Use of artificially designed novel NRG-1 in disease treatment

Abstract

The present invention provides an artificially designed novel NRG-1 nucleic acid molecule, a protein variant thereof, a vector, a vaccine, a cell, a pharmaceutical composition, and a protein composition. Also provided are a method for using the product to produce a protein and a primary RNA polynucleotide, and a use in preparation of a product for preventing, alleviating or treating cardiovascular diseases and neurological diseases, a product for maintaining normal functions of heart cells or tissues, and a vaccine.

Description

Application of a novel artificially designed NRG-1 in disease treatment Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of a novel artificially designed NRG-1 in disease treatment. Background Technology

[0002] Ischemic heart disease is one of the three leading causes of cardiovascular death in China, with myocardial infarction caused by coronary artery obstruction being the most common and severe form of ischemic heart disease. Because adult cardiomyocytes have very limited regenerative capacity, a large number of cardiomyocytes die after a myocardial infarction, leading to insufficient cardiomyocyte numbers, inflammation, and massive proliferation of fibroblasts, resulting in myocardial fibrosis. This causes poor ventricular remodeling, decreased myocardial contractility, and ultimately heart failure. Therefore, safe methods for protecting and replenishing cardiomyocytes are crucial for effectively preventing myocardial remodeling and heart failure after myocardial infarction.

[0003] Neuregulin-1 (NRG-1) is a growth factor expressed in capillary endothelial cells in the heart and acts on cardiomyocytes. NRG-1 exhibits excellent protective effects on the myocardium under pathological conditions; however, the short half-life of circulating NRG-1 severely limits its intravenous administration intervals in current clinical trials. Furthermore, high-dose intravenous infusion of NRG-1β3 can cause elevated liver transaminases and serum bilirubin levels in subjects, posing a significant challenge to the clinical application of NRG-1 due to potential hepatotoxicity. Therefore, efficiently expressing NRG-1 in the myocardium while avoiding hepatotoxicity is a crucial issue that needs to be addressed for the clinical application of NRG-1.

[0004] NRG-1 is a member of the epidermal growth factor (EGF) family, involved in regulating cell growth, division, differentiation, and survival. The NRG-1 gene has 33 exons and, through alternative splicing, produces various isoforms. Based on the alternative splicing at its N-terminus, these isoforms can be classified into types I-VI. Furthermore, based on the alternative splicing at the C-terminus of its active EGF domain, NRG-1 can be further divided into α and β isoforms. NRG-1β has 10-100 times the biological activity of NRG-1α and is crucial for cardiac growth and development and the maintenance of cardiac function. The NRG-1β3 isoform has a stop codon before its transmembrane domain, thus it is synthesized in a secreted form. In addition, types I-VI of NRG-1β are cell membrane anchoring molecules. Notably, type II NRG-1 has a Kringle sequence at its N-terminus, which mediates protein-protein or protein-matrix interactions and is essential for tissue repair and regeneration. Type III NRG-1 has a cysteine-rich domain (CRD) that is also inserted into the lipid bilayer. Summary of the Invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solution:

[0006] This invention provides a nucleic acid molecule that encodes the NRG1 gene, which is divided into type I NRG1-β1, type I NRG1-β2, and type II NRG1-β3, and the NRG1 gene has undergone codon optimization.

[0007] Furthermore, the nucleotide sequence encoding the nucleic acid molecule is as shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0008] Furthermore, the nucleic acid molecule encodes a nucleotide sequence as shown in any of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0009] Furthermore, the nucleic acid molecule also includes mutations in the cleavage sites of ADAM10, BACE1, and / or ADAM17 on the NRG1 gene, provided that the mutations do not affect the original function of the protein expressed by the NRG1 gene.

[0010] Furthermore, the nucleotide sequence encoding the nucleic acid molecule is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7.

[0011] Furthermore, the nucleic acid molecule encodes a nucleotide sequence of the amino acid sequence shown in either SEQ ID NO:6 or SEQ ID NO:7.

[0012] Furthermore, the nucleic acid molecule also includes a cardiomyocyte-specific promoter sequence.

[0013] Furthermore, the cardiomyocyte-specific promoters include hTNT, hBNP, α-myosin heavy chain promoter, early cardiomyocyte-specific promoter mNkx2.5, and ventricular myosin heavy chain gene promoter.

[0014] In some embodiments, the human troponin T (hTNT) or human brain natriuretic peptide (hBNP) promoters specifically initiate NRG-1 expression in cardiomyocytes or specific disease stages, and enhance promoter efficiency through enhancers.

[0015] Furthermore, the nucleotide sequence of the cardiomyocyte-specific promoter is as shown in SEQ ID NO:1 or SEQ ID NO:2, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0016] Furthermore, the cardiomyocyte-specific promoter is shown in either SEQ ID NO:1 or SEQ ID NO:2.

[0017] Furthermore, the cardiomyocyte-specific promoter sequence is sequentially linked to the nucleotide sequence encoding the NRG1 gene.

[0018] In some embodiments, the cardiomyocyte-specific promoter includes the promoter portion of any cytokine that is highly expressed in cardiomyocytes but poorly expressed or not expressed in other parts of the body, as is included in the prior art. In some embodiments, the cardiomyocyte-specific promoter includes the cardiomyocyte-specific promoter described in CN103173451A, comprising a portion of the Troponin I promoter and including an A / T-rich element (TATA / MEF-2), two GATA elements, and a cytosine-rich region (containing a CACC box and an Sp1 element), wherein the sequence of the Troponin I promoter is the sequence of the TNNI3 gene from position -1106 to position 67, wherein the transcription start site in the TNNI3 gene is position 1, and the position preceding the transcription start site is position -1.

[0019] Furthermore, the nucleic acid molecule also includes a WPRE sequence to maintain the stability of the overall sequence.

[0020] Furthermore, the nucleotide sequence encoding the NRG1 gene is sequentially linked to the WPRE sequence.

[0021] In this invention, codon optimization methods are known in the art and can be used as provided herein. In some embodiments, codon optimization can be used to match codon frequencies in the target and host organisms to ensure proper folding; to bias GC content to increase mRNA stability or reduce secondary structures; to minimize the execution of tandem repeat codons or bases that can impair gene structure or expression; to customize transcription and translation control regions; to insert or remove protein transport sequences; to remove / add post-translational modification sites (e.g., glycosylation sites) in encoded proteins; to add, remove, or replace protein domains; to insert or delete restriction sites; to modify ribosome binding sites and mRNA degradation sites; to modulate translation rates so that various protein domains can fold properly; or to reduce or eliminate problematic secondary structures within polynucleotides.

[0022] The terms “nucleic acid molecule,” “coding sequence,” “polynucleotide,” “nucleotide sequence,” “nucleic acid sequence,” and “nucleic acid” used in this invention are used interchangeably and include DNA, RNA, or their hybrids, which may be double-stranded or single-stranded.

[0023] The present invention provides an NRG1 protein variant comprising the amino acid sequence encoded by the aforementioned nucleic acid molecule.

[0024] Furthermore, the amino acid sequence of the NRG1 protein variant is as shown in SEQ ID NO:6 or SEQ ID NO:7, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7.

[0025] Furthermore, the amino acid sequence of the NRG1 protein variant is shown in either SEQ ID NO:6 or SEQ ID NO:7.

[0026] As used in this invention, the term "protein variant" refers to a polypeptide sequence that differs from the original or natural sequence, wherein one or more amino acid residues are deleted, substituted, or added. Variants can be naturally occurring allelic protein variants or non-natural protein variants. Variants may originate from the same species or from other species and may include homologs, paralogs, and orthologs. In some embodiments, protein variants of polypeptides useful in this invention have the same or similar biological activities as those of the parent polypeptide, including hormonal functions or activation-inhibition functions. The term "protein variant" with respect to (poly)peptides includes all forms of polypeptides as defined herein. The term "protein variant" includes naturally occurring polypeptides as well as recombinant and synthetically produced polypeptides.

[0027] The present invention provides a carrier comprising the nucleic acid molecules described above.

[0028] Furthermore, the vector includes plasmid vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, piggyBac vectors, or Sleeping Beauty transposable vectors.

[0029] Furthermore, the adeno-associated virus vector includes ssAAV, scAAV, and / or hybrid AAV subtypes.

[0030] Furthermore, the adeno-associated virus vector includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and / or AAV13.

[0031] In some implementations, the vector used in this invention has the function of carrying nucleic acid molecules, which is well known to the public. The technology, methods and materials required for transporting nucleic acid molecules can be obtained from the relevant technical documents. The technology includes, but is not limited to, vector construction, ligation of vector and target gene, vector transport and cell culture after transport.

[0032] In some embodiments, the vector may also contain other suitable "regulatory elements" or "regulatory sequences," including but not limited to enhancers; transcription factors; transcription terminators; efficient RNA processing signals, such as splicing and polyadenylation signals (polyA); sequences stabilizing cytoplasmic mRNA, such as the posttranscriptional regulatory element (WPRE) of whelping hepatitis virus (WHV); sequences enhancing translation efficiency (i.e., Kozak concordant sequences); sequences enhancing protein stability; and sequences enhancing the secretion of the encoded product when needed. In some embodiments, examples of polyA include SV40, bovine growth hormone (bGH), and TK polyA. In some embodiments, examples of enhancers include alpha-fetoprotein enhancers, TTR minimal promoter / enhancers, LSP (TH-binding globulin promoter / alpha-1-microglobulin / bikunin enhancer), and other enhancers.

[0033] In some embodiments, the adeno-associated virus used in this invention includes various types of registered AAVs, including but not limited to 13 different serotypes of AAVs in primates (i.e., AAV1-AAV13), of which AAV2, AAV3, and AAV9 are derived from humans. In some implementations, different serotypes of AAV can hybridize, and the hybridized AAV will possess characteristics of both heterozygous strains. Therefore, the adeno-associated virus used in this invention also includes AAV subtypes resulting from AVV hybridization, specifically including but not limited to rAAV2 / 1 (tissue affinity with the nervous system (high titer anterograde transsynapse), muscle, skeletal muscle, cardiac muscle, and smooth muscle), rAAV2 / 2 (tissue affinity with the retina, nervous system, muscle, liver, and vascular smooth muscle), rAAV2 / 3 (tissue affinity with muscle, liver, lung, and eye), rAAV2 / 4 (tissue affinity with the nervous system, muscle, eye, and brain), rAAV2 / 5 (tissue affinity with the nervous system, lung, retina, liver, and synovial joints), rAAV2 / 6 (tissue affinity with the nervous system, lung, muscle, and heart), rAAV2 / 7 (tissue affinity with muscle and liver), and rAAV2 / 8 (tissue affinity with the nervous system, liver, muscle, adipose tissue, pancreas, and retina). Tissue affinity), rAAV2 / 9 (tissue affinity with the nervous system, myocardium, lungs, retina, and skin), rAAV2-retro (tissue affinity with the nervous system (reverse non-transsynaptic), AAV-PHP.eB (tissue affinity across the blood-brain barrier), AAV-PHP.S (tissue affinity with the entire peripheral nervous system), AAV-PAN (tissue affinity with the pancreas), AAV-LUNG (tissue affinity with the lungs), AAV-DJ (with... AAV-7m8 (tissue affinity with retina, lung, kidney, and in vitro infected cells), AAV-ShH10Y (tissue affinity with retinal Muller cells), AAV-Rh10 (tissue affinity with liver, blood, heart, and in vitro infected cells), AAV-Anc80L65 (tissue affinity with inner ear, retina, skeletal muscle, and liver), and AAV-SCH9 (tissue affinity with SVZ region neural stem cells).

[0034] As used in this invention, the term "vector" refers to a vector nucleic acid molecule in which a nucleic acid sequence can be inserted for introduction into a cell, where it can replicate. The nucleic acid sequence can be "exogenous," meaning it is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence already present in the cell. Vectors include plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC).

[0035] The present invention provides a vaccine, which includes a viral vector vaccine, an RNA vaccine, a DNA vaccine, or a protein vaccine, and the vaccine includes an active ingredient.

[0036] Furthermore, the active ingredient includes the aforementioned nucleic acid molecule, the RNA polynucleotide encoded by the aforementioned nucleic acid molecule, and / or the protein encoded by the aforementioned nucleic acid molecule.

[0037] Furthermore, the viral vector vaccine includes the nucleic acid molecules described above.

[0038] Furthermore, the vector in the viral vector vaccine self-assembles into virus-like particles.

[0039] Furthermore, the carrier includes a self-deactivating carrier.

[0040] Furthermore, the viruses include lentiviruses, influenza viruses, hepatitis viruses, alpha viruses, filoviruses, adenoviruses, adeno-associated viruses, and / or flaviviruses.

[0041] In some embodiments, the viral vector is an enveloped virus or a non-enveloped virus. In some embodiments, examples of enveloped viruses are selected from families comprising the group consisting of: herpesviruses, poxviruses, hepatotropic DNA viruses, flaviviruses, encapsulated viruses, coronaviruses, hepatitis D viruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, Bunyaviruses, filoviruses, and retroviruses. In some embodiments, examples of non-enveloped viruses are selected from families comprising the group consisting of: adenoviruses, reoviruses, papillomaviruses, picornaviruses, and caliciviruses.

[0042] Furthermore, the virus is an adenovirus or adeno-associated virus.

[0043] Furthermore, the virus in question is an adeno-associated virus.

[0044] Furthermore, the adeno-associated virus includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and / or AAV13.

[0045] In some embodiments, the AAV virus includes an AAV core plasmid or a plasmid modified from an AAV core plasmid, and the structure of the AAV virus includes an AAV capsid and a vector genome packaged in the capsid.

[0046] Furthermore, the nucleotide sequence is codon-optimized or codon-degenerate.

[0047] Furthermore, the nucleotide sequence is codon-selected with changes from the natural type.

[0048] In some implementations, the viral vector vaccine is constructed using any suitable virus, including but not limited to: lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, influenza virus vectors, hepatitis virus vectors, alphavirus vectors, filovirus vectors, and / or flavivirus vectors.

[0049] Furthermore, the RNA vaccine comprises RNA polynucleotides encoded by the nucleic acid molecules described above.

[0050] In some implementations, the RNA vaccine is an RNA polynucleotide encoded by an open reading frame of a gene in the aforementioned nucleic acid molecule.

[0051] Furthermore, the open reading frames of the RNA polynucleotides are codon-optimized.

[0052] In some embodiments, codon-optimized RNA can be RNA in which the G / C level is enhanced. The G / C content of a nucleic acid molecule (e.g., mRNA) can affect RNA stability. RNA with increased amounts of guanine (G) and / or cytosine (C) residues can be functionally more stable than RNA containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. For example, WO02 / 098443 discloses a pharmaceutical composition containing mRNA stabilized in the translation region by sequence modification. Due to genetic code degradation, modification works by replacing existing codons with those that promote greater RNA stability without altering the resulting amino acids. The method is limited to the coding region of RNA.

[0053] Furthermore, the RNA polynucleotide contains at least one chemical modification.

[0054] Furthermore, the chemical modification includes methylation modification, pseudouridine (Ψ) modification, or hypoxanthine modification.

[0055] In some embodiments, chemical modification or being chemically modified refers to modification of at least one of the positions, patterns, percentages, or populations of adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytosine (C) ribonucleosides or deoxyribonucleosides. In some embodiments, polynucleotides may include, for example, any applicable modification to sugars, nucleobases, or inter-nucleoside linkages (e.g., to linked phosphate esters, phosphodiester bonds, or phosphodiester backbones). In some embodiments, modified nucleotide base pairings encompass not only standard adenosine-thymidine, adenosine-uridine, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides, including non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between non-standard bases and standard bases or between two complementary non-standard base structures.

[0056] Furthermore, the RNA polynucleotide also includes a 3'-UTR and / or at least one 5'-UTR.

[0057] Furthermore, the RNA polynucleotide also includes a 3'-UTR and at least one 5'-UTR.

[0058] Furthermore, each of the at least 3'-UTR and the at least 5'-UTR is heterogeneous to the other.

[0059] Furthermore, the at least one 3'-UTR is derived from a gene selected from the following: housekeeping genes, genes encoding membrane proteins, genes involved in cell metabolism, genes involved in transcription, translation and replication, genes involved in protein modification and genes involved in cell division.

[0060] Furthermore, at least one 5'-UTR is derived from a gene selected from the following: housekeeping genes, genes encoding membrane proteins, genes involved in cell metabolism, genes involved in transcription, translation and replication, genes involved in protein modification and genes involved in cell division.

[0061] Furthermore, the RNA polynucleotide is capped.

[0062] Furthermore, the capping includes cap0, cap1, and cap2.

[0063] Furthermore, the RNA polynucleotide is subjected to tailing treatment.

[0064] Furthermore, the tailing includes tailing via cleavage and polyadenylation of specific factors, cleavage stimulating factors, cleavage factor I and cleavage factor II, poly(A) polymerase, poly(A) binding protein, and paired protein.

[0065] Furthermore, the added tail includes a polyadenylated tail.

[0066] Furthermore, the RNA polynucleotide is subjected to capping and tailing treatment.

[0067] Furthermore, the RNA polynucleotide 5'-UTR, the RNA polynucleotide encoded by the aforementioned nucleic acid molecule, and the 3'-UTR are sequentially linked.

[0068] Furthermore, the RNA vaccine is multivalent.

[0069] Furthermore, the RNA vaccine is formulated and delivered on a payload.

[0070] Furthermore, the load includes dendritic cells, cationic nanoemulsions, cationic peptides and polymers, liposome polymers, liposome complexes, and lipid nanoparticles.

[0071] In some embodiments, the cationic nanoemulsion is referred to as cationic nanoemulsion (CNE). In some embodiments, the liposome polymer is referred to as lipopolyplex (LPR). In some embodiments, the lipid nanoparticles are referred to as lipid nanoparticles (LNP). In certain specific embodiments, the loading significantly enhances the efficacy of mRNA vaccines, including both chemically modified and unmodified mRNA vaccines.

[0072] Furthermore, the protein vaccine comprises the protein encoded by the nucleic acid molecule described above.

[0073] Furthermore, the protein vaccine is carried by a carrier comprising serum albumin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, diphtheria toxoid, genetically modified cross-reactive substances of diphtheria toxoid, CRM197, meningococcal outer membrane protein complex and Haemophilus influenzae protein D, rEPA, keyhole hemocyanin, and / or flagellin.

[0074] Furthermore, the vaccine also includes pharmaceutically acceptable adjuvants.

[0075] Furthermore, the pharmaceutically acceptable excipients include those required for intravenous infusion, subcutaneous injection, intravenous bolus injection, intravitreal injection, and intramuscular injection.

[0076] In some embodiments, the formulation is prepared by uniformly and tightly associating a viral vector, RNA polynucleotide, protein with a liquid carrier or a fine solid carrier, or both, and then shaping the product if necessary.

[0077] The term "pharmaceutically acceptable" as used in this invention refers to a carrier that is generally chemically and / or physically compatible with the other components constituting the formulation and physiologically compatible with its recipient. Pharmaceutically acceptable excipients used in the vaccine compositions of the present invention may include, but are not limited to, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous mediators (e.g., sodium chloride injection, Ringer's solution, isotonic glucose injection, sterile water injection, or Ringer's glucose and lactate injection), non-aqueous mediators (e.g., non-volatile plant oils, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents, isotonic agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citrate buffers), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / dispersing agents (e.g., sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween 80)), diluents, adjuvants, excipients, or non-toxic excipients, other components known in the art, or various combinations thereof.

[0078] The present invention provides a cell comprising the aforementioned nucleic acid molecule, the aforementioned NRG1 protein variant, and / or the aforementioned vector.

[0079] Furthermore, the cells include prokaryotic cells or eukaryotic cells.

[0080] Furthermore, the cells include artificial cell lines.

[0081] Furthermore, the artificial cell line includes the HEK293 cell line.

[0082] In some implementations, the artificial cell line refers to artificially modified cells capable of carrying nucleic acid molecules, secreting proteins, and expressing transport vectors.

[0083] In some embodiments, the artificial cell lines include, but are not limited to: HEK293 cell line, CHO cell line, COS cell line, NSO cell line, HeLa cell line, and BHK cell line.

[0084] The present invention provides a pharmaceutical composition comprising the aforementioned nucleic acid molecule, the aforementioned NRG1 protein variant, the aforementioned vector, the aforementioned vaccine, and / or the aforementioned cells.

[0085] The present invention also provides a pharmaceutical formulation comprising the pharmaceutical composition described above.

[0086] Furthermore, the dosage forms of the drug formulation include intravenous infusion formulations, subcutaneous injection formulations, intravenous bolus formulations, intramuscular injection formulations, virus-like particle formulations, self-inactivating carrier formulations, lipid nanoparticle (LNP) encapsulated formulations, cationic nanoemulsion formulations, liposome complex formulations, carrier-bound formulations, and / or suspension formulations.

[0087] The present invention also provides a kit comprising the aforementioned nucleic acid molecule, the aforementioned NRG1 protein variant, the aforementioned carrier, the aforementioned vaccine, the aforementioned cell, the aforementioned pharmaceutical composition, and / or the aforementioned pharmaceutical formulation.

[0088] The present invention provides a protein composition comprising a protein encoded by a nucleic acid molecule as described above and a detectable marker, wherein the protein and the detectable marker are directly or indirectly coupled to form a complex, and the coupling of the protein and the detectable marker does not affect the original function of the protein.

[0089] Furthermore, the detectable markers include fluorescent dyes, chemiluminescent compounds, radioactive isotopes, electron-dense reagents, enzymes, colored particles, or biotin.

[0090] The term "detectable marker" as used in this invention refers to a reagent that is detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful detectable markers include, but are not limited to, fluorescent dyes, chemiluminescent compounds, radioactive isotopes, electron-dense reagents, enzymes, colored particles, and biotin. Detectable markers often produce measurable signals, such as radioactivity, fluorescence, color, or enzyme activity. Antibodies conjugated to detectable reagents can be used for diagnostic or therapeutic purposes. Examples of detectable reagents include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions. Detectable substances can be directly linked to or conjugated with antibodies, or indirectly through intermediates such as known linkers, using techniques known in the art.

[0091] This invention provides any one of the following methods, the method comprising:

[0092] 1) A method for producing a protein, the method comprising: converting the aforementioned nucleic acid molecule or the aforementioned vector into cells, or directly using the aforementioned cells, culturing the cells, and isolating and purifying the protein encoded by the aforementioned nucleic acid molecule from the cell culture medium and / or cells.

[0093] 2) A method for producing primary RNA polynucleotides, the method comprising: transforming the aforementioned nucleic acid molecule or the aforementioned vector into cells, or directly using the aforementioned cells, culturing the cells, and isolating and purifying the primary RNA polynucleotide encoded by the aforementioned nucleic acid molecule from the cell culture medium and / or cells.

[0094] 3) A method for preventing, alleviating, or treating cardiovascular disease, the method comprising administering to a subject in need a preventive, alleviating, or therapeutically effective amount of the aforementioned nucleic acid molecule, the aforementioned NRG1 protein variant, the aforementioned vector, the aforementioned vaccine, the aforementioned cell, the aforementioned pharmaceutical composition, and / or the aforementioned pharmaceutical formulation.

[0095] Furthermore, the cardiovascular diseases include atherosclerosis, ischemic heart disease, coronary artery disease, hypertension, heart failure, arrhythmia, cardiomyopathy, endocarditis, peripheral artery disease, coronary artery bypass grafting, arteritis, myocarditis, cardiovascular disease, unstable angina, unstable refractory angina, stable angina, chronic stable angina, acute coronary syndrome, and / or myocardial infarction.

[0096] 4) A method for preventing, alleviating, or treating neurological diseases, the method comprising administering to a subject in need a preventive, alleviating, or therapeutically effective amount of the aforementioned nucleic acid molecule, the aforementioned NRG1 protein variant, the aforementioned vector, the aforementioned vaccine, the aforementioned cell, the aforementioned pharmaceutical composition, and / or the aforementioned pharmaceutical preparation.

[0097] Furthermore, the neurological diseases include Parkinson's disease, Huntington's disease, Alzheimer's disease, mild cognitive impairment, senile dementia and amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette syndrome, Friedrich's ataxia, Machado-Joseph disease, Lewy body dementia, dystonia, progressive supranuclear palsy and / or frontotemporal dementia.

[0098] 5) A method for improving the function of damaged cardiac cells or tissues, the method comprising administering to cardiac cells or tissues the aforementioned nucleic acid molecules, the aforementioned NRG1 protein variants, the aforementioned vectors, the aforementioned vaccines, the aforementioned cells, the aforementioned pharmaceutical compositions, and / or the aforementioned pharmaceutical preparations.

[0099] Furthermore, the cardiac cells or tissues may be in vivo or in vitro.

[0100] In some embodiments, the dosage and frequency (single or multiple doses) of the pharmaceutical composition or formulation administered to the subject may vary depending on a variety of factors, such as whether the mammal has another disease and the route of administration; the subject's age, sex, health status, weight, body mass index, and diet; the nature and severity of the symptoms of the disease being treated, the types of concurrent treatments, complications arising from the treated disease, or other health-related problems. Other treatment regimens or agents may be used in conjunction with the pharmaceutical compositions or formulations and treatment methods of the present invention as described above. Adjustments and manipulations of the established dosage (e.g., frequency and duration) are entirely within the capabilities of those skilled in the art.

[0101] This invention provides any of the following applications, the applications including:

[0102] 1) The use of the NRG1 gene, the aforementioned nucleic acid molecules, the aforementioned NRG1 protein variants, the aforementioned vectors, and / or the aforementioned cells in the preparation of products for the prevention, relief, or treatment of cardiovascular diseases.

[0103] Furthermore, the cardiovascular diseases include atherosclerosis, ischemic heart disease, coronary artery disease, hypertension, heart failure, arrhythmia, cardiomyopathy, endocarditis, peripheral artery disease, coronary artery bypass grafting, arteritis, myocarditis, cardiovascular inflammation, unstable angina, unstable refractory angina, stable angina, chronic stable angina, acute coronary syndrome, and myocardial infarction.

[0104] 2) The use of the NRG1 gene, the aforementioned nucleic acid molecules, the aforementioned NRG1 protein variants, the aforementioned vectors, and / or the aforementioned cells in the preparation of products that maintain the normal function of cardiac cells or tissues.

[0105] 3) The use of the NRG1 gene, the aforementioned nucleic acid molecules, the aforementioned NRG1 protein variants, the aforementioned vectors, and / or the aforementioned cells in vaccine preparation.

[0106] 4) The use of the NRG1 gene, the aforementioned nucleic acid molecules, the aforementioned NRG1 protein variants, the aforementioned vectors, and / or the aforementioned cells in the preparation of products for the prevention, relief, or treatment of neurological diseases;

[0107] Furthermore, the neurological diseases include Parkinson's disease, Huntington's disease, Alzheimer's disease, mild cognitive impairment, senile dementia and amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette syndrome, Friedrich's ataxia, Machado-Joseph disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and / or frontotemporal dementia.

[0108] Furthermore, the aforementioned NRG1 gene refers to the natural NRG1 gene sequence, with Gene ID: 3084.

[0109] The term "NRG1" as used in this invention, synonymous with "NRG-1," is intended to encompass its fragments, variants (e.g., allele variants), and derivatives. Representative human NRG1 cDNA and human NRG1 protein sequences are well known in the art and are publicly available from the NCBI website. For example, at least one human NRG1 isotype is known: human NRG1, Gene ID: 3084. Nucleic acid and polypeptide sequences of NRG1 orthologs in organisms other than humans are well known.

[0110] The term "ADAM10" as used in this invention is intended to include its fragments, variants (e.g., allele variants), and derivatives. Representative human ADAM10 cDNA and human ADAM10 protein sequences are well known in the art and are publicly available from the NCBI website. For example, at least one human ADAM10 isotype is known: human ADAM10, Gene ID: 102. Nucleic acid and polypeptide sequences of ADAM10 orthologs in organisms other than humans are well known.

[0111] The term "ADAM17" as used in this invention is intended to include fragments, variants (e.g., allele variants), and derivatives thereof. Representative human ADAM17 cDNA and human ADAM17 protein sequences are well known in the art and are publicly available from the NCBI website. For example, at least one human ADAM17 isotype is known: human ADAM17, Gene ID: 6868. Nucleic acid and polypeptide sequences of ADAM17 orthologs in organisms other than humans are well known.

[0112] The term "BACE1" as used in this invention is intended to include its fragments, variants (e.g., allele variants), and derivatives. Representative human BACE1 cDNA and human BACE1 protein sequences are well known in the art and are publicly available from the NCBI website. For example, at least one human BACE1 isotype is known: human BACE1, Gene ID: 23621. Nucleic acid and polypeptide sequences of BACE1 orthologs in organisms other than humans are well known.

[0113] The term "application" as used in this invention means by any means known in the art, specifically including: intravenous application, intramuscular application, intradermal application, subcutaneous application, transdermal application, mucosal application, or intralesional application.

[0114] The term "effective amount" used in this invention includes "therapeutic effective amount" and "preventive effective amount." Specifically, "therapeutic effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. "Therapeutic effective amount" can vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's immune system, the patient's general characteristics such as age, weight, and sex, the method of drug administration, and other concurrent treatments. Specifically, "preventive effective amount" refers to an amount sufficient to prevent, stop, or delay the onset of the disease.

[0115] The term "prevention, relief, or treatment" as used in this invention refers to the complete or partial improvement or reduction of a disease or condition, symptom, adverse effect or consequence, or associated phenotype. This includes, but is not limited to: preventing the onset or recurrence of disease, relieving symptoms, reducing any direct or indirect pathological consequences of disease, slowing the rate of disease progression, improving or mitigating the condition, and alleviating or improving prognosis.

[0116] The term "subject" as used in this invention includes both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles. In some embodiments, the "subject" is preferably a human.

[0117] Advantages and beneficial effects of the present invention:

[0118] This study creatively uses adeno-associated virus (AAV) as a gene therapy vector to specifically overexpress the cardiac endothelial cell protein NRG-1 in cardiomyocytes in a "reverse" manner, thereby increasing the local NRG-1 concentration in the myocardium and exerting its cardioprotective effect. This effectively treats heart failure caused by myocardial infarction and avoids liver toxicity, providing a new strategy and gene therapy drug for the clinical application of NRG-1 in the treatment of myocardial infarction.

[0119] The main technical problems to be solved include:

[0120] 1. By using adeno-associated virus (AAV) as a gene therapy vector, NRG-1 can be expressed in cardiomyocytes for a long time, thus solving the problem of the short half-life of circulating NRG-1 protein.

[0121] 2. By using AAV as a gene therapy vector, NRG-1 can be specifically expressed in cardiomyocytes in a "reverse" manner, thus solving the problem of insufficient penetration or low content of NRG-1 in the myocardium during NRG-1 treatment.

[0122] 3. By using AAV as a gene therapy vector, we can enhance cardiomyocyte-specific promoters and cardiac disease-responsive promoters, and optimize the codons of NRG-1β1, NRG-1β2, and NRG-1β3 to further enhance the expression of NRG-1 in cardiomyocytes, thus solving the problem of insufficient or low NRG-1 penetration in the myocardium during NRG-1 treatment.

[0123] 4. By using AAV as a gene therapy vector, NRG-1 can be specifically expressed in cardiomyocytes, increasing the local NRG-1 content in the myocardium and mitigating the risk of liver toxicity associated with high-dose intravenous infusion of NRG-1.

[0124] 5. By using AAV as a gene therapy vector, NRG-1 is specifically expressed in cardiomyocytes, promoting NRG-1-mediated intercellular interactions between cardiomyocytes and endothelial cells, and between cardiomyocytes, thereby enhancing its cardioprotective effect.

[0125] 6. Provides NRG-1β1 / NRG-1β2 with EGF cleavage site mutations, reducing its secretion into the blood, lowering the risk of liver toxicity, increasing the local NRG-1 concentration in the myocardium, and enhancing its cardioprotective effect. Attached Figure Description

[0126] Figure 1 is a statistical graph of body weight and ejection fraction (EF) in mice with myocardial infarction after injection of AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3.

[0127] Figure 2 is a statistical graph showing the measurements of diastolic left ventricular internal diameter (LVIDD), interventricular septal deviation (IVSD), and left ventricular posterior wall LVPWD in mice with myocardial infarction after injection of AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3.

[0128] Figure 3 is a statistical chart of body weight and cardiac index (heart weight / body weight, HW / BW) measurements in mice with myocardial infarction after injection of AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3.

[0129] Figure 4 shows the Masson's staining results of mice with myocardial infarction after injection of AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3;

[0130] Figure 5 shows the HE staining, Masson's staining, and transaminase results in mice after injection of AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3;

[0131] Figure 6 shows the TUNEL staining results of mice with myocardial infarction after injection of AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3. Detailed Implementation

[0132] Although specific terms are used herein, they are used only in a general and descriptive sense and not for limiting purposes. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several aspects without departing from the scope or substance of this disclosure.

[0133] It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods pertain. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having the same meaning as they have in the context of the specification and related art, and should not be interpreted as idealized or overly formal unless expressly defined herein.

[0134] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0135] Example

[0136] 1. Experimental materials

[0137] 1.1 Instrument Information

[0138] Specific instrument information is shown in Table 1.

[0139] Table 1

[0140] 1.2 Reagent Information

[0141] Specific reagent information is shown in Table 2.

[0142] Table 2

[0143] 2. Experimental Methods

[0144] 2.1 Sequence Construction

[0145] The sequences used in this study are shown in Table 3.

[0146] Table 3

[0147] 2.2 Plasmid Construction

[0148] The full-length AAV master plasmid and helper plasmids pAAV.hTNT.NRG-1.SV40 or pAAV.hTNT.NRG-1.SV40, pAAV2 / 1 or pAAV2 / 9n and pAdDeltaF6 were all synthesized after optimization of the cleavage site mutation.

[0149] 2.3 Virus Packaging and Purification

[0150] AAV was prepared according to the experimental method of Grieger et al. (Nat Protoc. 2006; 1(3): 1412-28).

[0151] 2.4 AAV Virus Gene Extraction and Titer Detection

[0152] Add 100 μl of DNase lysis buffer (50 U DNase I + 1 mL DNase lysis buffer) to 2 μL of virus solution.

[0153] Mix thoroughly for 2 seconds, being careful not to overmix to avoid damaging the viral capsid. Incubate in a water bath at 37°C for 1 hour.

[0154] Add 5 μL of EDTA, mix well, and incubate in a metal bath at 70 °C for 10 min.

[0155] Add 120 μL of protease lysis buffer (1.818 U proteinase K + 500 μL proteinase K solution), mix well, incubate briefly for 3 seconds, and treat in a metal bath at 55 °C for 2 hours.

[0156] The metal bath temperature was adjusted to 95℃ and treated for 10 minutes to inactivate the protease. Then it was placed on ice.

[0157] The obtained sample was diluted, and 3 μL was diluted 300 times for titer detection.

[0158] The extracted DNA was quantified using RT-PCR and standard plasmids to determine the AAV virus titer.

[0159] 2.5 Echocardiographic Examination

[0160] Myocardial infarction model: The day after the myocardial infarction surgery model, mice were injected with the virus AAV9.hTNT.NRG-1 at a dose of 1E11 vg per mouse. Echocardiography was performed weekly postoperatively to assess left ventricular function.

[0161] 2.6 Investigation into the mechanism by which NRG-1 exerts a protective effect on cardiomyocytes after myocardial infarction

[0162] The virus used was AAV9.hTNT.NRG-1, with an injection dose of 1E11 vg per mouse. Four weeks after injection, myocardial infarction surgery was performed. Ninety-six hours after surgery, the mice were sacrificed, and heart tissue sections were prepared for TUNEL staining to assess the degree of cardiomyocyte apoptosis.

[0163] 3. Experimental Results

[0164] 3.1 AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 can treat cardiac dysfunction after myocardial infarction and prevent it from developing into heart failure.

[0165] Eight-week-old male C57 mice underwent Sham surgery and MI surgery, respectively. Two days post-surgery, they were given 1×10 11 Mice were administered a viral load of vg / mouse via tail vein injection, followed by echocardiographic monitoring. Our results showed no significant difference in mouse body weight among the groups (Figure 1A). At 12 weeks post-MI, untreated mice exhibited significantly reduced cardiac ejection fraction, progressing to severe heart failure. Treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 significantly increased ejection fraction and prevented progression to heart failure (Figure 1B). These results demonstrate that treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 can effectively treat post-myocardial infarction cardiac dysfunction and prevent its progression to heart failure.

[0166] 3.2 AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 can prevent myocardial infarction from developing into heart failure and prevent cardiac chamber dilation.

[0167] The cardiac structure of mice 12 weeks after Sham and MI surgeries was examined by echocardiography. The results showed that the cardiac chambers dilated and the heart wall thinned after myocardial infarction in mice, and treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 could prevent this adverse decompensation to some extent (Figure 2A-C).

[0168] 3.3 Treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 can alleviate pathological myocardial hypertrophy caused by myocardial infarction.

[0169] Mice were sacrificed 12 weeks post-surgery for organ harvesting and weighing. Our results showed no significant difference in body weight among the four groups (Figure 3A). Myocardial infarction caused pathological hypertrophy of the heart in mice, while no significant hypertrophy was observed in the treatment group (Figure 3B).

[0170] 3.4 Treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 can effectively reduce the area of ​​myocardial infarction.

[0171] The hearts of mice sacrificed 12 weeks post-surgery were fixed, embedded in paraffin, and sectioned. Masson's staining was used to determine the area of ​​myocardial infarction. Our results showed that treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 effectively reduced the area of ​​myocardial infarction (Figure 4).

[0172] 3.5 Treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 did not cause liver damage.

[0173] The effects of AAV injection on the liver tissue of 8-week-old male C57 mice were investigated 4 weeks later. HE staining and Masson's staining results showed no cell death or fibrosis in the liver tissue (Figure 5A). Furthermore, we isolated mouse serum for transaminase measurement, and the results showed that injections of AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 did not cause an increase in serum transaminase levels (Figure 5B). No increase in transaminase levels was also found in the serum of mice in the MI surgery group and the treatment group (Figure 5C). These results indicate that AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 are not hepatotoxic and do not cause liver damage in mice.

[0174] 3.6 Treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 can significantly inhibit cardiomyocyte apoptosis after myocardial infarction.

[0175] Mice were first injected with AAV, and four weeks later underwent myocardial infarction surgery. Two days post-surgery, the mice were sacrificed, and TUNEL staining was performed. Our results showed that treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 effectively inhibited cardiomyocyte apoptosis (Figure 6). These results demonstrate that treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 can significantly inhibit cardiomyocyte apoptosis after myocardial infarction.

[0176] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes an NRG1 gene, the NRG1 gene is a type I NRG1-β1, a type I NRG1-β2 or a type II NRG1-β3, and the NRG1 gene is codon-optimized. The nucleic acid molecule according to claim 1, characterized in that The nucleotide sequence encoding the nucleic acid molecule is as shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the nucleotide sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule encodes a nucleotide sequence as shown in any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:

5. The nucleic acid molecule according to claim 1, characterized in that The nucleic acid molecule further comprises a mutation to the cleavage site of ADAM10, BACE1 and / or ADAM17 on the NRG1 gene, provided that the mutation does not affect the original function of the protein expressed by the NRG1 gene. The nucleic acid molecule according to claim 4, characterized in that The nucleotide sequence encoding the nucleic acid molecule is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 7, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO:

7. The nucleic acid molecule according to claim 5, characterized in that The nucleic acid molecule encodes a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO:

7. The nucleic acid molecule according to claim 1, characterized in that The nucleic acid molecule further comprises a myocardial cell-specific promoter sequence. The nucleic acid molecule according to claim 7, characterized in that The myocardial cell-specific promoter comprises hTNT, hBNP, a-myosin heavy chain promoter, myocardial cell early-specific promoter hNkx2.5, ventricular myosin heavy chain gene promoter and / or Snhg5 gene promoter. The nucleic acid molecule according to claim 8, characterized in that The nucleotide sequence of the myocardial cell-specific promoter is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2. The nucleic acid molecule according to claim 9, characterized in that The nucleotide sequence of the myocardial cell-specific promoter is as shown in SEQ ID NO: 1 or SEQ ID NO:

2. The nucleic acid molecule according to claim 7, characterized in that The myocardial cell-specific promoter sequence is sequentially connected to the nucleotide sequence encoding the NRG1 gene. The nucleic acid molecule according to claim 11, characterized in that The nucleic acid molecule further comprises a WPRE sequence for maintaining the stability of the overall sequence. The nucleic acid molecule according to claim 12, characterized in that The nucleotide sequence encoding the NRG1 gene is sequentially connected to the WPRE sequence. The nucleic acid molecule according to claim 1, characterized in that The nucleotide sequence of the nucleic acid molecule encoding the Type I NRG1-β1, Type I NRG1-β2, or Type II NRG1-β3 is set forth in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:

5. An NRG1 protein variant, characterized in that, The NRG1 protein variant comprises an amino acid sequence encoded by the nucleic acid molecule of any one of claims 1-14. The protein variant according to claim 15, characterized in that The amino acid sequence of the NRG1 protein variant is set forth in SEQ ID NO: 6 or SEQ ID NO: 7, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO:

7. The protein variant according to claim 16, characterized in that The amino acid sequence of the NRG1 protein variant is set forth in SEQ ID NO: 6 or SEQ ID NO:

7. A vector, characterized in that, The vector comprises the nucleic acid molecule of any one of claims 1-14. The carrier according to claim 18, characterized in that The vector comprises a plasmid vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a piggyBac vector, and / or a Sleeping Beauty transposon vector. The carrier according to claim 19, characterized in that The adeno-associated virus vector comprises a ssAAV, a scAAV, and / or a hybrid AAV subtype. The vector according to claim 19, characterized in that The adeno-associated virus vector comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and / or AAV13. A vaccine characterized in that, The vaccine comprises an active ingredient comprising the nucleic acid molecule of any one of claims 1-14, an RNA polynucleotide encoded by the nucleic acid molecule of any one of claims 1-14, and / or a protein encoded by the nucleic acid molecule of any one of claims 1-14. The vaccine according to claim 22, characterized in that The vaccine comprises a viral vector vaccine, an RNA vaccine, a DNA vaccine, or a protein vaccine. The vaccine according to claim 23, characterized in that The viral vector vaccine comprises the nucleic acid molecule of any one of claims 1-14. The vaccine according to claim 24, characterized in that The vectors in the viral vector vaccine self-assemble into virus-like particles. The vaccine according to claim 25, characterized in that The vectors are self-inactivating vectors. The vaccine according to claim 25, characterized in that The virus comprises a lentivirus, an influenza virus, a hepatitis virus, an alphavirus, a filovirus, an adenovirus, an adeno-associated virus, and / or a flavivirus. The vaccine according to claim 27, characterized in that The virus is an adenovirus and / or an adeno-associated virus. The vaccine according to claim 28, characterized in that The virus is an adeno-associated virus. The vaccine according to claim 29, characterized in that The adeno-associated virus comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and / or AAV13. The vaccine according to claim 22, characterized in that The nucleotide sequence of the nucleic acid molecule is codon-optimized or codon-degenerate. The vaccine according to claim 31, characterized in that The nucleotide sequence of the nucleic acid molecule is artificially selected for codon. The vaccine according to claim 22, characterized in that The RNA polynucleotide is codon-optimized. The vaccine according to claim 22, characterized in that The RNA polynucleotide comprises at least one chemical modification. The vaccine according to claim 34, characterized in that The chemical modification comprises a methylation modification, a pseudouridine (Ψ) modification, and / or a hypoxanthine modification. The vaccine according to claim 22, characterized in that The RNA polynucleotide further comprises at least one 3’-UTR and / or at least one 5’-UTR. The vaccine according to claim 36, characterized in that The at least one 3’-UTR and the at least one 5’-UTR are heterologous to each other. The vaccine of claim 36, characterized in that The at least one 3’-UTR is derived from a housekeeping gene, a gene encoding a membrane protein, a gene involved in cellular metabolism, a gene involved in transcription, translation, and replication processes, a gene involved in protein modification, and / or a gene involved in cell division. The vaccine of claim 36, characterized in that The at least one 5’-UTR is derived from a housekeeping gene, a gene encoding a membrane protein, a gene involved in cellular metabolism, a gene involved in transcription, translation, and replication processes, a gene involved in protein modification, and / or a gene involved in cell division. The vaccine according to claim 22, characterized in that The RNA polynucleotide is capped and / or tailed. The vaccine of claim 40, characterized in that The capping comprises cap0 capping, cap1 capping, and / or cap2 capping. The vaccine of claim 40, characterized in that The tailing comprises tailing by cleavage of polyadenylation specific factors, cleavage stimulating factors, cleavage factors I and II, poly(A) polymerase, poly(A) binding protein, and / or a coatomer. The vaccine of claim 40, characterized in that The tailing comprises a poly(A) tail. The vaccine of claim 40, characterized in that The RNA polynucleotide is capped and tailed. The vaccine according to claim 22, characterized in that The RNA polynucleotide is 5’-UTR, RNA polynucleotide encoded by the nucleic acid molecule of any one of claims 1-14, 3’-UTR in that order. The vaccine according to claim 23, characterized in that The RNA vaccine is multivalent. The vaccine of claim 46, characterized in that The RNA vaccine further comprises a carrier. The vaccine of claim 47, characterized in that The carrier comprises a dendritic cell, a cationic nanoemulsion, a cationic polypeptide and polymer, a liposome polymer, a liposome complex, and / or a lipid nanoparticle. The vaccine according to claim 23, characterized in that The protein vaccine comprises a protein encoded by the nucleic acid molecule of any one of claims 1-14. The vaccine of claim 49, characterized in that The protein vaccine is carried by a carrier, which comprises serum albumin, an immunoglobulin molecule, thyroglobulin, ovalbumin, tetanus toxoid, diphtheria toxoid, a genetically modified cross-reacting material of diphtheria toxoid, CRM197, a meningococcal outer membrane protein complex, and a Haemophilus influenza protein D, rEPA, keyhole limpet hemocyanin, and / or flagellin. The vaccine according to claim 22, characterized in that The vaccine further comprises a pharmaceutically acceptable adjuvant. The vaccine of claim 51, characterized in that The pharmaceutically acceptable adjuvant comprises an adjuvant required for intravenous drip, subcutaneous injection, intravenous bolus injection, intravitreal injection, and / or intramuscular injection. A cell, characterized in that, The cell comprises the nucleic acid molecule of any one of claims 1-14, the NRG1 protein variant of any one of claims 15-17, and / or the carrier of any one of claims 18-21. The cell of claim 53, wherein The cell comprises a prokaryotic cell or a eukaryotic cell. The cell of claim 53, wherein The cell comprises an artificial cell line. The cell of claim 55, wherein The artificial cell line comprises a HEK293 cell line. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the nucleic acid molecule of any one of claims 1-14, the NRG1 protein variant of any one of claims 15-17, the carrier of any one of claims 18-21, the vaccine of any one of claims 22-52, and / or the cell of any one of claims 53-56. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the pharmaceutical composition of claim 57. The pharmaceutical preparation according to claim 58, characterized in that The dosage form of the pharmaceutical preparation includes intravenous drip preparation, subcutaneous injection preparation, intravenous bolus preparation, intramuscular injection preparation, virus-like particle preparation, self-inactivating vector preparation, lipid nanoparticle (LNP) encapsulation preparation, cationic nanoemulsion preparation, liposome complex preparation, vector binding dosage form, and / or suspension preparation. A kit characterized in that, The kit comprises the nucleic acid molecule of any one of claims 1-14, the NRG1 protein variant of any one of claims 15-17, the vector of any one of claims 18-21, the vaccine of any one of claims 22-52, the cell of any one of claims 53-56, the pharmaceutical composition of claim 57, and / or the pharmaceutical preparation of claim 58 or 59. A protein composition characterized in that, The protein composition comprises the protein encoded by the nucleic acid molecule of any one of claims 1-14 and a detectable marker, the protein and the detectable marker being coupled directly or indirectly to form a complex, the protein and the detectable marker not affecting the original function of the protein after coupling. The protein composition of claim 61, wherein The detectable marker includes fluorescent dye, chemiluminescent compound, radioisotope, electron-dense reagent, enzyme, colored particle, and / or biotin. Any of the following methods characterized in that, The method comprises: 1) A method for producing a protein, characterized in that the method comprises: transforming the nucleic acid molecule of any one of claims 1-14 or the vector of any one of claims 18-21 into a cell, or directly using the cell of any one of claims 53-56, culturing the cell, and isolating and purifying the protein encoded by the nucleic acid molecule of any one of claims 1-14 from the cell culture solution and / or the cell; 2) A method for producing a primary RNA polynucleotide, characterized in that the method comprises: transforming the nucleic acid molecule of any one of claims 1-14 or the vector of any one of claims 18-21 into a cell, or directly using the cell of any one of claims 53-56, culturing the cell, and isolating and purifying the primary RNA polynucleotide encoded by the nucleic acid molecule of any one of claims 1-14 from the cell culture solution and / or the cell; 3) A method for preventing, alleviating, or treating cardiovascular disease, characterized in that the method comprises: administering to a subject in need thereof a prophylactically, alleviatingly, or therapeutically effective amount of the nucleic acid molecule of any one of claims 1-14, the NRG1 protein variant of any one of claims 15-17, the vector of any one of claims 18-21, the vaccine of any one of claims 22-52, the cell of any one of claims 53-56, the pharmaceutical composition of claim 57, and / or the pharmaceutical preparation of claim 58 or 59. 4) A method of preventing, alleviating or treating a neurological disease, the method comprising administering to a subject in need thereof a prophylactically, alleviating or therapeutically effective amount of the nucleic acid molecule of any one of claims 1-14, the NRG1 protein variant of any one of claims 15-17, the vector of any one of claims 18-21, the vaccine of any one of claims 22-52, the cell of any one of claims 53-56, the pharmaceutical composition of claim 57 and / or the pharmaceutical preparation of claim 58 or 59; 5) A method of improving impaired function of a cardiac cell or tissue, the method comprising administering to a cardiac cell or tissue the nucleic acid molecule of any one of claims 1-14, the NRG1 protein variant of any one of claims 15-17, the vector of any one of claims 18-21, the vaccine of any one of claims 22-52, the cell of any one of claims 53-56, the pharmaceutical composition of claim 57 and / or the pharmaceutical preparation of claim 58 or 59. The method of claim 63, wherein The cardiovascular disease comprises atherosclerosis, ischemic heart disease, coronary heart disease, hypertension, heart insufficiency, arrhythmia, cardiomyopathy, endocarditis, peripheral arterial disease, coronary artery bypass graft, arteritis, myocarditis, cardiovascular inflammation, unstable angina, unstable refractory angina, stable angina, chronic stable angina, acute coronary syndrome and / or myocardial infarction. The method of claim 63, wherein The neurological disease comprises Parkinson's disease, Huntington's disease, Alzheimer's disease, mild cognitive impairment, senile dementia and amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph disease, Lewy body dementia, dystonia, progressive supranuclear palsy and / or frontotemporal dementia. Any of the following applications characterized in that, The use comprises: 1) Use of the NRG1 gene, the nucleic acid molecule of any one of claims 1-14, the NRG1 protein variant of any one of claims 15-17, the vector of any one of claims 18-21 and / or the cell of any one of claims 53-56 for the manufacture of a product for preventing, alleviating or treating a cardiovascular disease; 2) Use of the NRG1 gene, the nucleic acid molecule of any one of claims 1-14, the NRG1 protein variant of any one of claims 15-17, the vector of any one of claims 18-21 and / or the cell of any one of claims 53-56 for the manufacture of a product for maintaining normal function of a cardiac cell or tissue; 3) Use of the NRG1 gene, the nucleic acid molecule of any one of claims 1-14, the NRG1 protein variant of any one of claims 15-17, the vector of any one of claims 18-21 and / or the cell of any one of claims 53-56 for the manufacture of a vaccine; 4) Use of the NRG1 gene, the nucleic acid molecule of any one of claims 1-14, the NRG1 protein variant of any one of claims 15-17, the vector of any one of claims 18-21, and / or the cell of any one of claims 53-56 in the manufacture of a product for preventing, alleviating or treating a neurological disease. The use according to claim 66, characterized in that The cardiovascular disease comprises atherosclerosis, ischemic heart disease, coronary heart disease, hypertension, heart insufficiency, arrhythmia, cardiomyopathy, endocarditis, peripheral arterial disease, coronary artery bypass graft, arteritis, myocarditis, cardiovascular inflammation, unstable angina, unstable refractory angina, stable angina, chronic stable angina, acute coronary syndrome and / or myocardial infarction. The use according to claim 66, characterized in that The neurological disease comprises Parkinson's disease, Huntington's disease, Alzheimer's disease, mild cognitive impairment, senile dementia and amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph disease, Lewy body dementia, dystonia, progressive supranuclear palsy and / or frontotemporal dementia.

Images