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 1X PBST (0.05%), 300 μL each time. The ELISA plate was blotted dry, and 300 μL / well of blocking buffer (5% skim milk) was added. Blocking was performed at 37°C for 1 hour. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 1X PBST (0.05%), 300 μL each time. The ELISA plate was blotted dry, and 100 μL / well of diluted sample was added. The sample was diluted with the coating buffer and incubated at 37°C for 1 hour. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 1X PBST (0.05%), 300 μL each time. The ELISA plate was then blotted dry. Human MYBPC3 detection antibody was diluted 1:3000 with blocking buffer, and 100 μL / well was added to the plate and incubated at 37°C for 1 hour. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 300 μL of 1XPBST (0.05%) each time. The ELISA plate was blotted dry. Anti-biotin detection antibody was diluted 1:4000 with blocking buffer, and 100 μL / well was added to the plate and incubated at 37°C for 1 hour. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 300 μL of 1XPBST (0.05%) each time. The ELISA plate was blotted dry. 100 μL / well of TMB chromogenic buffer was added, and the reaction was incubated at 37°C for 5-10 min. The reaction was stopped by adding 50 μL / well of 2N H2SO4, and the plate was read using an OD450 microplate reader. The MYBPC3 protein expression level (triple replicates) (ng / mg, representing the target protein expression level in a certain amount of total protein) was then calculated. The results are shown in Table 3.

[0185] Table 3

[0186] Example 6: Pharmacodynamic Experiment of Ocular Intravenous Administration in Mice

[0187] Experimental methods: Mice were C57BL\6 strain MYBPC3 gene knockout mice Mybpc3 KO (n=10). At two weeks of age, mice were administered AAV9-051-hTNNT2-501-MYBPC3 (constructed as in Example 5, hereinafter referred to as rAAV) via intraocular vein at a dose of 1E12vg / mouse. Cardiac function of mice was assessed by echocardiography every four weeks.

[0188] The experimental results of left ventricular mass / body weight ratio (LV Mass / body weight) and ejection fraction are shown in Tables 4A and 4B: After two-week-old homozygous knockout mice were injected with 1E12vg / mouse of rAAV virus encoding MYBPC3 via the orbital suture, the LV Mass / body weight ratio was significantly lower in the rAAV-treated group at 6 months compared with the control group (Mybpc3 KO) (Figure 4A); the cardiac ejection fraction was also increased at 6 months (Figure 4B).

[0189] Left ventricular wall thickness is used to assess the degree of myocardial hypertrophy. Increased wall thickness indicates myocardial hypertrophy. The results of left ventricular end-systolic posterior wall thickness (LVPW; s), left ventricular end-diastolic posterior wall thickness (LVPW; d), and interventricular septal thickness (IVS; s) are shown in Figures 4C, 4D, and 4E. Figure 4C shows that, compared to the control group (Mybpc3 KO), the left ventricular end-systolic posterior wall thickness in the rAAV-treated group decreased significantly at week 8; Figure 4D shows that, compared to the control group (Mybpc3 KO), the left ventricular end-diastolic posterior wall thickness in the rAAV-treated group decreased significantly from week 8; Figure 4E shows that, compared to the control group (Mybpc3 KO), the left ventricular interventricular septal thickness decreased significantly from week 4 in the rAAV-treated group.

[0190] Left ventricular volume (LVESV) is used to assess the degree of left ventricular dilation. It is the volume of the left ventricle at the end of systole and diastole; an increased volume indicates left ventricular dilation. Figure 4F shows that, compared to the control group (Mybpc3 KO), the rAAV-treated group had a significantly decreased left ventricular volume.

[0191] Example 7

[0192] A series of polynucleotide expression cassettes containing various combinations of regulatory elements and coding sequences were constructed using standard recombinant DNA cloning techniques or general molecular biology techniques (Figure 5). Recombinant plasmids containing the polynucleotide expression cassettes described in this figure were then constructed and cloned in *E. coli* using conventional DNA recombination and cloning techniques for subsequent preparation of recombinant AAV viruses. Figure 5 shows a schematic diagram of the recombinant plasmid expression cassette of the present invention, wherein, exemplary, the polynucleotide expression cassette is located between the inverted terminal repeat (ITR) sequences of adeno-associated virus serotype 9 (AAV9) and adeno-associated virus serotype 9 (AAV9-051), and the polynucleotide expression cassette contains the nucleic acid sequence described in SEQ ID NO: 15 from the 5' ITR to the 3' ITR.

[0193] Viral packaging of AAV9 or its mutants is generated by co-transfection of cells with three plasmids: a first plasmid containing an expression cassette with the target gene flanked by an ITR (as described above, containing different target genes); a mutant plasmid encoding the Rep / Cap gene constructed in Example 1 (AAV9-051 or N51, the second plasmid); and a third plasmid containing an adenovirus helper gene. The target gene plasmid / capsid protein plasmid / helper plasmid were mixed at a 1:2:1 ratio, filtered, and added to the packaging reagent. After mixing, the mixture was incubated at room temperature for 30 minutes and then added to suspended HEK293 cells. Cells were cultured at 120 rpm, 37°C, and 5% CO2 for 72 hours, then centrifuged. Cells were incubated at -80°C overnight, allowed to return to room temperature, and then lysed with acid. The pH was adjusted to neutral, filtered through a 0.22 μm filter, and purified using an AAVX purification column to obtain the virus (N51-SERCA2a+I-1c, AAV9-SERCA2a+I-1c). The viral titer was determined by PCR and used for later use.

[0194] Example 8: Pharmacological Study in a Mouse ISO Model

[0195] The ISO model induces cardiomyopathy by injecting animals with isoproterenol to simulate strong sympathetic activation and catechol surges.

[0196] The experimental procedure was as follows: Thirty-two male C57BL / 6J mice, aged 6-8 weeks, underwent echocardiography before grouping. Cardiac ejection fraction was the primary indicator, and body weight was a secondary indicator. Mice were randomly assigned to four groups of eight mice each, for a total of 32 mice. An osmotic pump containing either isoproterenol hydrochloride (ISO) or a blank solvent was implanted subcutaneously. ISO was administered at a dose of 50 mg / day to establish a chronic heart failure model; the day of administration was designated D0. Seven days after model establishment, the mice were administered via tail vein, including a control group (Sham), a model group (ISO), and experimental groups (N51-SERCA2a+I-1c, AAV9-SERCA2a+I-1c). The mice were administered 6E13 vg / kg, and echocardiography was performed to assess cardiac function. Echocardiography was performed every two weeks after administration. The results are shown in Figures 6A and 6B. The results showed that the N51-No.5 (SERCA2a+I-1c) administration group significantly increased cardiac ejection fraction and left ventricular mass ratio (LVdMass / body weight) compared with the AAV9-No.5 (SERCA2a+I-1c) administration group and the model group.

[0197] Example 9: Formulation of AAV drug formulation

[0198] Buffer solution preparation: Weigh appropriate amounts of sodium citrate, sodium chloride, and poloxamer 188, and prepare a buffer solution containing 40 mM sodium citrate buffer, 180 mM sodium chloride, and 0.005 wt% poloxamer 188 in water. Adjust the pH to approximately 6 ± 0.2 with dilute hydrochloric acid or sodium hydroxide solution.

[0199] Sample solution preparation: Take an appropriate amount of the re-prepared adeno-associated virus (AAV9-051-hTNNT2-501-MYBPC3) stock solution from Example 5, inject it into a dialysis card, and perform dialysis with buffer solution to be changed at a volume ratio of ≥100:1. The final concentration of rAAV in the sample solution is 1 x 10⁻⁶. 12 vg / ml.

[0200] Example 10

[0201] Buffer solution preparation: Weigh appropriate amounts of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and poloxamer 188, respectively, and use water as the solvent to prepare a buffer solution containing 30 mM disodium hydrogen phosphate, 15 mM sodium dihydrogen phosphate, 180 mM sodium chloride, and 0.005 wt% poloxamer 188. Adjust the pH to approximately 7 ± 0.2 with dilute hydrochloric acid or sodium hydroxide solution.

[0202] Sample solution preparation: Take an appropriate amount of the re-prepared adeno-associated virus (AAV9-051-hTNNT2-501-MYBPC3) stock solution from Example 5, inject it into a dialysis card, and perform dialysis with buffer solution to be changed at a volume ratio of ≥100:1. The final concentration of rAAV in the sample solution is 1 x 10⁻⁶. 12 vg / ml.

[0203] Example 11

[0204] Buffer preparation: Weigh appropriate amounts of Tris, sodium chloride, and poloxamer 188, using water as the solvent, to prepare a buffer solution containing 40 mM Tris buffer, 180 mM sodium chloride, and 0.005 wt% poloxamer 188. Adjust the pH to approximately 8 ± 0.2 using dilute hydrochloric acid or sodium hydroxide solution.

[0205] Sample solution preparation: Take an appropriate amount of the re-prepared adeno-associated virus (AAV9-051-hTNNT2-501-MYBPC3) stock solution from Example 5, inject it into a dialysis card, and perform dialysis with buffer solution to be changed at a volume ratio of ≥100:1. The final concentration of rAAV in the sample solution is 1 x 10⁻⁶. 12 vg / ml.

[0206] Example 12

[0207] Buffer solution preparation: Weigh appropriate amounts of sodium citrate, sucrose, aspartic acid, and poloxamer 188, respectively, and use water as the solvent to prepare a buffer solution containing 40 mM sodium citrate buffer, 5 wt% sucrose, 40 mM aspartic acid, and 0.005 wt% poloxamer 188. Adjust the pH to approximately 6 ± 0.2 with dilute hydrochloric acid or sodium hydroxide solution.

[0208] Sample solution preparation: Take an appropriate amount of the re-prepared adeno-associated virus (AAV9-051-hTNNT2-501-MYBPC3) stock solution from Example 5, inject it into a dialysis card, and perform dialysis with buffer solution to be changed at a volume ratio of ≥100:1. The final concentration of rAAV in the sample solution is 1 x 10⁻⁶. 12 vg / ml.

[0209] Example 13

[0210] Buffer solution preparation: Weigh appropriate amounts of sodium citrate, sucrose, arginine, and poloxamer 188, respectively, and use water as the solvent to prepare a buffer solution containing 40 mM sodium citrate buffer, 5 wt% sucrose, 40 mM arginine, and 0.005 wt% poloxamer 188. Adjust the pH to approximately 6 ± 0.2 with dilute hydrochloric acid or sodium hydroxide solution.

[0211] Sample solution preparation: Take an appropriate amount of the re-prepared adeno-associated virus (AAV9-051-hTNNT2-501-MYBPC3) stock solution from Example 5, inject it into a dialysis card, and perform dialysis with buffer solution to be changed at a volume ratio of ≥100:1. The final concentration of rAAV in the sample solution is 1 x 10⁻⁶. 12 vg / ml.

[0212] Example 14

[0213] Buffer solution preparation: Weigh appropriate amounts of sodium citrate, sucrose, aspartic acid, sodium chloride, and poloxamer 188, respectively, and use water as the solvent to prepare a buffer solution containing 20 mM sodium citrate buffer, 5 wt% sucrose, 20 mM aspartic acid, 100 mM sodium chloride, and 0.05 wt% poloxamer 188. Adjust the pH to approximately 6 ± 0.2 with dilute hydrochloric acid or sodium hydroxide solution.

[0214] Sample solution preparation: Take an appropriate amount of the re-prepared adeno-associated virus (AAV9-051-hTNNT2-501-MYBPC3) stock solution from Example 5, inject it into a dialysis card, and perform dialysis with buffer solution to be changed at a volume ratio of ≥100:1. The final concentration of rAAV in the sample solution is 1 x 10⁻⁶. 12 vg / ml.

[0215] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A mutant adeno-associated virus (AAV) capsid protein, characterized in that, The capsid protein is an insertion of a heterologous polypeptide comprising about 5 to 8 amino acids relative to the wild-type AAV capsid protein. The heterologous polypeptide includes an amino acid sequence selected from SVLMKEYD (SEQ ID NO:1), YMLDK (SEQ ID NO:3), or ARGNDYAR (SEQ ID NO:4), and the insertion site is located between amino acid positions 588 and 589 of the wild-type AAV9 capsid protein or at the corresponding position of other serum-type capsid proteins.

2. The mutant according to claim 1, characterized in that, The other serum capsid proteins are selected from AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, and AAVrh10.

3. A recombinant adeno-associated virus (rAAV) comprising: i. The modified capsid protein according to any one of claims 1-2; ii. Heterologous nucleic acids containing gene products.

4. The recombinant adeno-associated virus according to claim 3, characterized in that, The gene product is selected from one or two of the following and combinations thereof: i.MYBPC3, KCNH2, TRPM4, DSG2, ATP2A2 proteins; ii. CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A, SOS1, NPR1, ERBB4, VIP, MYH7 proteins; or iii. SERCA2a, I-1c protein.

5. The recombinant adeno-associated virus according to claim 4, characterized in that, The gene product is myosin-binding protein C (MYBPC3) or a functional variant thereof.

6. The recombinant adeno-associated virus according to claim 4, characterized in that, The MYBPC3 peptide sequence has at least 90%, 95%, 98%, 99%, or 100% homology with the polypeptide sequence of SEQ ID NO:

5.

7. The recombinant adeno-associated virus according to claim 4, characterized in that, The heterologous nucleic acid encoding MYBPC3 has at least 90%, 95%, 98%, 99%, or 100% homology with SEQ ID NO:

6.

8. The recombinant adeno-associated virus according to claim 4, characterized in that, The heterologous nucleic acid encoding the gene product is operatively linked to a promoter, wherein the promoter is: i. Muscle-specific promoters; and / or ii. Cardiac cell-specific promoters; and / or iii. Cardiac cell-specific promoters.

9. The recombinant adeno-associated virus according to claim 8, characterized in that, The promoter is the cardiac troponin T promoter.

10. The recombinant adeno-associated virus according to claim 9, characterized in that, The cardiac troponin T promoter is a natural human cardiac troponin T promoter or a truncated version thereof.

11. The recombinant adeno-associated virus according to claim 10, characterized in that, The cardiac troponin T promoter has SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or a sequence having at least 90%, 95%, 98%, 99% or 100% homology with it.

12. The recombinant adeno-associated virus according to claim 5, characterized in that, The recombinant adeno-associated virus comprises a polynucleotide expression cassette, which sequentially contains, from 5' ITR to 3' ITR: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) hTNNT2 promoter; (c) The encoding sequence of MYBPC3; (d) BGH polyadenylation signal; (e) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

13. The recombinant adeno-associated virus according to claim 12, characterized in that, The polynucleotide expression cassette contains the nucleic acid sequence described in SEQ ID NO:12 from 5'ITR to 3'ITR.

14. A recombinant adeno-associated virus (rAAV), characterized in that, The recombinant adeno-associated virus comprises a heterologous nucleic acid encoding MYBPC3 or a functional variant thereof, the heterologous nucleic acid being operatively linked to a promoter, the promoter being the cardiac troponin T promoter having a sequence having SEQ ID NO:9, SEQ ID NO:10 or a sequence having at least 90%, 95%, 98%, 99% or 100% homology thereto.

15. The recombinant adeno-associated virus according to claim 14, characterized in that, The recombinant adeno-associated virus comprises a polynucleotide expression cassette, which sequentially includes, from 5'ITR to 3'ITR, a 5'ITR, an hTNNT2 promoter, a heteronucleotide encoding MYBPC3, a BGH polyadenylation signal, and a 3'ITR.

16. The recombinant adeno-associated virus according to claim 15, characterized in that, The polynucleotide expression cassette contains the nucleic acid sequence described in SEQ ID NO:12 from 5'ITR to 3'ITR.

17. The recombinant adeno-associated virus according to claim 14, characterized in that, The MYBPC3 peptide sequence has at least 90%, 95%, 98%, 99%, or 100% homology with the polypeptide sequence of SEQ ID NO:

5.

18. The recombinant adeno-associated virus according to claim 14, characterized in that, The heterologous nucleic acid encoding MYBPC3 has at least 90%, 95%, 98%, 99%, or 100% homology with SEQ ID NO:

6.

19. A modified cardiac troponin T promoter, characterized in that, The promoters have sequences of SEQ ID NO:9, SEQ ID NO:10, or at least 90%, 95%, 98%, 99%, or 100% homology with them.

20. The recombinant adeno-associated virus according to claim 4, characterized in that, The gene product is SERCA2a and / or I-1c protein or a functional variant thereof, preferably SERCA2a and I-1c protein.

21. The recombinant adeno-associated virus according to claim 20, characterized in that, The SERCA2a and the polypeptide sequence of SEQ ID NO:13 have at least 90%, 95%, 98%, 99%, or 100% homology.

22. The recombinant adeno-associated virus according to claim 20, characterized in that, The I-1c peptide sequence has at least 90%, 95%, 98%, 99%, or 100% homology with the polypeptide sequence of SEQ ID NO:

14.

23. The recombinant adeno-associated virus according to claim 20, characterized in that, The recombinant adeno-associated virus comprises a polynucleotide expression cassette, which sequentially contains, from 5' ITR to 3' ITR: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) Polyadenylation signal; (c) SERCA2a encoded sequence; (d) miniCMV promoter; (e) CMV promoter; (f) PI introns; (i) I-1c encoded sequence; (j) Polyadenylation signal; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

24. The recombinant adeno-associated virus according to claim 23, characterized in that, The polynucleotide expression cassette contains the nucleic acid sequence described in SEQ ID NO:15 or SEQ ID NO:16 from 5'ITR to 3'ITR.

25. A pharmaceutical composition comprising: a) The recombinant adeno-associated virus according to any one of claims 3-18 or 20-24; b) Pharmaceutically acceptable excipients.

26. The pharmaceutical composition according to claim 26, characterized in that, The pharmaceutical composition is administered via intravenous, subcutaneous, intramuscular, or intracardiac injection; preferably, the recombinant adeno-associated virus or pharmaceutical composition is administered via intramuscular, intravenous, or intracardiac injection.

27. Use of the recombinant adeno-associated virus according to any one of claims 3-18 in the preparation of a medicament for the prevention or treatment of diseases or conditions caused by MYBPC3 mutations.

28. The use according to claim 27, characterized in that, The disease or condition caused by the MYBPC3 mutation is cardiomyopathy; preferably, the disease or condition caused by the MYBPC3 mutation is hypertrophic cardiomyopathy; more preferably, the prevention or treatment of the disease or condition caused by the MYBPC3 mutation includes expressing the MYBPC3 protein in the heart of the subject and / or increasing MYBPC3 activity and / or increasing cardiac function.

29. Use of the recombinant adeno-associated virus according to any one of claims 20-24 in the preparation of a medicament for the prevention or treatment of heart failure.

30. The use according to claim 27, characterized in that, The heart failure is caused by ischemia, arrhythmia, myocardial infarction, abnormal cardiac contractility, or abnormal cardiac function. 2+ Metabolic causes; preferably, the prevention or treatment of heart failure includes expressing the SERCA2a protein in the subject's heart and / or increasing SERCA2a activity.

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