Treatment method for promoting myocardial injury repair

Abstract

The invention provides a treatment method for promoting myocardial injury repair, and particularly provides an application of a Hand1 gene, or an encoding protein or an expression vector thereof in preparation of a composition or a preparation for promoting cardiac regeneration and repair after myocardial infarction. It is found for the first time that the Hand1 overexpression AAV9 vector driven by the NcTnT promoter can effectively promote repair after myocardial injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to a treatment method for promoting myocardial injury repair. Background Art

[0002] Myocardial cell loss caused by various injury or disease factors ultimately leads to heart failure, which is currently the leading cause of death. The current clinical treatment regimens cannot change the fundamental problem of heart failure caused by the reduction of myocardial cells after injury. The renewal rate of adult mammalian myocardial cells is extremely low and is not sufficient to repair myocardial injuries such as large-area myocardial infarction. It can only fill the injured area through fibrosis for scar repair and tissue remodeling. Scar tissue lacks contractile function, thus causing cardiac dysfunction and even heart failure.

[0003] Although the mammalian heart is not a post-mitotic organ, neonatal mammals have the ability to completely regenerate the myocardium through myocardial cell proliferation, although this ability significantly weakens in the first week after birth. And it has been reported that proliferating myocardial cells in the border zone of adult myocardial infarction are considered the source of heart regeneration. These myocardial cells have a very unique transcriptome, namely reduced mitochondrial gene expression, reduced mitochondrial activity expression, reduced mitochondrial activity, increased glycolytic gene expression, and increased glucose uptake, which is similar to the transcriptome characteristics of proliferating embryonic myocardial cells. The signaling network that drives embryonic heart development may control all aspects of postnatal heart regeneration. Therefore, a promising approach to improving the prognosis of heart failure is to extend this endogenous ability of embryonic and neonatal mammalian heart regeneration to the repair of myocardial injuries in adulthood.

[0004] The main mechanisms of current drug treatments for heart failure include reducing pressure afterload, appropriate diuresis, improving cardiac remodeling, and alleviating the hemodynamic pressure of neurohormones. Although treatments such as thrombolysis, percutaneous coronary intervention (PCI), and coronary artery bypass grafting (CAGB) have significantly reduced the mortality of myocardial infarction patients, the occurrence of complications is unpredictable, including bleeding, ischemia-reperfusion injury, and coronary restenosis, etc. Therefore, despite the rapidly developing treatment strategies, the prognosis of heart failure is still very poor, and the continuous development of heart failure increases the death risk of patients.

[0005] Currently, there are also no drugs or treatment means related to myocardial cell proliferation and differentiation clinically that can directly improve the progression of cardiac fibrosis after myocardial injury.

[0006] Therefore, there is an urgent need in this field to develop a new treatment method for promoting myocardial injury repair. Summary of the Invention

[0007] The object of the present invention is to provide a new treatment method for promoting myocardial injury repair.

[0008] In a first aspect of the present invention, there is provided a use of a Hand1 gene, or its encoded protein or its expression vector, for preparing a composition or preparation for promoting cardiac regeneration and repair after myocardial infarction.

[0009] In another preferred embodiment, the composition or preparation is used for promoting myocardial regeneration and repair in a population with myocardial infarction.

[0010] In another preferred embodiment, the composition or preparation is used for promoting regeneration and repair after cardiac trauma.

[0011] In another preferred embodiment, the composition or preparation is used for promoting myocardial cell proliferation.

[0012] In another preferred embodiment, the composition or preparation is further used for one or more uses selected from the following group:

[0013] (a) Improving cardiac dysfunction caused by myocardial infarction;

[0014] (b) Reducing the area of myocardial infarction and improving myocardial fibrosis;

[0015] (c) Promoting the proliferation of myocardial cells in the area surrounding the injury;

[0016] (d) Promoting the metabolic transformation of myocardial cells in the area surrounding the injury from fatty acid oxidation to glycolytic state, thereby improving myocardial injury after myocardial infarction.

[0017] In another preferred embodiment, the Hand1 is derived from a mammal; preferably, it is derived from a human, a mouse, a rat, or a rabbit; more preferably, it is derived from a human.

[0018] In another preferred embodiment, the Hand1 gene includes a wild-type Hand1 gene and a mutant Hand1 gene.

[0019] In another preferred embodiment, the mutant form includes a mutant form in which the function of the encoded protein does not change after mutation (i.e., the function is the same as or substantially the same as that of the wild-type encoded protein).

[0020] In another preferred embodiment, the polypeptide encoded by the mutant Hand1 gene is the same as or substantially the same as the polypeptide encoded by the wild Hand1 gene.

[0021] In another preferred embodiment, the mutant Hand1 gene includes a polynucleotide having a homology of ≥80% (preferably ≥90%, more preferably ≥95%, more preferably ≥98% or 99%) compared to the wild Hand1 gene.

[0022] In another preferred embodiment, the mutant Hand1 gene comprises a polynucleotide that is truncated or has 1 - 60 (preferably 1 - 30, more preferably 1 - 10) nucleotides added at the 5' end and / or 3' end of the wild-type Hand1 gene.

[0023] In another preferred embodiment, the Hand1 gene comprises a cDNA sequence, a genomic sequence, or a combination thereof.

[0024] In another preferred embodiment, the Hand1 protein comprises an active fragment of Hand1 or a derivative thereof.

[0025] In another preferred embodiment, the active fragment or its derivative has at least 90%, preferably 95%, more preferably 98% or 99% homology with Hand1.

[0026] In another preferred embodiment, the active fragment or its derivative has at least 80%, 85%, 90%, 95% or 100% of the Ptbp1 activity.

[0027] In another preferred embodiment, the amino acid sequence of the Hand1 protein is selected from the group consisting of:

[0028] (i) a polypeptide having the amino acid sequence shown in SEQ ID NO.: 1;

[0029] (ii) a polypeptide derived from (i) that has the function of the said protein and is formed by substitution, deletion or addition of one or several (such as 1 - 10) amino acid residues in the amino acid sequence shown in SEQ ID NO.: 1; or

[0030] (iii) a polypeptide having an amino acid sequence with ≥90% (preferably ≥95%, more preferably ≥98% or 99%) homology with the amino acid sequence shown in SEQ ID NO.: 1 and having the function of the said protein.

[0031] In another preferred embodiment, the nucleotide sequence of the Hand1 gene is selected from the group consisting of:

[0032] (a) a polynucleotide encoding the polypeptide shown in SEQ ID NO.: 1;

[0033] (b) a polynucleotide having the sequence shown in SEQ ID NO.: 2;

[0034] (c) a polynucleotide having a nucleotide sequence with ≥95% (preferably ≥98%, more preferably ≥99%) homology with the sequence shown in SEQ ID NO.: 2.

[0035] (d) A polynucleotide with 1 - 60 (preferably 1 - 30, more preferably 1 - 10) nucleotides truncated or added at the 5' end and / or 3' end of the polynucleotide shown in SEQ ID NO.:2;

[0036] (e) A polynucleotide complementary to the polynucleotide described in any one of (a) - (d).

[0037] In another preferred example, the Hand1 protein is as shown in SEQ ID NO.:1.

[0038] In another preferred example, the nucleic acid encoding the Hand1 protein is as shown in SEQ ID NO.:2.

[0039] In another preferred example, the concentration of the Hand1 gene, or its encoded protein, or its expression vector is 0.5 - 5×10^12 vg / mL, preferably 0.8 - 5×10^12 vg / mL, more preferably 1 - 5×10^12 vg / mL.

[0040] In another preferred example, the expression vector includes a viral vector.

[0041] In another preferred example, the viral vector is selected from the group consisting of: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, SV40, poxvirus, or a combination thereof.

[0042] In another preferred example, the vector is selected from the group consisting of: lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof, preferably the vector is adeno-associated virus (AAV).

[0043] In another preferred example, the vector includes AAV9.

[0044] In another preferred example, the expression vector further includes a myocardial-specific promoter for driving the expression of the Hand1 protein.

[0045] In another preferred example, the myocardial-specific promoter includes: cTnT promoter, Myh6, ɑ-SA.

[0046] In another preferred example, the composition includes a pharmaceutical composition.

[0047] In another preferred example, the composition contains (a) the Hand1 gene, or its encoded protein or its expression vector; and (b) a pharmaceutically acceptable carrier.

[0048] In another preferred example, the dosage form of the composition is selected from the group consisting of: freeze-dried preparation, liquid preparation, or a combination thereof.

[0049] In another preferred embodiment, the dosage form of the composition is a liquid preparation.

[0050] In another preferred embodiment, the dosage form of the composition is an injection dosage form.

[0051] In another preferred embodiment, in the composition, the component (a) accounts for 1-99 wt%, preferably 10-90 wt%, more preferably 30-70 wt% of the total weight of the pharmaceutical composition.

[0052] In another preferred embodiment, the composition further comprises other drugs for promoting cardiac regeneration and repair.

[0053] In another preferred embodiment, the other drugs for promoting cardiac regeneration and repair are selected from the group consisting of versican, Pkm2, or a combination thereof.

[0054] In another preferred embodiment, in the application of the composition or preparation for promoting myocardial regeneration and repair after myocardial infarction, it can be used alone or in combination.

[0055] In another preferred embodiment, the combined use includes: combined use with other drugs for promoting cardiac regeneration and repair.

[0056] The second aspect of the present invention provides a pharmaceutical composition, comprising:

[0057] (a1) A first active ingredient for promoting cardiac regeneration and repair after myocardial infarction, the first active ingredient comprising: the Hand1 gene, or its encoded protein, or its expression vector;

[0058] (a2) Optionally, a second active ingredient for promoting cardiac regeneration and repair after myocardial infarction, the second active ingredient comprising: other drugs for promoting cardiac regeneration and repair; and

[0059] (b) A pharmaceutically acceptable carrier.

[0060] In another preferred embodiment, in the pharmaceutical composition, the component (a1) accounts for 1-99 wt%, preferably 10-90 wt%, more preferably 30-70 wt% of the total weight of the pharmaceutical composition.

[0061] In another preferred embodiment, in the pharmaceutical composition, the component (a2) accounts for 1-99 wt%, preferably 10-90 wt%, more preferably 30-70 wt% of the total weight of the pharmaceutical composition.

[0062] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.

[0063] In another preferred embodiment, the other drugs that promote heart regeneration and repair are selected from the group consisting of versican, Pkm2, or a combination thereof.

[0064] In another preferred embodiment, the pharmaceutical composition may be a single compound or a mixture of multiple compounds.

[0065] In another preferred embodiment, the pharmaceutical composition is used for preparing a drug or preparation for promoting heart regeneration and repair after myocardial infarction.

[0066] In another preferred embodiment, the pharmaceutical dosage form is an injection or a syringe.

[0067] In another preferred embodiment, the total content of the active ingredient (a1) and the active ingredient (a2) is 1-99 wt% of the total weight of the composition, more preferably 5-90 wt%.

[0068] The third aspect of the present invention provides a medicine box, comprising:

[0069] (i) a first container, and the active ingredient (a1) Hand1 gene, or its encoded protein, or its expression vector, or a drug containing the active ingredient (a) located in the first container; and

[0070] (ii) an optional second container, and the active ingredient (a2) other drugs for promoting heart regeneration and repair, or a drug containing the active ingredient (a2) located in the second container.

[0071] In another preferred embodiment, the first container and the second container are the same or different containers.

[0072] In another preferred embodiment, the drug in the first container is a single preparation containing the Hand1 gene, or its encoded protein, or its expression vector.

[0073] In another preferred embodiment, the drug in the second container is a single preparation containing other drugs for promoting heart regeneration and repair.

[0074] In another preferred embodiment, the dosage form of the drug is an injection dosage form.

[0075] In another preferred embodiment, the kit further contains an instruction manual, and the instruction manual records the instructions for jointly administering the active ingredient (a1) and the active ingredient (a2) to promote heart regeneration and repair after myocardial infarction.

[0076] The fourth aspect of the present invention provides a use of the pharmaceutical composition according to the second aspect of the present invention or the medicine box according to the third aspect of the present invention for preparing a drug for promoting heart regeneration and repair after myocardial infarction.

[0077] The fifth aspect of the present invention provides a method for promoting cardiac regeneration and repair after myocardial infarction, comprising the steps of:

[0078] administering the Hand1 gene, or its encoded protein, or its expression vector, the pharmaceutical composition described in the second aspect of the present invention, or the kit described in the third aspect of the present invention, to a subject in need thereof.

[0079] In another preferred embodiment, the administration comprises injection (preferably intravenous injection).

[0080] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0081] In another preferred embodiment, the non-human mammal includes a rodent and a primate, preferably a mouse, a rat, a rabbit, or a monkey.

[0082] In another preferred embodiment, the administration frequency of the Hand1 gene, or its encoded protein, or its expression vector is 1 - 7 consecutive days per week, preferably 2 - 5 consecutive days per week, more preferably 2 - 3 consecutive days per week.

[0083] In another preferred embodiment, the administration time of the Hand1 gene, or its encoded protein, or its expression vector is 1 - 20 weeks, preferably 2 - 12 weeks, more preferably 4 - 8 weeks.

[0084] The sixth aspect of the present invention provides a method for screening a candidate drug for promoting cardiac regeneration and repair after myocardial infarction, comprising the steps of:

[0085] (a) In a test group, in a culture system, in the presence of a test substance, culturing cells expressing the Hand1 protein for a period of time T1, and detecting the expression level E1 and / or activity A1 of the Hand1 protein in the culture system of the test group;

[0086] And in a control group in the absence of the test substance and under the same other conditions, detecting the expression level E2 and / or activity A2 of the Hand1 protein in the culture system of the control group;

[0087] (b1) Comparing E1 and E2, if E1 is significantly higher than E2, it indicates that the test substance is a candidate drug for promoting cardiac regeneration and repair after myocardial infarction; and / or

[0088] (b2) Comparing A1 and A2, if A1 is significantly higher than A2, it indicates that the test substance is a candidate drug for promoting cardiac regeneration and repair after myocardial infarction.

[0089] In another preferred embodiment, the cells are mammalian cells.

[0090] In another preferred embodiment, the cells are selected from the group consisting of cardiomyocytes, fibroblasts, smooth muscle cells, or combinations thereof.

[0091] In another preferred embodiment, the cells are cells cultured in vitro.

[0092] In another preferred embodiment, "significantly higher than" means E1 / E2 ≥ 2, preferably ≥ 3, more preferably ≥ 4.

[0093] In another preferred embodiment, "significantly lower than" means A1 / A2 ≥ 2, preferably ≥ 3, more preferably ≥ 4.

[0094] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0095] In another preferred embodiment, the method includes step (c): administering the candidate drug determined in step (a) to a non-human mammal, so as to determine its effect on cardiac regeneration and repair after myocardial infarction in the non-human mammal.

[0096] In another preferred embodiment, the test substance is selected from the group consisting of small molecule compounds, antibodies, polypeptides, nucleic acids, or combinations thereof.

[0097] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 It shows that the expression of Hand1 increases in the peri-infarct area in the heart failure model after myocardial infarction; regulating Hand1 in cardiomyocytes to control cell proliferation and metabolic transformation after myocardial injury may promote myocardial injury repair.

[0099] Figure 2 It shows that in vivo infection of mice with an AAV9 vector overexpressing Hand1 driven by the cTnT promoter promotes repair after myocardial injury and increases cardiomyocyte proliferation.

[0100] Figure 3 It shows in vitro infection with an AAV9 vector overexpressing Hand1 and detecting its effect on cardiomyocyte proliferation. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0101] Through extensive and in-depth research, the present inventor unexpectedly discovered for the first time that the Hand1 gene, or its encoded protein or its expression vector, can be used to promote cardiac regeneration and repair after myocardial infarction, and can also promote cardiomyocyte proliferation. Specifically, the present inventor first discovered that an AAV9 vector overexpressing Hand1 driven by the NcTnT promoter can effectively promote repair after myocardial injury. On this basis, the present inventor completed the present invention.

[0102] Term Explanation

[0103] 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 this invention belongs.

[0104] As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0105] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0106] Heart failure

[0107] A pharmaceutical composition containing the Hand1 gene, or its encoded protein or its expression vector of the present invention, has a significant promoting effect on the repair after myocardial injury.

[0108] Heart failure is a syndrome mainly characterized by circulatory dysfunction caused by the inability of the heart to provide the required blood and oxygen to surrounding tissues to meet their metabolic needs due to myocardial diastolic or systolic dysfunction, manifested as a decrease (in heart failure with reduced ejection fraction) or preservation (in heart failure with preserved ejection fraction) of the left ventricular ejection fraction. Adult cardiomyocytes are particularly vulnerable to hypoxia after injury (such as myocardial infarction), which may lead to cell dysfunction and death, ultimately resulting in a large loss of cardiomyocytes and the occurrence of heart failure. Heart failure is almost the terminal outcome of the development of all heart diseases and seriously affects human health.

[0109] In the present invention, diseases related to heart failure caused by ischemic myocardial injury are not particularly limited, including various diseases related to ischemic myocardial injury known in the art. Representative examples of diseases related to ischemic myocardial injury include (but are not limited to): stable angina pectoris, unstable angina pectoris, ischemic cardiomyopathy, asymptomatic coronary heart disease, etc.

[0110] The Hand1 gene and its encoded protein

[0111] Hand1 is a member of the bHLH (basic helix-loop-helix) transcription factor family and is one of the regulators of cardiac progenitor cell fate. The cardiac expression of Hand1 was first detected in the posterior ventricle of mouse embryos at E8.5 days. During embryonic development, myocardial energy metabolism mainly relies on glycolysis, and Hand1 is highly expressed during this period. Studies have found that the levels of cardiac Hand1 mRNA and protein decline immediately after birth. Hand1 itself inhibits the expression of many genes involved in myocardial cell lipid metabolism.

[0112] In the present invention, the terms "Hand1 gene" and "Hand1 polynucleotide" are used interchangeably and both refer to a nucleic acid sequence having the Hand1 nucleotide sequence.

[0113] The full-length human Hand1 gene genome is 1701 bp (Gene ID: 9421), located at Chromosome 5, NC_000005.10.

[0114] The full-length mouse Hand1 gene genome is 1898 bp (Gene ID: 15110), located at Chromosome 11, NC_000077.7.

[0115] The similarity between human and mouse Hand1 at the DNA level is 82%, and the protein sequence similarity is 92%. It should be understood that nucleotide substitutions in codons are acceptable when encoding the same amino acid. Additionally, it should be understood that nucleotide changes are also acceptable when conservative amino acid substitutions are produced by nucleotide substitutions.

[0116] When the amino acid fragment of Hand1 is obtained, a nucleic acid sequence encoding it can be constructed based on it, and specific probes can be designed according to the nucleotide sequence. The full-length nucleotide sequence or its fragment can usually be obtained by PCR amplification, recombination, or artificial synthesis methods. For the PCR amplification method, primers can be designed according to the Hand1 nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequence. When the sequence is long, it is often necessary to perform PCR amplification two or more times, and then splice the fragments amplified each time together in the correct order.

[0117] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods.

[0118] In addition, synthetic methods can also be used to synthesize relevant sequences, especially when the fragment length is short. Generally, by first synthesizing multiple small fragments and then ligating them, fragments with very long sequences can be obtained.

[0119] Currently, it is already possible to obtain the DNA sequence encoding the protein (or its fragment, derivative) of the present invention entirely by chemical synthesis. Then, this DNA sequence can be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0120] Through conventional recombinant DNA techniques, the polynucleotide sequence of the present invention can be used to express or produce recombinant VEGFA polypeptide. Generally, the following steps are involved:

[0121] (1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the human Hand1 polypeptide of the present invention, or with a recombinant expression vector containing this polynucleotide;

[0122] (2). Culture the host cell in a suitable medium;

[0123] (3). Isolate and purify the protein from the medium or the cells.

[0124] In the present invention, the Hand1 polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is usually that it contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0125] Methods well-known to those skilled in the art can be used to construct an expression vector containing the Hand1-encoding DNA sequence and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The said DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0126] In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0127] A vector containing the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence can be used to transform a suitable host cell so that it can express the protein.

[0128] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, etc.

[0129] Transformation of the host cell with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, and the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0130] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0131] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic lysis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0132] In a preferred embodiment, the sequence of the Hand1 protein is as shown in SEQ ID NO.1:

[0133] MNLVGSYAHHHHHHHSHPPHPMLHEPFLFGPASRCHQERPYFQSWLLSPADAAPDFPAGGPPPTTAVAAAAYGPDARPSQSPGRLEALGSRLPKRKGSGPKKERRRTESINSAFAELRECIPNVPADTKLSKIKTLRLATSYIAYLMDVLAKDAQAGDPEAFKAELKKTDGGRESKRKRELPQQPESFPPASGPGEKRIKGRTGWPQQVWALELNQ(SEQ ID NO.1)

[0134]

[0135] Adeno-associated virus

[0136] Due to the characteristics that adeno-associated virus (AAV) is smaller than other viral vectors, non-pathogenic, and can transfect both dividing and non-dividing cells, gene therapy methods based on AAV vectors for genetic diseases have received extensive attention.

[0137] Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus Dependovirus of the Parvoviridae family. It is a single-stranded DNA defective virus with the simplest structure discovered so far and requires a helper virus (usually adenovirus) to participate in replication. It encodes the cap and rep genes in the inverted terminal repeat (ITR) sequences at both ends. The ITRs play a decisive role in virus replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.

[0138] Recombinant adeno-associated virus vector (rAAV) is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, and long-term expression of foreign genes in vivo, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially a lot of data has been accumulated on its application effects in various cells, tissues, and in vivo experiments. In medical research, rAAV is used in the research of gene therapy for various diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, constructing disease models, preparing gene knockout mice, etc.

[0139] In a preferred embodiment of the present invention, the vector is a recombinant AAV vector. AAV is a relatively small DNA virus that can integrate into the genome of the cells it infects in a stable and site-specific manner. They can infect a large series of cells without having any effect on cell growth, morphology, or differentiation, and they do not seem to be involved in human pathology. The AAV genome has been cloned, sequenced, and characterized. AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of replication of the virus. The rest of the genome is divided into two important regions with encapsidation functions: the left part of the genome containing the rep gene involved in virus replication and virus gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.

[0140] AAV vectors can be prepared using standard methods in the art. Any serotype of adeno-associated virus is suitable. Methods for purifying the vectors can be found, for example, in U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT / US2005 / 027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for in vitro and in vivo gene transfer has been described (see, for example, International Patent Application Publication Nos. WO91 / 18088 and WO93 / 09239; U.S. Patent Nos. 4,797,368, 6,596,535, and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety). These patent publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced with the gene of interest, and the use of these constructs for transferring the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). Replication-deficient recombinant AAV can be prepared by co-transfecting a cell line infected with a human helper virus (such as adenovirus) with a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.

[0141] In some embodiments, the recombinant vector is encapsulated into viral particles (such as AAV viral particles including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). Accordingly, the present disclosure includes recombinant viral particles containing any of the vectors described herein (which are recombinant because they contain a recombinant polynucleotide). Methods for generating such particles are known in the art and are described in U.S. Patent No. 6,596,535.

[0142] Adeno-associated virus (AAV) has currently become an important vector in gene therapy for cardiovascular diseases. AAV is a single-stranded linear DNA virus. Since it is a defective virus that cannot replicate on its own, AAV is generally considered non-pathogenic and thus a feasible and promising gene therapy approach in clinical treatment. Phase I and Phase II clinical trials (CUPIDI) of gene therapy for heart failure by introducing the SERCA2a gene into the myocardium via an AAV vector have been carried out abroad. No AAV-related side effects were found during the trial, indicating good biosafety of AAV gene therapy. Different AAV serotypes have different cell tropisms. Small animal experiments have shown that AAV9 has the highest transfection efficiency in cardiomyocytes, and this type of AAV can be used as an effective vector for gene therapy targeting cardiomyocytes.

[0143] Construct an adeno-associated virus overexpressing Hand1 (AAV9-cTnTp-mHand1-EGFP) or a control virus vector (AAV9-cTnTp-EGFP) driven by the cardiac-specific gene cTnT promoter, and inject it via the tail vein at a virus dose of 4×10 11 units per animal. Collect the heart 14 days after AAV injection to detect the expression of Hand1 in cardiomyocytes.

[0144] As used herein, the "AAV9 vector overexpressing Hand1 driven by the cTnT promoter" is a small molecule or macromolecule compound that specifically highly expresses Hand1 in cardiomyocytes, promotes the conversion of the metabolic state of cardiomyocytes from fatty acid oxidation to glycolysis, and promotes the proliferation of endogenous cardiomyocytes.

[0145] In a preferred embodiment, the AAV9 vector overexpressing Hand1 driven by the cTnT promoter is an effective solution for targeted treatment of myocardial infarction. The schematic diagram of its construction is as follows:

[0146]

[0147] Drug composition

[0148] The present invention also provides a pharmaceutical composition, which comprises (a) the Hand1 gene of the present invention, or its encoded protein or its expression vector; and (b) a safe and effective pharmaceutically acceptable carrier. The severity of the disease to be treated in the patient, the patient's body weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg - 50 mg / kg of animal body weight per day (preferably 0.0001 mg - 25 mg / kg of animal body weight) (the dosage for experimental mice is 25 mg / kg), satisfactory effects can be obtained. For example, depending on the urgency of the treatment situation, several separate doses can be administered per day, or the dose can be proportionally reduced. In addition, the Hand1 gene of the present invention, or its encoded protein or its expression vector can be used alone or in combination with other therapeutic agents (such as formulated in the same pharmaceutical composition).

[0149] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. This term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not overly toxic after administration. These carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, adjuvants and combinations thereof.

[0150] The pharmaceutically acceptable carrier in the therapeutic composition may contain liquids, such as water, saline, glycerol and ethanol. Additionally, auxiliary substances may also be present in these carriers, such as wetting agents or emulsifying agents, pH buffering substances, etc.

[0151] Generally, the therapeutic composition can be made into an injectable form, such as a liquid solution or suspension; it can also be made into a solid form that is suitable for formulation into a solution or suspension in a liquid carrier before injection.

[0152] Once the composition of the present invention is formulated, it can be administered by conventional routes, including (but not limited to): intratumoral, intramuscular, intravenous, subcutaneous, intradermal, or topical administration. The subject to be prevented or treated can be an animal; especially a human.

[0153] When the pharmaceutical composition of the present invention is used for actual treatment, pharmaceutical compositions of various different dosage forms can be adopted according to the usage. Preferably, it is an intravenous preparation.

[0154] These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.

[0155] For example, the preparation of eye drops can be carried out as follows: The Hand1 gene of the present invention, or its encoded protein or its expression vector is dissolved in sterile water (in which a surfactant is dissolved) together with a basic substance, the osmotic pressure and pH are adjusted to physiological conditions, and suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and thickeners can be optionally added, and then it is completely dissolved.

[0156] The pharmaceutical compositions of the present invention can also be administered in the form of sustained-release agents. For example, the Hand1 gene of the present invention, or its encoded protein or its expression vector can be incorporated into pills or microcapsules with a sustained-release polymer as a carrier, and then the pills or microcapsules are surgically implanted into the tissue to be treated. Examples of sustained-release polymers include ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, lactic acid-glycolic acid copolymers, etc. Preferably, biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers can be exemplified.

[0157] When the pharmaceutical compositions of the present invention are used for actual treatment, the dose of the Hand1 gene of the present invention, or its encoded protein or its expression vector as an active ingredient can be reasonably determined according to the weight, age, gender and symptom degree of each patient to be treated.

[0158] The main advantages of the present invention include:

[0159] (1) The present invention unexpectedly discovers for the first time that the Hand1 gene, or its encoded protein or its expression vector can be used to promote cardiac regeneration and repair after myocardial infarction, and can also promote myocardial cell proliferation. Specifically, the present invention discovers for the first time that the NcTnT promoter-driven Hand1 overexpression AAV9 vector can effectively promote repair after myocardial injury.

[0160] (2) The present invention discovers for the first time that overexpression of Hand1 can effectively promote repair after myocardial injury.

[0161] (3) The present invention discovers for the first time that the present invention can promote the transformation of the metabolic state of myocardial cells to the glycolysis direction by overexpressing Hand1 in myocardial cells, and promote myocardial cell proliferation.

[0162] (4) The present invention provides a new method for promoting cardiomyocyte proliferation and thus improving myocardial injury repair.

[0163] (5) The method of the present invention is simple to operate, simple to prepare drugs, and has high efficiency.

[0164] (6) The present invention can be delivered to cardiomyocytes through AAV9 for targeted therapy.

[0165] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0166] Unless otherwise specified, the reagents and materials used in the embodiments of the present invention are all commercially available products.

[0167] Example 1

[0168] The expression of Hand1 increases in the border area of myocardial infarction; regulating Hand1 in cardiomyocytes to control cell proliferation and metabolic transformation after myocardial injury may promote myocardial injury repair

[0169] 1.1 The expression of Hand1 in cardiomyocytes in the border area of infarction increases: C57BL / 6JSlac mice (bred in the animal house of Tongji University, purchased from Slac Experimental Animal Center) were subjected to ligation of the left anterior descending coronary artery to induce ischemic myocardial injury (myocardial infarction). Primary cardiomyocytes in different regions of the sham operation group and the myocardial infarction group were isolated and protein quantification was performed. The Western-blot results showed that compared with the sham operation group (sham), the expression of Hand1 in cardiomyocytes in the border zone (BZ) of infarction was relatively high ( Figure 1 A). The change in cardiomyocyte proliferation mainly occurs at the junction of injured and normal myocardium, that is, the border area of myocardial infarction. It is indicated that the high expression of Hand1 in the border area of myocardial infarction during ischemic myocardial injury may play a role in the process of myocardial injury repair.

[0170] 1.2 Knockdown of Hand1 in NMCMs (primary neonatal mouse cardiomyocytes) significantly reversed the upregulation of glycolytic genes and the downregulation of lipid metabolism genes, and reduced the proliferation of cardiomyocytes

[0171] Newborn mice born on days P1-P0 (C57BL / 6JSlac mice raised in the animal house of Tongji University and purchased from Slac Laboratory Animal Center) were selected. After isolating NMCMs, they were transiently transfected with Hand1-siRNA (5’-CCAAGCUCUCCAAGAUCAATT-3’, 5’-UUGAUCUUGGAGAGCUUGGTT-3’). After 48 hours of transient transfection, the protein and mRNA expressions of Hand1 were detected to confirm the knockdown efficiency ( Figure 1 B-C). Subsequently, the expressions of glycolysis-related genes and lipid metabolism-related genes at the mRNA level were detected ( Figure 1 D). Meanwhile, 48 hours after transient transfection with Hand1-siRNA, after fixation with 4% PFA, the proliferation of NMCMs in the control group and the Hand1-siRNA group was detected by co-immunostaining of Edu and PH3 with a-SA ( Figure 1 E-F). The results showed that: in the Hand1 knockdown group in NMCMs, glycolysis genes were significantly downregulated, while the expressions of lipid metabolism genes were significantly upregulated; Hand1 knockdown significantly reduced the proliferation of cardiomyocytes. This suggests that Hand1 may regulate cardiomyocyte proliferation by regulating the metabolic state of cardiomyocytes.

[0172] Example 2

[0173] Infection with the AAV9 vector overexpressing Hand1 driven by the cTnT promoter in mice promotes repair after myocardial injury and increases cardiomyocyte proliferation

[0174] 2.1 The AAV9 vector overexpressing Hand1 driven by the cTnT promoter promotes myocardial injury repair:

[0175] The AAV9 vector overexpressing Hand1 driven by the cTnT promoter (AAV9-cTnTp-mHand1-EGFP) (purchased from Hanheng Biotechnology Co., Ltd.) (the titer of the AAV9 vector was 1.3*10^12 vg / ml) was injected into the body of mice (C57BL / 6JSlac mice raised in the animal house of Tongji University and purchased from Slac Laboratory Animal Center) via the tail vein. After 14 days, the hearts were collected, frozen sections were made, and co-fluorescent staining with the myocardial marker ɑ-SA was performed to detect the overexpression level of Hand1 ( Figure 2 A), and the heart tissues were collected to detect the expressions of Hand1 at the protein level and mRNA level ( Figure 2 B-C); after infection with AAV9-cTnTp-mHand1-EGFP, myocardial infarction was modeled. Echocardiography results 28 days after the operation found that overexpression of Hand1 in cardiomyocytes improved cardiac function (EF, FS) after myocardial infarction ( Figure 2D-E); Hearts were collected 28 days after myocardial infarction, paraffin sections were made, and after Masson staining, it was found that the overexpression group of Hand1 reduced the degree of cardiac fibrosis ( Figure 2 F), These results suggest that enhancing the expression of Hand1 in cardiomyocytes during myocardial infarction may promote the repair after myocardial injury.

[0176] 2.2 The overexpression AAV9 vector of Hand1 driven by the cTnT promoter increases the proliferation of cardiomyocytes: After infecting with adeno-associated virus, myocardial infarction was modeled, hearts were collected 14 days after myocardial infarction, paraffin sections were made, and the proliferation of cardiomyocytes was detected by fluorescence co-staining of proliferation-related markers (Edu, Ki67, PH3 and Aurora B) with the myocardial marker ɑ-SA ( Figure 2 G-J). The results suggest that overexpression of Hand1 in cardiomyocytes significantly increases the proliferation of cardiomyocytes after myocardial infarction and may promote the repair after myocardial injury.

[0177] The results suggest that after overexpression of Hand1 in cardiomyocytes, cardiac function can be improved and the infarct area after myocardial infarction can be reduced; this effect may be achieved by promoting the proliferation of endogenous cardiomyocytes.

[0178] Example 3

[0179] In vitro infection with the overexpression AAV9 vector of Hand1, and detection of its effect on the proliferation of cardiomyocytes

[0180] 3.1 After isolating primary neonatal rat cardiomyocytes, infect with AAV9-cTnTp-mHand1-EGFP:

[0181] After infecting primary neonatal rat cardiomyocytes (NMCMs) (raised in the animal house of Tongji University, C57BL / 6JSlac mice purchased from Slac Laboratory Animal Center) with AAV9-cTnTp-mHand1-EGFP for 48 h ( Figure 3 A), compared with the control group, high expression at the mRNA and protein levels was observed in the group infected with the AAV9-Hand1 overexpression viral vector ( Figure 3 B-C). This reflects the high efficiency of the expression of AAV9-cTnTp-mHand1-EGFP in primary cardiomyocytes.

[0182] 3.2 Overexpression of Hand1 in NMCMs increases the proliferation of cardiomyocytes:

[0183] After infecting primary neonatal rat cardiomyocytes (NMCMs) with AAV9-cTnTp-mHand1-EGFP for 48 h, Edu was observed by immunofluorescence co-staining of Edu and PH3 with ɑ-SA + 、PH3 +The number of cardiomyocytes in the AAV9-cTnTp-mHand1-EGFP group was significantly increased compared with the control group ([ Figure 3 Figure 3 D-E). In vitro experiments also demonstrated that overexpression of Hand1 in cardiomyocytes significantly promoted cardiomyocyte proliferation.

[0184] All documents mentioned in the present invention are incorporated herein by reference as if each individual document was specifically and individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of a Hand1 gene, or its encoded protein or its expression vector, characterized in that, For preparing a composition or preparation for promoting cardiac regeneration and repair after myocardial infarction.

2. The use according to claim 1, characterized in that, The composition or preparation is for promoting regenerative repair after cardiac trauma.

3. The use according to claim 1, wherein The composition or preparation is for promoting cardiomyocyte proliferation.

4. The use according to claim 1, characterized in that, The composition or preparation is also for one or more uses selected from the following group: (a) Improving cardiac dysfunction caused by myocardial infarction; (b) Reducing the area of myocardial infarction and improving myocardial fibrosis; (c) Promoting the proliferation of cardiomyocytes in the peripheral region of injury; (d) Promoting the metabolic conversion of cardiomyocytes in the peripheral region of injury from fatty acid oxidation to glycolytic state, thereby improving myocardial injury after myocardial infarction.

5. The use according to claim 1, wherein The amino acid sequence of the Hand1 protein is selected from the following group: (i) A polypeptide having the amino acid sequence shown in SEQ ID NO.: 1; (ii) A polypeptide derived from (i) formed by substitution, deletion or addition of one or several (such as 1-10) amino acid residues of the amino acid sequence shown in SEQ ID NO.: 1 and having the function of the said protein; or (iii) A polypeptide having an amino acid sequence with a homology of ≥90% (preferably ≥95%, more preferably ≥98% or 99%) to the amino acid sequence shown in SEQ ID NO.: 1 and having the function of the said protein.

6. The use according to claim 1, wherein The concentration of the Hand1 gene, or its encoded protein, or its expression vector is 0.5-5×10^12 vg / mL, preferably 0.8-5×10^12 vg / mL, more preferably 1-5×10^12 vg / mL.

7. A pharmaceutical composition, characterized in that, Comprising: (a1) A first active ingredient for promoting cardiac regeneration and repair after myocardial infarction, the first active ingredient comprising: the Hand1 gene, or its encoded protein, or its expression vector; (a2) Optionally, a second active ingredient for promoting cardiac regeneration and repair after myocardial infarction, the second active ingredient comprising: other drugs for promoting cardiac regeneration and repair; and (b) A pharmaceutically acceptable carrier.

8. A medicine box, characterized in that, Comprising: (i) A first container, and the active ingredient (a1) Hand1 gene, or its encoded protein, or its expression vector located in the first container, or a drug containing the active ingredient (a); and (ii) Optionally, a second container, and the active ingredient (a2) other drugs for promoting cardiac regeneration and repair located in the second container, or a drug containing the active ingredient (a2).

9. Use of the pharmaceutical composition according to claim 7 or the kit according to claim 8, characterized in that For preparing a drug for promoting cardiac regeneration and repair after myocardial infarction.

10. A method for screening candidate drugs for promoting cardiac regeneration and repair after myocardial infarction, characterized in that, Comprising the steps of: (a) In a test group, in a culture system, in the presence of a test substance, culturing cells expressing the Hand1 protein for a period of time T1, and detecting the expression level E1 and / or activity A1 of the Hand1 protein in the culture system of the test group; And in a control group where the test substance is absent and other conditions are the same, detecting the expression level E2 and / or activity A2 of the Hand1 protein in the culture system of the control group; (b1) Comparing E1 and E2, if E1 is significantly higher than E2, it indicates that the test substance is a candidate drug for promoting cardiac regeneration and repair after myocardial infarction; and / or (b2) Compare A1 and A2. If A1 is significantly higher than A2, it indicates that the test substance is a candidate drug for promoting cardiac regeneration and repair after myocardial infarction.

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