Application of a new artificially designed NRG-1 in disease treatment

Abstract

The present invention discloses an application of an artificially designed novel NRG-1 in disease treatment. The present invention provides a nucleic acid molecule, an NRG1 protein variant, a vector, a vaccine, a cell, a pharmaceutical composition and a protein composition. The present invention also provides the following methods: a method for producing a protein, a method for producing a primary RNA polynucleotide. The present invention also provides the following applications: application in preparing a product for preventing, alleviating or treating cardiovascular diseases, application in preparing a product for preventing, alleviating or treating neurological diseases, application in preparing a product for maintaining the normal function of cardiac cells or tissues, and application in preparing a vaccine.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of an artificially designed novel NRG-1 in disease treatment. Background Art

[0002] Ischemic heart disease is one of the three leading causes of cardiovascular death in China, and myocardial infarction (MI) caused by coronary artery obstruction is the most common and severe form of ischemic heart disease. Due to the limited regenerative capacity of adult cardiomyocytes, a large number of cardiomyocytes undergo necrosis after MI, resulting in insufficient cardiomyocyte numbers, inflammation, and massive fibroblast proliferation, leading to myocardial fibrosis. This leads to adverse ventricular remodeling, decreased myocardial contractility, and ultimately heart failure. Therefore, safe cardiomyocyte protection and supplementation are important means to effectively prevent myocardial remodeling and heart failure after MI.

[0003] Neuregulin-1 (NRG-1) is a growth factor that is expressed in capillary endothelial cells in the heart and acts on cardiomyocytes. NRG-1 has a very superior protective effect on the myocardium under pathological conditions, but due to the short half-life of circulating NRG-1, its intravenous administration interval is very limited in existing clinical trials. In addition, high-dose intravenous infusion of NRG-1β3 can cause elevated liver transaminases and serum bilirubin in subjects. The potential liver toxicity makes the clinical therapeutic application of NRG-1 a huge challenge. Therefore, how to efficiently express NRG-1 in the myocardium and avoid liver toxicity is an urgent problem to be solved in the clinical application of NRG-1.

[0004] NRG-1, a member of the epidermal growth factor (EGF) family, is involved in regulating cell growth, division, differentiation, and survival. The NRG-1 gene has 33 exons and produces multiple isoforms through alternative splicing. These isoforms are classified as types I through VI based on alternative splicing at their N-terminus. NRG-1 is further divided into α and β isoforms based on alternative splicing at the C-terminus of its active EGF domain. NRG-1β is 10-100 times more bioactive than NRG-1α and is crucial for cardiac growth, development, and maintenance of cardiac function. The NRG-1β3 isoform has a stop codon before the transmembrane domain, resulting in its secreted form. Furthermore, all types I through VI of NRG-1β are membrane-anchored molecules. In particular, type II NRG-1 possesses a Kringle sequence at its N-terminus, which mediates protein-protein or protein-matrix interactions and is crucial 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 solutions:

[0006] The present invention provides a nucleic acid molecule encoding an NRG1 gene. The NRG1 gene is divided into type I NRG1-β1, type I NRG1-β2, and type II NRG1-β3. The NRG1 gene is codon-optimized.

[0007] Furthermore, the nucleotide sequence encoded by the nucleic acid molecule has at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 97%, preferably at least 98%, and preferably at least 99% identity with the nucleotide sequence shown in any one of SEQ ID NOs: 3-5.

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

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

[0010] Furthermore, the nucleotide sequence encoded by the nucleic acid molecule has at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 97%, preferably at least 98%, and preferably at least 99% identity with the nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NO: 6 and SEQ ID NO: 7.

[0011] Furthermore, the nucleic acid molecule encodes a nucleotide sequence of an amino acid sequence as shown in any one of SEQ ID NO: 6 and 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, cardiomyocyte early-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) promoter is used as a promoter to specifically promote the expression of NRG-1 in cardiomyocytes or at special disease stages, and the efficiency of the promoter is increased by an enhancer.

[0015] Furthermore, the cardiomyocyte-specific promoter has at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 97%, preferably at least 98%, and preferably at least 99% identity with the nucleotide sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:2.

[0016] Furthermore, the cardiomyocyte-specific promoter is shown in any one of SEQ ID NO: 1 and SEQ ID NO: 2.

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

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

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

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

[0021] In the present 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 the codon frequencies in the target and host organisms to ensure proper folding; bias the GC content to increase mRNA stability or reduce secondary structure; minimize tandemly repeated codons or base runs that can impair gene structure or expression; customize transcription and translation control regions; insert or remove protein trafficking sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; add, remove or replace protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust the translation rate so that the various domains of the protein can fold properly; or reduce or eliminate problematic secondary structures within the polynucleotide.

[0022] The terms "nucleic acid molecule", "coding sequence", "polynucleotide", "nucleotide sequence", "nucleic acid sequence" and "nucleic acid" used in the present invention are used interchangeably and include DNA, RNA or hybrids thereof, which can be double-stranded or single-stranded.

[0023] The present invention provides an NRG1 protein variant, which includes the amino acid sequence encoded by the above-mentioned nucleic acid molecule.

[0024] Furthermore, the amino acid sequence of the NRG1 protein variant is at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 97%, preferably at least 98%, and preferably at least 99% identical to the amino acid sequence shown in any one of SEQ ID NO:6 and 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] The term "protein variant" used in the present invention refers to a polypeptide sequence that is different from the original or native sequence, in which one or more amino acid residues are deleted, substituted or added. The variant can be a naturally occurring allele protein variant or a non-naturally occurring protein variant. The variant may be from the same species or from other species and may include homologues, paralogues and orthologues. In certain embodiments, the protein variants of the polypeptides useful in the present invention have biological activities that are the same or similar to those of the parent polypeptide, including hormone functions or activation inhibitory functions. The term "protein variant" for (poly)peptides includes all forms of polypeptides defined herein. The term "protein variant" includes naturally occurring polypeptides, as well as polypeptides produced by recombinant and synthetic methods.

[0027] The present invention provides a vector comprising the aforementioned nucleic acid molecule.

[0028] Furthermore, the vector includes a plasmid vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a piggyBac vector or a Sleeping Beauty transposase vector.

[0029] Furthermore, the adeno-associated viral 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 AAV13.

[0031] In some embodiments, the function of the vector used in the present invention to carry nucleic acid molecules is well known to the public, and the technologies, methods and materials required for transporting nucleic acid molecules are publicly available in technical documents in the corresponding fields. The technologies include but are not limited to vector construction, connection between the vector and the target gene, transport of the vector, and cell culture after transport.

[0032] In some embodiments, the vector may also contain other appropriate "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 that stabilize cytoplasmic mRNA, such as the woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product. In certain embodiments, examples of polyA include SV40, bovine growth hormone (bGH), and TK polyA. In certain embodiments, examples of enhancers include the α-fetoprotein enhancer, the TTR minimal promoter / enhancer, LSP (TH-binding globulin promoter / α1-microglobulin / bikunin enhancer), and other enhancers.

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

[0034] As used herein, the term "vector" refers to a carrier nucleic acid molecule into 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 that it is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the cell. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).

[0035] The present invention provides a vaccine, which includes a viral vector vaccine, an RNA 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 molecule described above.

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

[0039] Furthermore, the vector includes a self-inactivating vector.

[0040] Furthermore, the virus includes lentivirus, influenza virus, hepatitis virus, alphavirus, filovirus, adenovirus, adeno-associated virus and / or flavivirus.

[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 the family consisting of herpesviruses, poxviruses, hepadnaviruses, flaviviruses, togaviruses, coronaviruses, hepatitis D, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, filoviruses, and retroviruses. In some embodiments, examples of non-enveloped viruses are selected from the family consisting of adenoviruses, reoviruses, papillomaviruses, picornaviruses, and caliciviruses.

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

[0043] Furthermore, the virus 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 AAV13.

[0045] In some embodiments, the AAV virus includes an AAV virus core plasmid or a plasmid modified from an AAV virus 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 codon usage of the nucleotide sequence is altered from the natural type.

[0048] In some embodiments, any suitable virus is used to construct a viral vector vaccine, including but not limited to

[0049] Furthermore, the RNA vaccine includes the RNA polynucleotide encoded by the nucleic acid molecule described above.

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

[0051] Furthermore, the open reading frame of the RNA polynucleotide is codon-optimized.

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

[0053] Furthermore, the RNA polynucleotide comprises 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 chemically modified refers to the modification of at least one of the position, pattern, percentage or population of adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytosine (C) ribonucleosides or deoxyribonucleosides. In some embodiments, polynucleotides may include any applicable modification, for example, to the sugar, nucleobase or internucleoside linkage (e.g., to the attached phosphate, to the phosphodiester bond or to the phosphodiester backbone). In some embodiments, modified nucleotide base pairing encompasses not only standard adenosine-thymine, adenosine-uracil 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 the non-standard base and the standard base or between two complementary non-standard base structures.

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

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

[0058] Further, each of the at least one 3'-UTR and the at least one 5'-UTR is heterologous to each other.

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

[0060] Furthermore, the at least one 5'-UTR is derived from a gene selected from the group consisting of housekeeping genes, genes encoding membrane proteins, genes involved in cell metabolism, genes involved in transcription, translation and replication processes, 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 tailed.

[0064] Furthermore, the tailing includes tailing by cleavage and polyadenylation 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 tailing includes a poly(A) tail.

[0066] Furthermore, the RNA polynucleotide is capped and tailed.

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

[0068] Furthermore, the RNA vaccine is multivalent.

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

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

[0071] In some embodiments, the cationic nanoemulsion is referred to as a cationic nanoemulsion, CNE. In some embodiments, the liposome polymer is referred to as a lipopolyplex, LPR. In some embodiments, the lipid nanoparticle is referred to as a lipid nanoparticle, LNP. In certain specific embodiments, the carrier significantly enhances the effectiveness of mRNA vaccines, including chemically modified and unmodified mRNA vaccines.

[0072] Furthermore, the protein vaccine includes the protein encoded by the aforementioned nucleic acid molecule.

[0073] Furthermore, the protein vaccine is carried by a carrier, which includes 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 limpet hemocyanin, and / or flagellin.

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

[0075] Furthermore, the pharmaceutically acceptable adjuvants include adjuvants required for intravenous drip, subcutaneous injection, intravenous push injection, intravitreal injection, and intramuscular injection.

[0076] In some embodiments, the formulations are prepared by uniformly and intimately bringing into association the viral vector, RNA polynucleotide, protein with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0077] As used herein, the term "pharmaceutically acceptable" designates a carrier that is generally chemically and / or physically compatible with the other ingredients making up the formulation and physiologically compatible with the recipient thereof. Pharmaceutically acceptable adjuvants used in the vaccine compositions of the present invention may include, but are not limited to, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles (e.g., sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection or Ringer's dextrose and lactate injection), non-aqueous vehicles (e.g., fixed oils of plant origin, 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 buffer), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose 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 auxiliary substances, 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 HEK293 cell line.

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

[0083] 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 cell.

[0084] The present invention provides a protein composition, which includes the protein encoded by the nucleic acid molecule described above and a detectable marker, wherein the protein and the detectable marker are directly or indirectly coupled to form a complex, and the protein and the detectable marker do not affect the original function of the protein after coupling.

[0085] Furthermore, the detectable label includes fluorescent dyes, chemiluminescent compounds, radioactive isotopes, electron-dense reagents, enzymes, colored particles, or biotin.

[0086] As used herein, the term "detectable label" refers to an agent detectable by spectroscopy, photochemistry, biochemistry, immunochemistry, or chemical means. Useful detectable labels include, but are not limited to, fluorescent dyes, chemiluminescent compounds, radioisotopes, electron-dense reagents, enzymes, colored particles, and biotin. Detectable labels often produce a measurable signal, such as radioactivity, fluorescence, color, or enzymatic activity. Antibodies coupled to detectable agents can be used for diagnostic or therapeutic purposes. Examples of detectable agents 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 or coupled to antibodies, or indirectly through intermediates such as linkers known in the art, using techniques known in the art.

[0087] The present invention provides any of the following methods, comprising:

[0088] 1) A method for producing a protein, 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 protein encoded by the aforementioned nucleic acid molecule from the cell culture fluid and / or cells.

[0089] 2) A method for producing a primary RNA polynucleotide, 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 fluid and / or cells.

[0090] 3) A method for preventing, alleviating or treating cardiovascular disease, comprising administering to a patient the aforementioned nucleic acid molecule, the aforementioned NRG1 protein variant, the aforementioned vector, the aforementioned vaccine, the aforementioned cell, and / or the aforementioned pharmaceutical composition.

[0091] Furthermore, the cardiovascular diseases include atherosclerosis, ischemic heart disease, coronary heart disease, hypertension, heart failure, arrhythmia, cardiomyopathy, endocarditis, peripheral artery disease, coronary artery bypass grafting, arteritis, myocarditis, angiocarditis, unstable angina, unstable refractory angina, stable angina, chronic stable angina, acute coronary syndrome, and myocardial infarction.

[0092] 4) A method for preventing, alleviating or treating neurological diseases, comprising administering to a patient the aforementioned nucleic acid molecule, the aforementioned NRG1 protein variant, the aforementioned vector, the aforementioned vaccine, the aforementioned cell, and / or the aforementioned pharmaceutical composition.

[0093] 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, Friedreich's ataxia, Machado-Joseph disease, dementia with Lewy bodies, dystonia, progressive supranuclear palsy, and / or frontotemporal dementia.

[0094] 5) A method for improving damaged function of cardiac cells or tissues, the method comprising administering the aforementioned nucleic acid molecule, the aforementioned NRG1 protein variant, the aforementioned vector, the aforementioned vaccine, the aforementioned cell, and / or the aforementioned pharmaceutical composition to the cardiac cells or tissues.

[0095] Furthermore, the cardiac cells or tissues include in vivo or in vitro.

[0096] The present invention provides any of the following applications, comprising:

[0097] 1) Use of the aforementioned nucleic acid molecule, the aforementioned NRG1 protein variant, the aforementioned vector, and / or the aforementioned cell in the preparation of a product for preventing, alleviating or treating cardiovascular disease.

[0098] Furthermore, the cardiovascular diseases include atherosclerosis, ischemic heart disease, coronary heart disease, hypertension, heart failure, arrhythmia, cardiomyopathy, endocarditis, peripheral artery disease, coronary artery bypass grafting, arteritis, myocarditis, angiocarditis, unstable angina, unstable refractory angina, stable angina, chronic stable angina, acute coronary syndrome, and myocardial infarction.

[0099] 2) Use of the aforementioned nucleic acid molecule, the aforementioned NRG1 protein variant, the aforementioned vector, and / or the aforementioned cell in the preparation of a product for maintaining the normal function of cardiac cells or tissues.

[0100] 3) Use of the aforementioned nucleic acid molecule, the aforementioned NRG1 protein variant, the aforementioned vector, and / or the aforementioned cell in the preparation of a vaccine.

[0101] 4) Use of the aforementioned nucleic acid molecules, the aforementioned NRG1 protein variants, the aforementioned vectors, and / or the aforementioned cells in the preparation of products for preventing, alleviating or treating neurological diseases;

[0102] 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, Friedreich's ataxia, Machado-Joseph disease, dementia with Lewy bodies, dystonia, progressive supranuclear palsy, and / or frontotemporal dementia.

[0103] As used herein, the term "NRG1" is intended to encompass fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human NRG1 cDNA and human NRG1 protein sequences are well known in the art and are publicly available on the NCBI website. For example, at least one human NRG1 isoform is known: human NRG1, Gene ID: 3084. Nucleic acid and polypeptide sequences of NRG1 orthologs in organisms other than humans are well known.

[0104] The term "ADAM10" as used in the present invention is intended to encompass fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human ADAM10 cDNA and human ADAM10 protein sequences are well known in the art and are publicly available on the NCBI website. For example, at least one human ADAM10 isoform is known: human ADAM10, Gene ID: 102. The nucleic acid and polypeptide sequences of ADAM10 orthologs in organisms other than humans are well known.

[0105] The term "ADAM17" as used in the present invention is intended to include fragments, variants (e.g., allelic 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 isoform is known: human ADAM17, Gene ID: 6868. The nucleic acid and polypeptide sequences of ADAM17 orthologs in organisms other than humans are well known.

[0106] As used herein, the term "BACE1" is intended to encompass fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human BACE1 cDNA and human BACE1 protein sequences are well known in the art and are publicly available on the NCBI website. For example, at least one human BACE1 isoform is known: human BACE1, Gene ID: 23621. The nucleic acid and polypeptide sequences of BACE1 orthologs in organisms other than humans are well known.

[0107] Advantages and beneficial effects of the present invention:

[0108] This study creatively used adeno-associated virus (AAV) as a gene therapy vector to "reversely" overexpress the cardiac endothelial cell protein NRG-1 in cardiomyocytes, thereby increasing the local NRG-1 concentration in the myocardium and exerting its myocardial protective effect, thereby effectively treating heart failure caused by myocardial infarction and avoiding liver toxicity, providing a new strategy and gene therapy drug for the clinical application of NRG-1 to treat myocardial infarction.

[0109] The main technical problems solved include:

[0110] 1. Using adeno-associated virus (AAV) as a gene therapy vector, NRG-1 can be expressed in cardiomyocytes for a long time, solving the problem of the short half-life of circulating NRG-1 protein.

[0111] 2. Using AAV as a gene therapy vector, NRG-1 is specifically expressed in cardiomyocytes "inversely", solving the problem of insufficient myocardial NRG-1 penetration or low content in NRG-1 therapy.

[0112] 3. Using AAV as a gene therapy vector, enhance the cardiomyocyte-specific promoter and heart disease-responsive promoter, and optimize the NRG-1β1, NRG-1β2, and NRG-1β3 codons to further enhance the expression of NRG-1 in cardiomyocytes and solve the problem of insufficient myocardial NRG-1 penetration or low content in NRG-1 therapy.

[0113] 4. Using AAV as a gene therapy vector, NRG-1 is specifically expressed in cardiomyocytes, increasing the local NRG-1 content in the myocardium and solving the liver toxicity risk of high-dose intravenous infusion of NRG-1.

[0114] 5. Using AAV as a gene therapy vector, NRG-1 is specifically expressed in cardiomyocytes, promoting NRG-1-mediated cell-to-cell interactions between cardiomyocytes and endothelial cells, and between cardiomyocytes, thereby enhancing its myocardial protective effect.

[0115] 6. Provide NRG-1β1 / NRG-1β2 with mutations in the EGF cleavage site to reduce its secretion into the blood, reduce the risk of liver toxicity, increase the local NRG-1 concentration in the myocardium, and enhance its myocardial protective effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1 This is a statistical graph of body weight and EF in mice with myocardial infarction after injection of AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3;

[0117] Figure 2This is a statistical graph showing the measurement of diastolic left ventricular internal diameter (LVIDD), interventricular septum (IVSD), and left ventricular posterior wall (LVPWD) in mice with myocardial infarction after injection of AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3;

[0118] Figure 3 This is a statistical graph of body weight and cardiac index (heart weight / body weight, HW / BW) measurements after myocardial infarction mice were injected with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3;

[0119] Figure 4 This is the Masson's staining result after myocardial infarction mice were injected with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3;

[0120] Figure 5 Figures are HE staining, Masson's staining, and transaminase results of mice before / after injection of AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3;

[0121] Figure 6 The figure shows the results of TUNEL staining after injection of AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 into mice with myocardial infarction. DETAILED DESCRIPTION

[0122] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. 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 or combined with features from any of the other several aspects without departing from the scope or spirit of this disclosure.

[0123] It should also be understood that the terminology used herein is for the purpose of describing particular aspects 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 belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the specification and the relevant art, and should not be interpreted as idealized or overly formal unless expressly defined herein.

[0124] The specific embodiments of the present invention are further described in detail below with reference to the examples.

[0125] Example

[0126] 1. Experimental Materials

[0127] 1.1 Instrument Information

[0128] The specific instrument information is shown in Table 1.

[0129] Table 1

[0130] Instruments and Equipment Country manufacturers model anesthesia machine Shanghai Ren Yi MSS-3 Stereoscope AMSCOPE SM-4TZ-144-10M Ventilator Harvard University MiniVentTYPE845 Ultra-high-resolution small animal ultrasound VisualSonics Canada Vevo2100 ultracentrifuge American Beckman Optimal-100XP Biological safety cabinets Suzhou Thermal Power 1389 Clean bench Shanghai Zhicheng ZHJH-C1112B CO2 incubator American Thermoelectric 150i Biochemical incubator Beijing Zhongyi Guoke SPX-70 Constant temperature oscillator Shanghai Zhicheng ZWY-240 vacuum pump Jiangsu Haimen Qilin Bell GL-802B Inverted fluorescence microscope Olympus Japan IX73 Nucleic acid protein analyzer American Thermoelectric nanoONE PCR instrument American Bole S1000 pipette Eppendorf, Germany ResearchPlus Electric suction device Eppendorf, Germany Easypet3 electronic pipettes Eppendorf, Germany MultipetteE3 High-speed refrigerated centrifuge American Thermoelectric ST16R metal bath Hangzhou Aosheng K30 Water bath (3 holes) Shanghai Yiheng DK-8D vortexer Jiangsu Haimen Qilin Bell VOTEX5 analytical balance Shanghai Mettler Toledo ME204 / 02 Heating magnetic stirrer Jiangsu Haimen Qilin Bell GL-3250A pH meter Shanghai Mettler Toledo FE28-Standard

[0131] 1.2 Reagent Information

[0132] The specific reagent information is shown in Table 2.

[0133] Table 2

[0134]

[0135]

[0136] 2. Experimental methods

[0137] 2.1 Sequence construction

[0138] The sequences used in this study are listed in Table 3.

[0139] Table 3

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] 2.2 Plasmid construction

[0146] The AAV main plasmid after cleavage site mutation optimization and the auxiliary plasmids pAAV.hTNT.NRG-1.SV40 or pAAV.hTNT.NRG-1.SV40, pAAV2 / 1 or pAAV2 / 9n and pAdDeltaF6 were all synthesized in full length.

[0147] 2.3 Virus packaging and purification

[0148] AAV was prepared according to the experimental method of Grieger et al. (Nat Protoc. 2006; 1(3): 1412-28).

[0149] 2.4 AAV virus gene extraction and titer detection

[0150] Add 100 μl of DNase lysis solution (50 U DNase I + 1 mL DNase lysis solution) to 2 μl of virus solution.

[0151] Mix thoroughly for 2 seconds, being careful not to damage the viral coat. Incubate in a 37°C water bath for 1 hour.

[0152] Add 5 μL of EDTA, mix well, and place in a metal bath at 70°C for 10 min.

[0153] Add 120 μL of protease lysis solution (1.818 U proteinase K + 500 μL proteinase K solution), mix well, centrifuge for 3 seconds, and treat in a 55°C metal bath for 2 hours.

[0154] The metal bath temperature was adjusted to 95°C for 10 min to inactivate the protease and the cells were placed on ice.

[0155] The obtained sample was diluted, and 3 μL was taken and diluted 300 times for titer detection.

[0156] The extracted DNA was quantified by RT-PCR and standard plasmids to determine the AAV virus titer.

[0157] 2.5 Echocardiography

[0158] Myocardial Infarction Model: AAV9.hTNT.NRG-1 was injected into each mouse at a dose of 1E11 vg the day after myocardial infarction surgery. Echocardiography was performed weekly postoperatively to assess left ventricular function.

[0159] 2.6 Mechanism of protective effect of NRG-1 in cardiomyocytes after myocardial infarction

[0160] The virus injected was AAV9.hTNT.NRG-1, with an injection volume of 1E11 vg per mouse. Four weeks after injection, myocardial infarction surgery was performed. 96 hours after surgery, the mice were sacrificed, and cardiac tissue sections were sectioned and TUNEL staining was performed to assess the extent of cardiomyocyte apoptosis.

[0161] 3. Experimental results

[0162] 3.1AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 can treat cardiac dysfunction after myocardial infarction and prevent it from developing into heart failure.

[0163] Eight-week-old C57 male mice underwent sham surgery and MI surgery, and 1×10 11 The mice were injected with 1000 vg of virus per mouse by tail vein and monitored by echocardiography. Our results showed that there was no significant difference in the body weight of mice among the groups ( Figure 1A). Twelve weeks after MI surgery, the ejection fraction of untreated mice was significantly reduced, and they had developed severe heart failure. However, after treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3, the ejection fraction of mice was significantly improved, and the progression to heart failure was prevented ( Figure 1 B). The above results indicate that AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 can effectively treat cardiac dysfunction after myocardial infarction and prevent it from developing into heart failure.

[0164] 3.2AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 can prevent myocardial infarction from developing into heart failure and prevent cardiac chamber expansion.

[0165] The cardiac structure of mice was examined by echocardiography 12 weeks after sham surgery and MI surgery. The results showed that the heart chambers of mice were dilated and the heart walls were thinned after myocardial infarction, and AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 treatment could prevent this adverse decompensation to a certain extent ( Figure 2 AC).

[0166] 3.3AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 treatment can alleviate pathological myocardial hypertrophy caused by myocardial infarction.

[0167] The mice were killed 12 weeks after surgery, and the organs were collected and weighed. Our results showed that there was no significant difference in the body weight of the four groups of mice ( Figure 3 A). Myocardial infarction caused cardiac hypertrophy in mice, while in the treatment group, there was no obvious cardiac hypertrophy ( Figure 3 B).

[0168] 3.4AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 treatment can effectively reduce the area of ​​myocardial infarction.

[0169] The hearts of mice sacrificed 12 weeks after surgery were fixed, paraffin-embedded, and sectioned. The area of ​​myocardial infarction was determined by Masson's staining. Our results showed that the area of ​​myocardial infarction was effectively reduced after treatment with AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 ( Figure 4 ).

[0170] 3.5AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 treatment does not cause liver damage.

[0171] The effects of AAV injection on liver tissue in 8-week-old C57 male mice were examined 4 weeks later. HE staining and Masson's staining showed no cell death or tissue fibrosis in the liver tissue. Figure 5 A). We further isolated mouse serum for transaminase assay, and the results showed that AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 injection did not cause an increase in serum transaminase ( Figure 5 B) No increase in transaminase was found in the serum of mice in the MI surgery group and the treatment group ( Figure 5 C). The above results indicate that AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 are non-hepatotoxic and do not cause liver damage in mice.

[0172] 3.6AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 treatment can significantly inhibit myocardial cell apoptosis after myocardial infarction.

[0173] First, mice were injected with AAV, and 4 weeks later, myocardial infarction surgery was performed. Two days after surgery, the mice were sacrificed and TUNEL staining was performed. Our results showed that AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 treatment can effectively inhibit cardiomyocyte apoptosis ( Figure 6 The above results indicate that AAV9.hTnT-NRG-1β1 and AAV9.hTnT-NRG-1β3 treatment can significantly inhibit cardiomyocyte apoptosis after myocardial infarction.

[0174] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the amino acid sequence shown in SEQ ID NO:

6.

2. The nucleic acid molecule according to claim 1, characterized in that The nucleic acid molecule further includes a cardiomyocyte-specific promoter sequence.

3. The nucleic acid molecule according to claim 2, characterized in that The myocardial cell specific promoter includes hTNT, hBNP, α-myosin heavy chain promoter, myocardial cell early specific promoter hNkx2.5, ventricular myosin heavy chain gene promoter or Snhg5 gene promoter.

4. The nucleic acid molecule according to claim 3, characterized in that The cardiomyocyte-specific promoter is shown in either SEQ ID NO: 1 or SEQ ID NO:

2.

5. The nucleic acid molecule according to claim 2, characterized in that The cardiomyocyte-specific promoter sequence is sequentially linked to the nucleotide sequence of the nucleic acid molecule.

6. The nucleic acid molecule according to claim 5, characterized in that The nucleic acid molecule further comprises a WPRE sequence for maintaining the stability of the overall sequence.

7. The nucleic acid molecule according to claim 6, characterized in that The nucleotide sequence of the nucleic acid molecule is sequentially linked to the WPRE sequence.

8. A NRG1 protein variant, characterized in that The amino acid sequence of the NRG1 protein variant is shown in SEQ ID NO:

6.

9. A carrier, characterized in that The vector comprises the nucleic acid molecule according to any one of claims 1 to 7.

10. The carrier according to claim 9, characterized in that The vector includes a plasmid vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a piggyBac vector or a Sleeping Beauty transposable vector.

11. The carrier according to claim 10, characterized in that The adeno-associated viral vectors include ssAAV, scAAV and / or hybrid AAV subtypes.

12. The carrier according to claim 11, characterized in that The adeno-associated virus vector includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13.

13. A cell, characterized in that The cell comprises the nucleic acid molecule of any one of claims 1 to 7, the NRG1 protein variant of claim 8, and / or the vector of any one of claims 9 to 12.

14. The cell according to claim 13, characterized in that The cells include prokaryotic cells or eukaryotic cells.

15. The cell according to claim 13, characterized in that The cells include artificial cell lines.

16. The cell according to claim 15, characterized in that The artificial cell line includes the HEK293 cell line.

17. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nucleic acid molecule according to any one of claims 1 to 7, the NRG1 protein variant according to claim 8, the vector according to any one of claims 9 to 12, and / or the cell according to any one of claims 13 to 16.

18. A protein composition, characterized in that The protein composition comprises a protein encoded by the nucleic acid molecule according to any one of claims 1 to 7 and a detectable marker, wherein the protein and the detectable marker are directly or indirectly coupled to form a complex, and the protein and the detectable marker do not affect the original function of the protein after coupling.

19. The protein composition according to claim 18, characterized in that The detectable labels include fluorescent dyes, chemiluminescent compounds, radioisotopes, electron-dense reagents, enzymes, colored particles or biotin.

20. A method for producing a protein, characterized in that: The method comprises: transforming the nucleic acid molecule of any one of claims 1 to 7 or the vector of any one of claims 9 to 12 into cells, or directly using the cells of any one of claims 13 to 16, culturing the cells, and isolating and purifying the protein encoded by the nucleic acid molecule of any one of claims 1 to 7 from the cell culture fluid and / or cells.

21. A method for producing a primary RNA polynucleotide, characterized in that The method comprises: transforming the nucleic acid molecule according to any one of claims 1 to 7 or the vector according to any one of claims 9 to 12 into cells, or directly using the cells according to any one of claims 13 to 16, culturing the cells, and isolating and purifying the primary RNA polynucleotide encoded by the nucleic acid molecule according to any one of claims 1 to 7 from the cell culture medium and / or cells.

22. Use of the nucleic acid molecule according to any one of claims 1 to 7, the NRG1 protein variant according to claim 8, the vector according to any one of claims 9 to 12, and / or the cell according to any one of claims 13 to 16 in the preparation of a product for preventing, alleviating or treating cardiovascular disease; The cardiovascular disease is myocardial infarction.

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