Targeted SGCA polypeptide and application thereof in cardiac specific drug delivery

Abstract

The invention relates to a polypeptide targeting SGCA and application of the polypeptide in cardiac specific drug delivery. Specifically, the invention relates to a polypeptide capable of being specifically combined with myocardial cell surface SGCA protein, a coding nucleic acid of the polypeptide, and a polypeptide conjugate containing the targeting peptide. The invention further relates to application of the SGCA targeting peptide in preparation of drugs or reagents for detecting, preventing, relieving or treating cardiovascular diseases, for example, the SGCA targeting peptide can be coupled with the drugs or the detection reagents to form a targeting delivery system, and therefore efficient and specific delivery of the drugs or the detection reagents to myocardial cells is achieved. The targeting peptide disclosed by the invention has excellent heart targeting property and cell internalization capability, and meanwhile, the treatment effect of the medicine can be remarkably improved, and systemic toxic and side effects can be reduced.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to a polypeptide targeting SGCA and its application in cardiac-specific drug delivery. Background Technology

[0002] Cardiovascular disease is a leading cause of death and disability worldwide. Its pathological processes often involve damage and dysfunction of cardiomyocytes, and systemic drug administration is a common treatment approach. However, the heart has a unique physiological environment: rapid blood perfusion, high intracardiac shear stress, and cardiomyocytes are densely encapsulated by the extracellular matrix, making it difficult for drug molecules to effectively accumulate and penetrate. Therefore, systemic drug administration results in low accumulation efficiency in diseased cardiac tissues and a lack of tissue targeting. This leads to most drugs being distributed in non-target tissues and organs, limiting therapeutic efficacy and potentially causing systemic off-target effects and dose-limiting toxicities, posing potential safety risks.

[0003] Existing targeting proteins have low targeting efficiency, making them difficult to widely use in clinical practice. Therefore, developing a targeted delivery system that can specifically recognize and bind to cardiomyocytes and deliver therapeutic drugs efficiently and accurately to the site of cardiac lesions is a key technical problem that urgently needs to be solved in the field of cardiovascular disease treatment.

[0004] It should be noted that the methods described in this section are not necessarily methods that had been previously conceived or adopted. Unless otherwise specified, no method described in this section should be assumed to be prior art simply because it is included in this section. Similarly, unless otherwise specified, the issues mentioned in this section should not be considered to be accepted in any prior art. Summary of the Invention

[0005] This application aims to provide a novel polypeptide that can specifically target cardiomyocytes, and a delivery system based on the polypeptide, which can be used for the efficient and low-toxicity treatment or diagnosis of cardiovascular diseases.

[0006] Specifically, the beneficial effects of this application include at least the following five aspects: First, high specificity and affinity: SGCA is a protein specifically and highly expressed on the cardiomyocyte membrane, especially under pathological conditions such as myocardial infarction, where its expression level is significantly upregulated, making it a highly promising drug delivery target. The SGCA-targeting peptides (SGCA-8 and SGCA-9) obtained in this application through phage display technology can specifically and with high affinity bind to SGCA proteins on the surface of cardiomyocytes, while exhibiting extremely low non-specific binding to other major organs and tissues.

[0007] Second, excellent cardiac targeting: In vivo experiments have shown that SGCA-targeting peptides can be rapidly and specifically enriched in the cardiac region after intravenous injection, demonstrating excellent cardiac targeting ability, especially SGCA-9.

[0008] Third, effective cell internalization function: The SGCA-targeting peptide provided in this application can be internalized by cardiomyocytes and is mainly located in lysosomes, indicating that it can effectively deliver the therapeutic drugs it carries into the cell to exert their effects.

[0009] Fourth, significant therapeutic effects: In animal models of myocardial infarction, siRNA delivered via SGCA-targeting peptides significantly reduced the infarct area and improved cardiac function, demonstrating the effectiveness and therapeutic potential of this delivery system.

[0010] Fifth, high safety: The SGCA-targeting peptide provided in this application can significantly reduce the distribution of the drug in non-target tissues by improving the cardiac targeting of the drug, thereby potentially reducing systemic toxic side effects and improving the therapeutic index.

[0011] According to one embodiment of this application, this application provides a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] According to one embodiment of this application, a polynucleotide is also provided, the polynucleotide comprising a nucleotide sequence encoding the polypeptide described in this application.

[0013] According to one embodiment of this application, an expression vector is also provided, the expression vector comprising the polynucleotide described in this application. According to one embodiment of this application, a polypeptide conjugate is also provided, the polypeptide conjugate comprising the polypeptide described in this application, and further comprising at least one of a drug or a detectable marker.

[0014] According to one embodiment of this application, a pharmaceutical composition is also provided, the pharmaceutical composition comprising the polypeptide, the polynucleotide, the expression vector, and / or the polypeptide conjugate described in this application.

[0015] According to one embodiment of this application, a kit is also provided, the kit comprising the polypeptide, the polynucleotide, the expression vector, and / or the polypeptide conjugate described in this application.

[0016] According to one embodiment of this application, the use of the polypeptide, polynucleotide, expression vector, and / or polypeptide conjugate described in this application in the preparation of medicaments, reagents, or kits for detecting, preventing, alleviating, or treating diseases is also provided.

[0017] According to one embodiment of this application, the use of the polypeptide, polynucleotide, expression vector, and / or polypeptide conjugate described in this application in the preparation of a medicament or diagnostic reagent for delivery to cardiac cells or cardiac tissue is also provided.

[0018] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0019] The accompanying drawings exemplify embodiments and form part of the specification, serving together with the textual description to explain exemplary implementations of the embodiments. The illustrated embodiments are for illustrative purposes only and do not limit the scope of the claims. Throughout the drawings, the same reference numerals refer to similar but not necessarily identical elements.

[0020] Figure 1 The results of Western Blot analysis and statistical analysis of the expression level of SGCA protein in different mouse tissues in Example 1.1 are shown.

[0021] Figure 2 This is the Western Blot result and statistical result of the changes in SGCA protein expression level in the heart tissue of mice in the central infarction model group (MI) and control group mice (Sham) in Example 1.2.

[0022] Figure 3 The results are from the phage pool ELISA detection in Example 2, which used SGCA as the target and employed phage display peptide library technology for four rounds of screening.

[0023] Figure 4 The results of ELISA detection of phage clones in the third round of enrichment products (R3) obtained in Example 2 are shown.

[0024] Figure 5 The results of phage-level ELISA detection for the nine potential positive plasmids (SGCA-1, SGCA-5, SGCA-7, SGCA-8, SGCA-9, SGCA-14, SGCA-17, SGCA-34, and SGCA-54) obtained in Example 2 are shown.

[0025] Figure 6 The results of ELISA detection of protein levels of the eight successfully expressed recombinant proteins (SGCA-5, SGCA-7, SGCA-8, SGCA-9, SGCA-14, SGCA-17, SGCA-34, and SGCA-54) obtained in Example 2 are shown.

[0026] Figure 7 This is an in vivo imaging image of mice after Cy5.5-labeled SGCA-8 peptide (S-8) and SGCA-9 peptide (S-9) were injected via the inner canthus vein in Example 3.1.

[0027] Figure 8 The image shows the flow cytometry results and statistical graphs of the Rhodamine B-labeled SGCA-9 polypeptide (S-9) co-incubated with H9C2 cardiomyocytes in Example 3.2.

[0028] Figure 9 This is a confocal microscope image of FAM-labeled SGCA-9 polypeptide (S-9) co-incubated with iPSC-derived cardiomyocytes (iPSC-CM) in Example 3.2.

[0029] Figure 10 The results and statistical graphs of the cardioprotective effect of SGCA-9 peptide (S-9) mediated by Camk2d siRNA on a mouse model of myocardial infarction in Example 4 include myocardial infarction area and cardiac function indicators. Detailed Implementation

[0030] Unless otherwise stated, all figures used in this specification and claims to represent content, concentration, proportion, mass, volume, time, temperature, thickness, technical effect, etc., should in any instance be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. They can vary for those skilled in the art depending on the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by those skilled in the art.

[0031] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​presented in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain some errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range falling within that wider range, as if these narrower numerical ranges were explicitly stated herein.

[0032] Unless otherwise stated or contradicted by the context, the terms or expressions used herein should be read in the context of the document and as understood by one of ordinary skill in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0033] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0034] When used in this document, the terms “include,” “contain,” and “comprising” mean that other elements are not excluded in addition to the listed elements.

[0035] The terms “nucleic acid” and “polynucleotide” used in this application are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogues.

[0036] In this application, the terms "polypeptide" and "peptide" are used interchangeably and refer to a polymer of amino acids of any length. Therefore, polypeptides, oligopeptides, proteins, antibodies, and enzymes are all included in the definition of polypeptide.

[0037] It should be noted that, in the context of this application, upstream refers to the 5' end of the nucleic acid or the N-terminus of the polypeptide, and downstream refers to the 3' end of the nucleic acid or the C-terminus of the polypeptide. The direction from upstream to downstream is from the 5' end to the 3' end or from the N-terminus to the C-terminus.

[0038] The term "vector" as used in this application refers to a self-replicating DNA molecule that transfers a foreign target gene into a host organism, and is often in the form of a circular double-stranded DNA molecule. Typical vectors include plasmids, viruses, bacteriophages, kinases, and mini-chromosomes. Among these, plasmids are the most common form of vector, referring to circular double-stranded DNA that can accept foreign nucleic acid fragments and replicate in prokaryotic or eukaryotic cells.

[0039] In this application, the terms "expression vector" and "recombinant vector" are used interchangeably, referring to a vector containing a foreign gene and also including regulatory elements for expression in a specified host organism. Introducing the expression vector into a suitable host organism enables it to express the inserted target gene.

[0040] The term "detectable marker" as used in this application refers to any substance that can be detected by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, or chemistry. Such markers are well known in the art, and examples include, but are not limited to: enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes, chemiluminescent substances (such as acridine esters), or biotin.

[0041] The terms "inhibit expression," "reduce expression," "downregulate expression," and "silence" used in this application, when referring to Camk2d, mean that the expression of the Camk2d gene is at least partially inhibited, and the degree of inhibition can be tested according to methods known in the art. In some embodiments, the degree of inhibition is manifested as a reduction in the amount of Camk2d mRNA isolated from a first cell or cell population relative to a second cell or cell population, wherein: the first cell or cell population refers to experimental group cells that have been treated to the point that the expression of the Camk2d gene is inhibited; the second cell or cell population is substantially the same as the first cell or cell population, but it is a control group cell that has not undergone the above treatment, and the expression of its Camk2d gene is not inhibited. In other embodiments, the degree of inhibition can be given based on changes in parameters related to the expression function of the Camk2d gene, including but not limited to a decrease in the content of the protein encoded by the Camk2d gene in the cell. Those skilled in the art will be able to determine the degree of inhibition of Camk2d gene expression using any appropriate experiments.

[0042] The terms "double-stranded ribonucleic acid," "double-stranded RNA," or "dsRNA" as used herein refer to a complex of one or more ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands. These two strands forming the double-stranded structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules. In the case of separate RNA molecules, such dsRNA is generally referred to as "short interfering RNA (siRNA)"; when the two strands are part of a larger molecule, and thus linked by an uninterrupted nucleotide chain between the 3' end of the first strand and the 5' end of the second strand to form the double-stranded structure, this linking RNA chain is referred to as "short hairpin RNA (shRNA)." Those skilled in the art will understand that siRNA or shRNA is responsible for RNA interference, a pathway for sequence-specific posttranscriptional gene silencing in animals and plants. siRNA is produced by ribonuclease III from the cleavage of a longer double-stranded RNA (dsRNA) homologous to the silenced gene, or by delivering synthetic RNA to the cell. Techniques for designing such molecules for targeted gene expression repression are well known to those skilled in the art.

[0043] The term "antibody" as used in this application refers to a specific immunoglobulin targeting an antigenic site. Antibodies can be manufactured according to methods known in the art. Antibodies can take the form of polyclonal or monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (e.g., bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, fully human antibodies, human antibodies, fusion proteins containing an antigen-binding site, and any other modified immunoglobulin molecule containing an antigen-binding site, provided that the antibody exhibits the desired biobinding activity.

[0044] The term "antigen-binding fragment" as used in this application refers to one or more portions of an antibody that retain the specificity for binding to a target antigen. Antigen-binding fragments include, but are not limited to, VHH fragments and fragments containing CDRs.

[0045] The term "diagnosis" as used in this application includes the detection or identification of a subject's disease state or condition, determining the likelihood that a subject will develop a specific disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis (or possible progression or regression) of a subject with a disease or condition, or determining the effect of treatment on a subject with a disease or condition.

[0046] The terms "relief" and "treatment" as used in this application, and their synonyms, refer to the improvement of a disease, symptom, and / or condition. "Relief" and "treatment" can be an improvement in at least one measurable physical parameter, which is not necessarily identifiable by the patient. "Relief" and "treatment" can also be the suppression of the progression of a disease, symptom, and / or condition physically (e.g., stabilizing identifiable symptoms), physiologically (e.g., stabilizing physical parameters), or both. "Relief" and "treatment" can also be the slowing of the progression of a disease, symptom, and / or condition or its reversal.

[0047] The term “prevention” as used in this application and its synonyms refer to delaying the onset of a particular disease, condition and / or symptom or related symptoms of such disease, condition and / or symptom or reducing the risk of acquiring such disease, condition and / or symptom.

[0048] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below.

[0049] Polypeptides, polynucleotides, expression vectors According to one embodiment of this application, this application provides a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0050] In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 2.

[0051] According to one embodiment of this application, a polynucleotide is also provided, the polynucleotide comprising a nucleotide sequence encoding the polypeptide described in this application.

[0052] Due to the degeneracy of codons, those skilled in the art will understand that there are various nucleic acid sequences capable of encoding the polypeptide provided in this application, and no limitation is made here. Those skilled in the art can perform appropriate codon optimization and select suitable nucleic acid sequences for expressing the SGCA-targeting polypeptide provided in this application, depending on the intended use or the host.

[0053] According to one embodiment of this application, an expression vector is also provided, the expression vector comprising the polynucleotide described in this application.

[0054] The expression vector can be of any suitable type, such as plasmids, viruses, bacteria, bacteriophages, and insertable DNA fragments, etc., and is not limited thereto. In some preferred embodiments, the expression vector includes, but is not limited to, plasmid vectors or viral vectors. Those skilled in the art can select a suitable expression vector for expressing the polynucleotides provided in this application, depending on the expression purpose or the host.

[0055] The expression vector of this application can be constructed using methods known in the art. For example, appropriate restriction enzyme sites can be added to both ends of the polynucleotide of this application according to the restriction enzyme sites contained in the backbone vector used, and then loaded into the backbone vector.

[0056] In some embodiments, the polynucleotide or the expression vector further comprises a promoter. The promoter can be any suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by cells expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the antibody. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected expressing cells, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins or polypeptides that are homologous or heterologous to those of the expressing cells.

[0057] In some embodiments, the polynucleotide or the expression vector further includes a transcription termination sequence. The transcription termination sequence is a sequence that can be recognized by the expressing cells to terminate transcription. In some embodiments, the transcription termination sequence is operatively attached to the 3' end of a nucleic acid sequence encoding a protein or polypeptide. Any terminator that can function in the selected expressing cells can be used in this application.

[0058] peptide conjugates According to one embodiment of this application, a polypeptide conjugate is also provided, the polypeptide conjugate comprising the polypeptide described in this application, and further comprising at least one of a drug or a detectable marker.

[0059] Because the polypeptides described in this application have specific targeting properties, they can be conjugated to any type of drug or detectable marker and enriched at a specific site. In some embodiments, the detectable markers include chemiluminescent compounds, bioluminescent compounds, radioisotopes, or contrast agents. Those skilled in the art can select any suitable drug or detectable marker as needed.

[0060] Depending on the specific type of the selected drug or detectable marker, those skilled in the art can also use appropriate methods to link the peptide provided in this application with the drug or detectable marker, and these linking methods are all known in the art. In some embodiments, the peptide and the drug or detectable marker form the peptide conjugate through chemical coupling, physical encapsulation, or adsorption.

[0061] The polypeptide described in this application can be conjugated with a detectable marker for the detection of diseases.

[0062] In some embodiments, the detectable marker is a luminescent compound. In some embodiments, the detectable marker includes fluorescein, rhodamine, cyanine, or a fluorescent protein. Examples include, but are not limited to, 5. Carboxyfluorescein, 5 fluorescein isothiocyanate, 6 Carboxyfluorescein, Tetramethylrhodamine 6 Isothiocyanate, 5 Carboxytetramethylrhodamine, 5 Carboxylated p-aminophenol, tetramethyl and tetraethyl rhodamine, diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthalene rhodamine, rhodamine 101 sulfonyl chloride, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, indocyanine green, IR800CW, GFP, RFP, 6 FAM, FAM, fluorescein, or other substances known in the art that can be used for labeling.

[0063] In some embodiments, the detectable marker is a radioactive isotope. Examples include, but are not limited to, those mentioned above. 43 Sc、 44 Sc、 51 Mn, 52 Mn, 64 Cu、 67 Ga、 68 Ga、 86 Y、 89 Zr、 94 mTc, 99 mTc, 111 In、 152 Tb, 155 Tb, 201 Tl、 203 Pb, 18 F, 76 Br、 77 Br、 123 I, 124 I, 125 I.

[0064] The polypeptides described in this application can also be conjugated with drugs for the prevention, relief, or treatment of diseases.

[0065] In some embodiments, the drug comprises at least one of polypeptides, polynucleotides, polynucleotides, organic compounds, inorganic compounds, and / or natural products.

[0066] The drug may be a drug for treating a specific disease. In some embodiments, the drug includes a drug for treating cardiovascular disease. In some embodiments, the drug includes calmodulin-dependent kinase inhibitors, antiplatelet drugs, beta-blockers, aldosterone receptor antagonists, SGLT2 inhibitors, statins, positive inotropic agents, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, sodium channel blockers, potassium channel blockers, gene editing reagents, or cell therapy modifying molecules, or drugs known in the art for treating cardiovascular disease.

[0067] The drug may be a drug targeting a specific target gene or target protein. In some embodiments, the drug is used to inhibit the transcription and / or translation of the nucleic acid encoding the target gene. In some other embodiments, the drug is used to cause deletion and / or mutation of the nucleic acid encoding the target gene. In some other embodiments, the drug is used to cause degradation of the target protein. In some other embodiments, the drug is used to inhibit the activity of the target protein. Those skilled in the art can select suitable peptides, polynucleotides, polynucleotides, organic compounds, inorganic compounds, and / or natural products from the prior art as needed, and conjugate the corresponding drug molecules with the peptide provided in this application, thereby delivering them to a specific site to exert their effect.

[0068] For example, the drug may be an siRNA used to inhibit or reduce the expression of a target gene; to improve the stability of expression in vivo, the drug may also be a short hairpin RNA (shRNA) with a circular structure, which can also inhibit or reduce the expression of a target gene. In some embodiments, the drug further includes antisense oligonucleotides (ASO), microRNAs (miRNAs), or transcription inhibitors. These types of drugs can all be obtained and prepared by methods known in the art. In some embodiments, the siRNA is chemically synthesized. In some embodiments, siRNA can be produced by cleaving long dsRNAs (e.g., dsRNAs longer than about 25 nucleotides) using E. coli RNase III or Dicer enzymes, which process dsRNAs into biologically active siRNAs. In some embodiments, after the siRNA is introduced into target cells in the form of shRNA via an adeno-associated virus vector, it is catalyzed by RNA polymerase III and Dicer enzymes within the target cells to produce biologically active siRNA. See, for example... Proc.Natl.Acad.Sci.USA , 99:9942-9947 (2002); Proc.Natl.Acad.Sci.USA , 99:14236(2002); Ambion TechNotes , 10(1): 4-6(2003); Nucleic Acids Res. , 31: 981-987 (2003); Science , 293: 2269-2271 (2001); and J.Biol.Chem. These publicly available contents are incorporated into this paper in their entirety by reference (243:82(1968)).

[0069] In some specific embodiments, the drug is a double-stranded ribonucleic acid (BRNA) for inhibiting Camk2d expression, the BRNA comprising a sense strand and an antisense strand. In some specific embodiments, the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 4, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 5.

[0070] Those skilled in the art can also use the sequence of Camk2d and its encoding nucleic acid, and apply methods known in the art to prepare other suitable drugs to inhibit or reduce the expression level of Camk2d or its encoding nucleic acid, thereby achieving the same or similar effects, including but not limited to gene knockout, antisense nucleic acid, or interfering RNA technology. Techniques for designing such molecules for targeted inhibition at the nucleic acid level are well known to those skilled in the art.

[0071] Furthermore, the target gene or target protein is not limited to Camk2d. Those skilled in the art can also target other targets and conjugate their corresponding drug molecules with the peptides provided in this application, thereby delivering them to specific sites to exert their effects, which will not be elaborated here.

[0072] In addition to the peptide conjugates disclosed above, the peptides disclosed herein can also be conjugated with other factors by chemical methods or through genetic engineering methods, which will enhance or provide other properties to the peptides.

[0073] Drug compositions, reagent kits According to one embodiment of this application, a pharmaceutical composition is also provided, characterized in that the pharmaceutical composition comprises the polypeptide, the polynucleotide, the expression vector, and / or the polypeptide conjugate described in this application.

[0074] In some embodiments, the pharmaceutical composition is administered via intravenous injection, subcutaneous injection, intramuscular injection, or local injection.

[0075] In some embodiments, the pharmaceutical composition further includes a pharmaceutically or physiologically acceptable carrier. The carrier may be any compatible, physiologically acceptable, non-toxic substance suitable for delivering the polypeptide, polynucleotide, or recombinant vector provided in this application into a mammal (e.g., a human).

[0076] "Pharmaceutically acceptable carrier" refers to a carrier, diluent, or adjuvant used in the formulation or administration of the polypeptide, polynucleotide, or recombinant carrier provided in this application, which is not an essential active ingredient and does not cause excessive toxicity after administration. Suitable pharmaceutically acceptable carriers are well known to those skilled in the art.

[0077] "Physiologically acceptable carrier" refers to a carrier, diluent, or adjuvant that does not cause significant irritation to an organism and does not eliminate the pharmaceutical activity and properties of the peptide, polynucleotide, or recombinant carrier provided in this application. Suitable physiologically acceptable carriers are also well known to those skilled in the art.

[0078] In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients. In some embodiments, the excipients include at least one selected from solubilizers, disintegrants, wetting agents, stabilizers, thickeners, diluents, buffers, and flavoring agents.

[0079] In some non-limiting embodiments, the carrier and / or excipients used in the pharmaceutical compositions of this application may comprise, for example, liquid, gel or solid carriers, aqueous mediators, non-aqueous mediators, antimicrobial agents, isotonic agents, buffers, antioxidants, suspending agents, dispersants, chelating agents, diluents, adjuvants, excipients or non-toxic excipients, other components known in the art, or various combinations thereof.

[0080] According to one embodiment of this application, a kit is also provided, the kit comprising the polypeptide, the polynucleotide, the expression vector, and / or the polypeptide conjugate described in this application.

[0081] Uses and methods According to one embodiment of this application, the use of the polypeptide, polynucleotide, expression vector, and / or polypeptide conjugate described in this application in the preparation of medicaments, reagents, or kits for detecting, preventing, alleviating, or treating diseases is also provided.

[0082] According to one embodiment of this application, the use of the polypeptide, polynucleotide, expression vector, and / or polypeptide conjugate described in this application in the preparation of a medicament or diagnostic reagent for delivery to cardiac cells or cardiac tissue is also provided.

[0083] In some implementations, the disease is associated with elevated SGCA expression levels.

[0084] In some embodiments, the disease includes cardiovascular disease. In some embodiments, the disease includes myocardial infarction, heart failure, myocardial hypertrophy, hypertension, arrhythmia, myocarditis, myocardial fibrosis, and myocardial ischemia-reperfusion injury. In some embodiments, the myocardial infarction includes ischemic myocardial infarction.

[0085] When preparing drugs, reagents, or kits for the detection, prevention, relief, or treatment of diseases, appropriate excipients, pharmaceutically acceptable carriers, physiologically acceptable carriers, etc., may be added according to what is known in the art, and instructions for use may also be attached if necessary.

[0086] When used for prevention, relief, or treatment, the dosage of the peptide conjugate provided in this application may depend on several factors, including the severity and responsiveness of symptoms, route of administration, duration of treatment (from days to months to years), and time to symptom improvement. Those skilled in the art can adjust the dosage regimen to provide a therapeutic response based on the patient's specific circumstances. For example, a single dose may be administered, several separate doses may be administered over a predetermined time period, or the dose may be reduced or increased as indicated by the treatment outcome. The dosage specification is determined by the specific therapeutic effect to be achieved. The dosage value may also vary depending on the type and severity of the condition to be relieved. For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs and the professional judgment of the treating clinician.

[0087] When used for detection purposes, the polypeptides disclosed in this application can be chemically conjugated to a detectable moiety or genetically engineered to provide a detectable polypeptide. The detectable polypeptide includes a detectable moiety, which includes, but is not limited to, chemiluminescent compounds, bioluminescent compounds, radioactive isotopes, contrast agents, enzymes, and prosthetic groups.

[0088] In some embodiments, the detection methods include enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, and / or immunostaining. Those skilled in the art can select appropriate methods as needed; the implementation steps of these detection methods are known in the art and are not limited herein.

[0089] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory), and all embodiments formed by such combinations are considered as part of the disclosure of this application.

[0090] The exemplary embodiments of this application will now be described with reference to the accompanying drawings, including various details of the embodiments to aid understanding. It should be understood that these are merely exemplary and are in no way intended to limit the scope of protection of this application. The scope of protection of this application is defined only by the claims. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0091] Example Unless otherwise specified in this embodiment, the techniques or conditions described in the literature in this field or in accordance with the product instructions shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0092] Example 1: Validation of SGCA as a cardiac target 1.1 Tissue distribution of SGCA protein in normal mice This experiment used male C57BL / 6 mice aged 10 to 12 weeks, all purchased from Beijing Weishang Lide Biotechnology Co., Ltd. (product number: VSM10001). Animals were housed in a specific pathogen-free (SPF) environment with a temperature maintained at 20-25℃ and relative humidity controlled at 30-70%, using a 12-hour light / 12-hour dark cycle. Throughout the experiment, mice had free access to standard feed and water. The expression of SGCA protein in the heart, liver, spleen, lung, kidney, and skeletal muscle tissues of normal mice was detected using Western blotting.

[0093] The specific experimental steps are as follows: Approximately 50 mg of heart, liver, spleen, lung, kidney, and skeletal muscle tissue from normal mice was collected. After homogenization with tissue protein extraction buffer, the tissues were incubated on ice for 30 minutes to achieve complete lysis. The supernatant was collected after centrifugation at 12,000 rpm for 15 minutes at 4°C; this supernatant was the tissue protein. The protein concentration was determined using a BCA protein concentration assay kit, and the protein was diluted with 5× loading buffer and then denatured by heating at 100°C for 10 minutes. 60 μg of deformed tissue protein was separated by SDS-PAGE gel electrophoresis and transferred to a 0.45 μm nitrocellulose membrane. The membrane was incubated at room temperature for 60 minutes with 1×TBST buffer containing 5% skim milk powder, followed by overnight incubation at 4°C with diluted SGCA-specific antibody (1:1000, Abcam, ab189254). The next day, after washing three times with 1×TBST buffer, the membrane was incubated at room temperature for 1 hour with horseradish peroxidase-labeled secondary antibody under light-protected conditions. After color development, scanning and band brightness analysis were performed using an Amersham Image Quant 800 protein blot imaging system. After washing away the original bound antibody with protein blot membrane regeneration buffer, the incubation and detection of the internal control GAPDH antibody were repeated. Changes in SGCA protein expression levels were normalized using GAPDH as an internal control.

[0094] The results are as follows Figure 1 As shown, SGCA protein is specifically highly expressed in cardiac tissue, but hardly expressed in other major organs.

[0095] 1.2 Expression of SGCA protein in a myocardial infarction model mouse Male C57BL / 6 mice aged 10-12 weeks were randomly divided into two groups: a control group (Sham) and a myocardial infarction model group (MI). Mice in the model group underwent thoracotomy via the left third intercostal space after anesthesia with 2-3% isoflurane inhalation, and the left anterior descending coronary artery (LAD) was ligated with 6-0 silk suture. ST segment elevation and pale myocardial color on electrocardiogram were used as markers of successful ischemia modeling. Mice in the control group underwent only thoracotomy without LAD ligation.

[0096] On day 1 (MI1d) and day 3 (MI3d) after modeling, heart tissue was collected for Western blotting to detect the expression of SGCA protein in the heart of the control group and the model group. The specific experimental procedure for Western blotting was the same as in Example 1.1.

[0097] The results are as follows Figure 2 As shown, compared with the control group (Sham), the expression level of SGCA protein in the cardiac tissue of the myocardial infarction model groups (MI1d, MI3d) was significantly upregulated. These results indicate that SGCA is an ideal targeted delivery site for cardiovascular diseases.

[0098] Example 2: Screening and Identification of SGCA-Targeting Peptides Phage display peptide library technology was used for biopanning. Using ArSyNal synthesized peptide libraries (version 1.0), recombinant SGCA protein coated on ELISA plate wells was used as the solid-phase target, and a four-round "adsorption-washing-elution-amplification" panning process was performed. A negative control (e.g., wells coated only with mFc tag protein or BSA) was included in each panning round to reduce non-specific binding.

[0099] Phage pool ELISA assay: To evaluate the enrichment effect of each round of panning, the amplified phage pools after each round of panning were used for ELISA assay. The specific method is as follows: SGCA antigen and negative control antigen (e.g., mFc) were coated separately onto 96-well plates with carbonate buffer (pH 9.6) and incubated overnight at 4°C. The coating solution was discarded, and 300 μL of PBST (containing 0.05% Tween-20) containing 5% skim milk powder (PHUGENE, catalog number PH1519) was added to each well for blocking, and the plates were incubated at 37°C for 2 hours. After blocking, the plates were washed three times with PT buffer (containing 0.05% Tween-20). Subsequently, 100 μL of appropriately diluted phage pools from each round (usually diluted with blocking buffer) was added to each well, and the plates were incubated at 37°C for 1 hour. After incubation, the plates were washed five times with PT buffer to remove non-specifically bound phages. Next, add 100 μL of HRP-labeled M13 phage secondary antibody (SinoBiological, catalog number 11973-MM05T-H) diluted 1:5000 with blocking buffer to each well and incubate at 37°C in the dark for 1 hour. After washing 5 times, add 100 μL of TMB chromogenic solution (Hubei Yingchuang, catalog number TMB-S-003) to each well for colorimetric reaction and incubate at room temperature in the dark for 10-15 minutes. Finally, add 50 μL of 2 M sulfuric acid to each well to stop the reaction, and immediately measure the absorbance (OD450) at 450 nm using a microplate reader. The results are as follows. Figure 3 As shown, the phage pool of the third-round enrichment product (R3) showed a significant binding signal (high OD450 value) to the SGCA target, and the ratio of the signal to the background signal of the negative control was the highest, indicating that effective enrichment was achieved in this round. Therefore, R3 was selected for subsequent monoclonal screening.

[0100] Identification of 55 positive clones: 96 single-clone phages were randomly selected from R3 and amplified. Initial screening was performed using a similar phage cloning ELISA method as described above. The phage supernatant from each clone was reacted with wells coated with the SGCA / mFc fusion protein and control wells coated only with mFc or BSA. The "specific binding signal" of a clone was defined as (OD450 value of SGCA / mFc well) / (OD450 value of control well). Clones with a ratio greater than or equal to 5 were considered positive clones. The initial screening results are as follows: Figure 4 As shown, a total of 55 positive clones were obtained.

[0101] Positive clone sequence analysis and phage-level validation: Sequencing was performed on the 55 positive clones, yielding nine unique amino acid sequences, named SGCA-1, SGCA-5, SGCA-7, SGCA-8, SGCA-9, SGCA-14, SGCA-17, SGCA-34, and SGCA-54. To confirm the specificity of these unique sequence clones, the corresponding nine monoclonal phages were validated again using phage-level ELISA. The experimental method was similar to that of phage pool ELISA, with three replicates for each clone, including wells containing the SGCA target and control wells containing irrelevant proteins. Results are as follows: Figure 5 As shown, all nine clones exhibited specific binding to the SGCA target, while the binding signal with the control protein was extremely low, verifying their specificity.

[0102] Protein-level ELISA validation: To eliminate phage background interference and confirm the properties of the peptides themselves, genes encoding the above nine unique sequences were cloned into expression vectors, induced for expression in an *E. coli* system, and purified to obtain recombinant proteins. Eight of these (except SGCA-1) were successfully expressed and purified. Protein-level ELISA validation was performed: The purified recombinant proteins (concentration uniformly 10 μg / mL) were used as primary antibodies and added to wells coated with SGCA antigen and control antigen, respectively. Subsequent steps used HRP-labeled secondary antibodies against the corresponding tags (e.g., His tags) and a TMB colorimetric system for detection. Results are as follows: Figure 6 As shown, SGCA-8 and SGCA-9 proteins exhibited the highest SGCA binding signal and the lowest background signal.

[0103] Based on the above results, SGCA-8 (corresponding to SEQ ID NO: 1) and SGCA-9 (corresponding to SEQ ID NO: 2) were finally identified as positive targeting peptides with high specificity and affinity, which will be used for subsequent in vivo and in vitro functional studies.

[0104] Example 3: Validation of the in vitro and in vivo targeting and delivery function of the targeting peptide 3.1 In vivo targeting validation Peptide Synthesis: SGCA-8 and SGCA-9 peptides were prepared by Beijing Zhongke Yaguang Biotechnology Co., Ltd. using the Fmoc solid-phase synthesis method. The crude peptides were purified by Varian ProStar 218 high-performance liquid chromatography (HPLC) using an Aglient VenusilMP C18 reversed-phase column. Elution was performed using a linear gradient of water and acetonitrile containing 0.05% trifluoroacetic acid (1 mL / min), monitored at 220 nm. The purified peptides were identified by Voyager-DE STR mass spectrometry, confirming that their molecular weights were consistent with theoretical values. The peptides were dissolved in deionized water to a concentration of 20 mg / mL, aliquoted, and stored at -20°C for later use.

[0105] Peptide labeling: The purified peptides were covalently linked to the near-infrared fluorescent dye Cy5.5NHS ester (Lumiprobe, catalog number 27020). The specific steps were as follows: the peptides were dissolved in sodium bicarbonate buffer at pH 8.5, and a 5-fold molar excess of Cy5.5 NHS ester was added. The reaction was carried out at room temperature in the dark for 2 hours. After the reaction was complete, unreacted dye was removed by HPLC or a desalting column (such as a PD-10 column) to obtain purified Cy5.5-labeled peptides (Cy5.5-SGCA-8 and Cy5.5-SGCA-9). The labeling efficiency was confirmed by mass spectrometry or UV-Vis spectrophotometry.

[0106] Animal experiments: Male C57BL / 6 mice aged 10-12 weeks (purchased from Beijing Weishang Lide Biotechnology Co., Ltd.) were used. Each mouse was injected with 100 μL of PBS buffer (containing 50 μg Cy5.5-labeled peptide) via the inner canthus vein. One hour after injection, the mice were perfused with physiological saline and the hearts were removed. Imaging was performed using the Xenogen IVIS imaging system to quantitatively analyze the distribution of fluorescence signals. Imaging conditions were: excitation filter 640 nm, emission filter 700 nm, and exposure time set automatically or uniformly.

[0107] The results are as follows Figure 7 As shown, Cy5.5-SGCA-8 and Cy5.5-SGCA-9 exhibited significant specific enrichment signals in the cardiac region one hour after injection. The cardiac enrichment signal intensity of Cy5.5-SGCA-9 was significantly higher than that of Cy5.5-SGCA-8, demonstrating that SGCA-9 has superior cardiac targeting capability.

[0108] 3.2 In vitro cell internalization verification (1) Fluorescent labeling of the target peptide: Rhodamine B labeling: The SGCA-9 peptide was labeled with rhodamine B isothiocyanate (Sigma-Aldrich, catalog number #283924). The labeling reaction was carried out in borate buffer at pH 9.0, with a peptide to dye molar ratio of 1:5, and incubated overnight at 4°C in the dark. The labeled product was purified by desalting column to obtain RhB-SGCA-9.

[0109] FAM labeling: The FAM-labeled SGCA-9 peptide (FAM-SGCA-9) was prepared and supplied by Beijing Zhongke Yaguang Biotechnology Co., Ltd., which introduced 5(6)-carboxyfluorescein (5(6)-FAM) at the N-terminus during solid-phase synthesis. The product was purified by HPLC and identified by mass spectrometry.

[0110] (2) Cell experiments: Flow cytometry analysis: H9C2 rat cardiomyocytes (purchased from ATCC, USA, catalog number CRL-1446) were used. Cells were cultured at 2 × 10⁻⁶ cells / mL. 5 / wells were seeded in 6-well plates and cultured for 24 hours. Medium containing 0 (control), 0.05, and 0.1 μg / μL RhB-SGCA-9 was added, and the cells were incubated at 37°C for 4 hours. After incubation, the cells were washed three times with PBS, trypsinized, and resuspended in PBS. Analysis was performed using a flow cytometer (e.g., BD LSR Fortessa), with excitation using a 488 nm laser and fluorescence detection via the 585±21 nm (PE channel). 10,000 cell events were collected from each sample. Results are as follows: Figure 8 As shown, the average fluorescence intensity of cells increases with increasing RhB-SGCA-9 concentration, demonstrating that its internalization is dose-dependent.

[0111] Confocal microscopy observation: Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs, purchased from FujiFilm Cellular Dynamics, iCell® Cardiomyocytes) were used. Cells were seeded in confocal culture dishes. During the experiment, culture medium containing 5 μM FAM-SGCA-9 was added, and the cells were incubated at 37°C for 2 hours. Thirty minutes before the end of incubation, lysosomal red fluorescent probe (LysoTracker™ Deep Red, Thermo Fisher, catalog number #L12492) was added for co-staining. After incubation, the cells were washed three times with pre-warmed PBS. Observation was performed using a confocal microscope (OLYMPUS FV3000). The excitation wavelength of FAM was 488 nm, and the emission detection band was 500-550 nm; the excitation wavelength of LysoTracker Red was 577 nm, and the emission detection band was 590-650 nm. Results are as follows: Figure 9 As shown, the signals of FAM-SGCA-9 (green fluorescence) and lysosomal probe (red fluorescence) highly overlap (appearing yellow), proving that the polypeptide is mainly located in lysosomes after being endocytosed by cells.

[0112] Conclusion: The above in vitro and in vivo experiments collectively demonstrate that the SGCA-9 peptide possesses excellent cardiac tissue targeting and effective cell internalization capabilities, and that it is primarily transported via the lysosomal pathway after entering cells. This provides crucial evidence for its potential as a cardiac-specific drug delivery carrier, delivering therapeutic or diagnostic payloads into cardiomyocytes and exerting its effects.

[0113] Example 4: Evaluation of the therapeutic effect of targeted drug complex 4.1 Construction of the targeting peptide-siRNA complex The siRNA targeting Camk2d (siR-Camk2d) and its control siRNA (siR-NC) were commercially available. Specifically: the nucleic acid sequence of the sense strand of siR-Camk2d is shown in SEQ ID NO: 4, and the nucleic acid sequence of the antisense strand of siR-Camk2d is shown in SEQ ID NO: 5; the nucleic acid sequence of the sense strand of siR-NC is shown in SEQ ID NO: 6, and the nucleic acid sequence of the antisense strand of siR-NC is shown in SEQ ID NO: 7.

[0114] A control peptide (NT) of the same length as the SGCA-9 peptide but without targeting activity was commercially available, and its amino acid sequence is shown in SEQ ID NO: 3.

[0115] The S-9 / siR-Camk2d complex (a complex of SGCA-9 and siR-Camk2d) is constructed as follows: First, a maleimide reactive group is chemically introduced into the C-terminus of the SGCA-9 peptide. Simultaneously, a thiol (-SH) group is commercially synthesized and introduced into the 3' end (sense or antisense strand) of the siR-Camk2d double strand. Subsequently, the modified peptide and modified siRNA are mixed in degassed PBS buffer at pH 7.2-7.4 and reacted at room temperature in the dark for 2-4 hours, forming a stable covalent thioether bond through a specific click chemistry reaction between the maleimide and the thiol group. The reaction product is purified by size exclusion chromatography (SEC) or ultrafiltration centrifugation to remove unreacted peptide and siRNA, yielding the stoichiometric S-9 / siR-Camk2d conjugate.

[0116] The S-9 / siR-NC (a complex of SGCA-9 and siR-NC) and NT / siR-Camk2d complex (a complex of the control peptide and siR-Camk2d) used as controls were also prepared using the exact same chemical modification and coupling methods as the S-9 / siR-Camk2d complex.

[0117] 4.2 Establishment of animal models and group treatment The method for constructing the myocardial infarction (MI) model is the same as in Example 1.2. After successful modeling, the mice were randomly divided into the following four groups, with 6 mice in each group (n=6): ①Sham surgery group: only open chest surgery, no LAD ligation.

[0118] ② Control group 1 (S-9 / siR-NC): Injected with SGCA-9 peptide-conjugated control siRNA complex.

[0119] ③ Control group 2 (NT / siR-Camk2d): Injection of a control peptide-conjugated target Camk2d siRNA complex.

[0120] ④ Treatment group (S-9 / siR-Camk2d): Injection of SGCA-9 peptide-conjugated target Camk2d siRNA complex.

[0121] All treatment interventions were performed 30 minutes after successful model establishment. Each group of mice received a single injection of 100 μL of PBS buffer via the medial canthal vein, containing 2.5 nmol of siRNA from the complex per mouse.

[0122] 4.3 Efficacy Evaluation Indicators Myocardial infarction area determination: On day 1 after modeling, mice in each group were randomly selected and sacrificed. Hearts were removed, rinsed with pre-cooled PBS, and then uniformly sliced ​​(approximately 1-2 mm thick) perpendicular to the long axis of the heart. Heart slices were immersed in 1% 2,3,5-triphenyltetrazolium chloride (TTC) staining solution (Sigma-Aldrich, catalog number T8877) and incubated at 37°C in the dark for 15 minutes. Normal myocardium, containing active dehydrogenases, stained brick red, while the infarcted area appeared pale white due to loss of enzyme activity. After staining, the slices were fixed with 4% paraformaldehyde, photographed, and the percentage of the infarct area (pale area) to the total ventricular area was calculated using ImageJ software.

[0123] Cardiac function was assessed by echocardiography on day 14 post-modeling using a small animal-specific high-frequency ultrasound imaging system (VisualSonics Vevo 2100). Mice were anesthetized with 1.5-2% isoflurane and placed in the left lateral decubitus position. Left ventricular motion curves were recorded using a 30 MHz linear probe in M-mode ultrasound on the parasternal long and short axis sections of the left ventricle. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (FS), among other cardiac function parameters, were measured and calculated. All measurements were performed independently by at least two researchers unaware of the group assignments.

[0124] The results are as follows Figure 10 As shown, the phenotypes of the treated mice were significantly improved compared to the control group, including myocardial necrosis and cardiac function. Specifically, compared with the S-9 / siR-NC group and the NT / siR-Camk2d group, the treated group (S-9 / siR-Camk2d) mice showed a significant reduction in myocardial infarction area and a significant improvement in cardiac function parameters (left ventricular ejection fraction, LVEF). This indicates that Camk2d siRNA delivered by the SGCA-9 peptide can efficiently target diseased hearts and exert a significant cardioprotective effect.

[0125] It should be noted that the above are merely preferred embodiments of this application and are not intended to limit the application. Various modifications and variations are possible for those skilled in the art. Although specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents of the above embodiments may exist or be unforeseeable to the applicant or other those skilled in the art. Therefore, the appended claims and any possible amendments to the claims are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents. Importantly, as technology evolves, many elements described herein can be replaced by equivalent elements appearing after this application.

Claims

1. A polypeptide, characterized in that, The polypeptide contains an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A polynucleotide, characterized in that, The polynucleotide comprises a nucleotide sequence encoding the polypeptide of claim 1.

3. An expression carrier, characterized in that, The expression vector comprises the polynucleotide of claim 2.

4. A polypeptide conjugate, characterized in that, The polypeptide conjugate comprises the polypeptide of claim 1, and further comprises at least one of a drug or a detectable marker.

5. The polypeptide conjugate according to claim 4, wherein, The detectable markers include chemiluminescent compounds, bioluminescent compounds, radioactive isotopes, or contrast agents.

6. The polypeptide conjugate according to claim 5, wherein the detectable marker comprises fluorescein, rhodamine, cyanine, or a fluorescent protein.

7. The polypeptide conjugate according to claim 4, wherein the drug comprises at least one selected from polypeptides, polynucleotides, polynucleotides, organic compounds, inorganic compounds, and / or natural products.

8. The polypeptide conjugate according to claim 7, wherein the drug is used for at least one of the following: This causes deletion and / or mutation of the coding nucleic acid of the target gene; Inhibit the transcription and / or translation of the target gene's encoded nucleic acid; Inhibit the activity of target proteins; and / or It causes the target protein to degrade.

9. The polypeptide conjugate according to claim 8, wherein the drug comprises antisense oligonucleotide (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antibody or antigen-binding fragment thereof, or small chemical molecule inhibitor.

10. The polypeptide conjugate of claim 9, wherein the drug is a double-stranded ribonucleic acid for inhibiting Camk2d expression, the double-stranded ribonucleic acid comprising a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO:

5.

11. The polypeptide conjugate of claim 7, wherein the drug comprises a drug for treating cardiovascular diseases.

12. The polypeptide conjugate of claim 11, wherein the drug comprises calmodulin-dependent kinase inhibitors, antiplatelet drugs, beta-blockers, aldosterone receptor antagonists, SGLT2 inhibitors, statins, positive inotropic agents, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, sodium channel blockers, potassium channel blockers, gene editing reagents, or cell therapy modifying molecules.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polypeptide of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, and / or the polypeptide conjugate of any one of claims 4-12.

14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is administered by intravenous injection, subcutaneous injection, intramuscular injection, or local injection.

15. A reagent kit, characterized in that, The kit comprises the polypeptide of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, and / or the polypeptide conjugate of any one of claims 4-12.

16. Use of the polypeptide of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, and / or the polypeptide conjugate of any one of claims 4-12 in the preparation of a medicament, reagent, or kit for detecting, preventing, alleviating, or treating a disease.

17. The use according to claim 16, wherein the disease is associated with elevated SGCA expression levels.

18. The use according to claim 16, wherein the disease includes cardiovascular disease.

19. The use according to claim 18, wherein the disease includes myocardial infarction, heart failure, myocardial hypertrophy, hypertension, arrhythmia, myocarditis, myocardial fibrosis, and myocardial ischemia-reperfusion injury.

20. Use of the polypeptide of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, and / or the polypeptide conjugate of any one of claims 4-12 in the preparation of a medicament or diagnostic reagent for delivery to cardiac cells or cardiac tissue.

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