Use of enhanced tbx1 expression in repairing cardiac tissue damage

Abstract

The application discloses a use of an active ingredient in repairing heart tissue damage. Specifically, the application discloses an active ingredient which can be used for repairing heart tissue damage and treating diseases related to lymphatic vessel abnormalities, and the active ingredient comprises a TBX1 protein, a coding sequence of the TBX1 protein, or a promoter of the TBX1 protein, or a combination thereof. The TBX1 protein can promote the proliferation of heart lymphatic vessel endothelial cells, play an immune regulation role, promote the establishment of an immune inhibition microenvironment in myocardium after heart tissue damage, inhibit an autoimmune reaction after the heart tissue damage, relieve concurrent heart tissue inflammation, and promote the repair and regeneration of the damaged heart tissue.

Description

Technical Field

[0001] This invention relates to the field of cardiovascular disease treatment, and more particularly to the use of TBX1 in repairing cardiac tissue damage. Background Technology

[0002] Cardiovascular disease is the leading cause of death in my country, and the mortality rate continues to rise in recent years. In mammals, cardiomyocytes have proliferative capacity only for a brief window after birth; once the heart muscle matures, it exits the cell cycle. Therefore, myocardial necrosis is usually irreversible, has a poor prognosis, and can lead to serious consequences. For example, up to 60% of patients with ischemic myocardial infarction develop heart failure within six years of their initial attack. Currently, treatment for myocardial infarction mainly focuses on reducing myocardial ischemia and necrosis through vascular recanalization and slowing adverse ventricular remodeling by regulating neurohumoral mechanisms with medication. However, these treatments cannot completely block ventricular remodeling after myocardial infarction, and a significant proportion of patients progress to heart failure within a short period without further ischemic damage.

[0003] Therefore, there is an urgent need in this field to develop new treatments for heart disease. Summary of the Invention

[0004] The purpose of this invention is to provide a new active ingredient and its use for the effective treatment of cardiac diseases and / or lymphatic abnormalities associated with heart damage.

[0005] A first aspect of the present invention provides the use of an active ingredient for preparing a formulation or drug, said formulation or drug being used for:

[0006] (i) Activates the proliferation of cardiac lymphatic endothelial cells;

[0007] (ii) Suppressing cardiac autoimmune responses;

[0008] (iii) Promote the transformation of M1 macrophages into M2 macrophages;

[0009] (iv) Repairing heart tissue damage;

[0010] (v) Treatment of heart disease associated with damage to cardiac tissue; and

[0011] (vi) Treatment of diseases related to lymphatic vessel abnormalities;

[0012] The active ingredients include: TBX1 protein, its coding sequence, or its promoters, or a combination thereof.

[0013] In another preferred embodiment, the coding sequence includes DNA, cDNA, and mRNA.

[0014] In another preferred embodiment, the "promoting the transformation of M1 macrophages into M2 macrophages" includes: promoting the transformation of M1 macrophages (promoting inflammatory response) into M2 macrophages (promoting tissue repair) in myocardial tissue.

[0015] In another preferred embodiment, the “cardiac disease associated with cardiac tissue damage” is a cardiac disease associated with an immune response (especially an autoimmune response).

[0016] In another preferred embodiment, the cardiac disease associated with cardiac tissue damage is selected from the group consisting of: myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, cardiac injury caused by novel coronavirus pneumonia (COVID-19), or a combination thereof.

[0017] In another preferred embodiment, the lymphatic vessel abnormality-related disease is selected from the group consisting of: primary lymphedema caused by lymphatic vessel dysplasia, secondary lymphedema caused by lymphatic vessel rupture or obstruction, lymphedema presenting as a genetic syndrome such as Turner syndrome, yellow nail syndrome, and Hennekam syndrome, or a combination thereof.

[0018] In a second aspect, the present invention provides the use of TBX1 protein or its coding sequence or its detection reagent for preparing a diagnostic reagent or diagnostic kit, said diagnostic reagent or diagnostic kit for prognostic assessment of cardiac diseases or lymphatic abnormalities related to cardiac tissue damage.

[0019] In another preferred embodiment, the detection reagent is selected from the group consisting of primers, probes, chips, antibodies, or combinations thereof.

[0020] In another preferred embodiment, if the expression level and / or activity of TBX1 is increased compared with normal populations or controls, it suggests a good prognosis for the diagnosed subject.

[0021] In another preferred embodiment, a decrease in TBX1 expression and / or activity compared to normal individuals or controls suggests a poor prognosis for the diagnosed individual.

[0022] In another preferred embodiment, the diagnosed subject has or is suspected of having heart disease or lymphatic abnormality-related disease associated with cardiac tissue damage.

[0023] In another preferred embodiment, the method for prognosing cardiac diseases or lymphatic abnormality-related diseases associated with cardiac tissue damage includes: providing a subject sample, detecting the expression level T1 of TBX1 protein in the subject sample, and comparing it with the average expression level T0 of TBX1 protein in the cardiac lymphatic vessels of patients with myocardial infarction.

[0024] If T1 is greater than T0, it indicates that the prognosis of the subject is good;

[0025] If T1 is less than T0, it indicates that the subject has a poor prognosis.

[0026] In another preferred embodiment, the method for prognosing cardiac disease or lymphatic vessel abnormality-related disease associated with cardiac tissue damage includes: providing a subject sample and detecting whether there are TBX1 coding and / or regulatory sequence variations in the subject sample; if the TBX1 variation is a variation that leads to loss or decrease of TBX1 function, it indicates a poor prognosis for the subject; if the TBX1 variation is a variation that leads to enhanced TBX1 function, it indicates a good prognosis for the subject.

[0027] In another preferred embodiment, the TBX1 function refers to promoting lymphatic vessel regeneration and the formation of an immunosuppressive microenvironment.

[0028] In another preferred embodiment, the cardiac disease associated with cardiac tissue damage includes myocardial infarction.

[0029] In another preferred embodiment, the subject suffered from myocardial infarction.

[0030] In another preferred embodiment, the subject is a human or a non-human mammal.

[0031] In another preferred embodiment, the sample includes a cardiac tissue sample, a cardiac cell sample, a lymphatic endothelial cell sample, a lymph fluid sample, peripheral blood cell sample, or a combination thereof.

[0032] In another preferred embodiment, the sample includes a cell sample, a DNA sample, an RNA sample, or a combination thereof.

[0033] In another preferred embodiment, the kit further includes a detection reagent for detecting additional biomarkers selected from the group consisting of Top2a, Mki67, Cenpe, Aurkb, or combinations thereof.

[0034] In another preferred embodiment, an increase in the expression level (or amount) of these additional markers suggests that the TBX1 variant is a variant that leads to enhanced TBX1 function.

[0035] In another preferred embodiment, if the expression level (or amount) of these additional biomarkers increases compared to normal individuals or controls, it suggests a good prognosis for the diagnosed individual.

[0036] In another preferred embodiment, a decrease in the expression level (or amount) of these additional biomarkers compared to normal individuals or controls suggests a poor prognosis for the diagnosed individual.

[0037] A third aspect of the present invention provides an active ingredient that can be used to repair cardiac tissue damage and treat lymphatic vessel abnormality-related diseases, said active ingredient comprising: TBX1 protein, its coding sequence, or an promoter thereof, or a combination thereof.

[0038] In another preferred embodiment, the active ingredient is a promoter of the TBX1 protein.

[0039] In another preferred embodiment, the promoter promotes the expression of TBX1 protein in lymphatic vessels of the heart and other parts of the body.

[0040] In another preferred embodiment, the promoter upregulates the function of TBX1 protein in lymphatic vessels in the heart and other parts of the body.

[0041] In a fourth aspect, the present invention provides an expression vector containing an expression cassette for expressing the TBX1 protein.

[0042] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, plasmids, eukaryotic expression vectors, prokaryotic expression vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, transposons, or combinations thereof.

[0043] In another preferred embodiment, the expression vector specifically infects lymphatic endothelial cells.

[0044] In another preferred embodiment, the expression box has a structure of Formula I from the 5' end to the 3' end:

[0045] Z0-Z1-Z2(I)

[0046] In the formula,

[0047] Each "-" independently represents a chemical bond or nucleotide linking sequence;

[0048] Z0 is either an empty or 5'UTR sequence;

[0049] Z1 is the nucleotide sequence encoding the TBX1 protein; and

[0050] Z2 is either an empty or 3'UTR sequence.

[0051] In another preferred embodiment, the expression cassette further includes a lymphatic endothelial-specific promoter operatively linked to the coding sequence of the TBX1 protein.

[0052] In another preferred embodiment, the length of each nucleotide linker sequence is 1-30 nt, more preferably 1-15 nt, and even more preferably 3-6 nt.

[0053] In another preferred embodiment, the nucleotide sequence encoding the TBX1 protein is derived from a human or a non-human mammal.

[0054] In another preferred embodiment, the nucleotide sequence is as shown in SEQ ID NO.1.

[0055] In another preferred embodiment, the nucleotide sequence is as shown in SEQ ID NO.2.

[0056] In a fifth aspect, the present invention provides a host cell containing the expression vector described in the fourth aspect of the present invention.

[0057] In another preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell.

[0058] In another preferred embodiment, the host cell is selected from the group consisting of bacterial cells, yeast cells, or mammalian cells.

[0059] In another preferred embodiment, the host cell is a lymphatic endothelial cell.

[0060] In another preferred embodiment, the host cell is a cardiac lymphatic endothelial cell.

[0061] In a sixth aspect, the present invention provides a pharmaceutical formulation comprising (a) the active ingredient described in the third aspect of the present invention, or the expression vector described in the fourth aspect of the present invention, or the cell described in the fifth aspect of the present invention, and (b) a pharmaceutically acceptable carrier, excipient, or diluent.

[0062] In another preferred embodiment, the dosage form of the pharmaceutical preparation is selected from the group consisting of lyophilized preparations, liquid preparations, or combinations thereof.

[0063] In another preferred embodiment, the pharmaceutical preparation is an injection solution.

[0064] In another preferred embodiment, the pharmaceutical preparation further comprises: additional active ingredients;

[0065] Preferably, the additional active ingredients include: BMP4, CCL28, anti-CD8 antibody, or a combination thereof.

[0066] A seventh aspect of the present invention provides the use of the active ingredient described in the third aspect of the present invention, or the expression vector described in the fourth aspect of the present invention, or the cell described in the fifth aspect of the present invention, or the pharmaceutical preparation described in the sixth aspect of the present invention, in the preparation of a medicament for treating heart disease and / or lymphatic vessel abnormality-related diseases associated with cardiac injury.

[0067] In another preferred embodiment, the cardiac disease associated with cardiac injury is selected from the group consisting of: myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, cardiac injury caused by novel coronavirus pneumonia (COVID-19), or a combination thereof.

[0068] In another preferred embodiment, the lymphatic vessel abnormality-related disease is selected from the group consisting of: primary lymphedema caused by lymphatic vessel dysplasia, secondary lymphedema caused by lymphatic vessel rupture or obstruction, lymphedema presenting as a genetic syndrome such as Turner syndrome, yellow nail syndrome, and Hennekam syndrome, or a combination thereof.

[0069] An eighth aspect of the present invention provides a method for screening promoters of TBX1 protein, comprising the steps of: administering a candidate drug to in vitro cultured lymphatic endothelial cells and / or experimental animals; if the TBX1 mRNA level or TBX1 protein level in the in vitro cultured lymphatic endothelial cells or lymphatic tissue in the experimental animals is increased relative to a control group not treated with the candidate drug, or the TBX1 protein function is enhanced relative to a control group not treated with the candidate drug, then the candidate drug can be used as a promoter of TBX1 protein.

[0070] In another preferred embodiment, the promoter of the TBX1 protein can be used to treat heart disease associated with cardiac tissue damage.

[0071] In another preferred embodiment, the promoter of the TBX1 protein can be used to treat lymphatic vessel abnormalities-related diseases.

[0072] A ninth aspect of the present invention provides a method for treating heart disease associated with cardiac tissue damage, the method comprising: applying the active ingredient of the third aspect of the present invention or the pharmaceutical preparation of the sixth aspect of the present invention to a subject in need.

[0073] In another preferred embodiment, the subject in need suffers from heart disease associated with damage to cardiac tissue.

[0074] In another preferred embodiment, the cardiac disease associated with cardiac injury is selected from the group consisting of: myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, cardiac injury caused by novel coronavirus pneumonia (COVID-19), or a combination thereof.

[0075] In a tenth aspect of the present invention, a method for treating lymphatic vessel abnormality-related diseases is provided, the method comprising: applying the active ingredient described in the third aspect of the present invention or the pharmaceutical preparation described in the sixth aspect of the present invention to a subject in need.

[0076] In another preferred embodiment, the subject in need suffers from heart disease associated with lymphatic abnormalities.

[0077] In another preferred embodiment, the lymphatic vessel abnormality-related disease is selected from the group consisting of: primary lymphedema caused by lymphatic vessel dysplasia, secondary lymphedema caused by lymphatic vessel rupture or obstruction, lymphedema presenting as a genetic syndrome such as Turner syndrome, yellow nail syndrome, and Hennekam syndrome, or a combination thereof.

[0078] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0079] Figure 1 The images show the activation and expression of Tbx1 in lymphoendothelial cells after myocardial infarction. (A) Tbx1 expression in the heart at 3 and 7 days post-myocardial infarction. Undamaged heart (baseline, n=2), day 3 (n=2), day 4 (n=2), and day 7 (n=6) post-myocardial infarction. (B) X-gal staining of heart sections with Vegfr3 immunostaining at day 7 post-myocardial infarction (n=3). This shows that Tbx1 is mainly expressed in lymphatic endothelial cells. (C) Whole heart X-gal staining at days 14 (n=3) and 28 (n=3) post-myocardial infarction. dpMI: day number post-myocardial infarction.

[0080] Figure 2This study demonstrates how endothelial cell-specific knockout of Tbx1 leads to abnormal lymphatic regeneration and impaired repair after myocardial infarction. (A) Schematic diagram of Tbx1-deficient mouse construction, where the functional genomic sequence was knocked out upon Cre activation; (B) Ventral view of the heart 28 days after myocardial infarction. Black arrow: left ventricular anterior wall aneurysm. Green arrow: postoperative adhesion; (C) HE staining of cardiac sections 14 days after myocardial infarction (n=3), green arrow: mononuclear infiltrating inflammatory cells, blue arrow: hemorrhage points; (D) Changes in ejection fraction (EF) in the control group and Tbx1Cko group after myocardial infarction (n=7), data are presented as mean ± standard error, *, P<0.05. Mann-Whitney U test; (E) Changes in left ventricular end-diastolic volume (LVEDV) in the control group and Tbx1Cko group after myocardial infarction (n=7), data are presented as mean ± standard error, *, p<0.05, **, p<0.01, Mann-Whitney U test. (F, G) Vegfr3 staining shows the regeneration status of cardiac lymphatic endothelium at the site of myocardial infarction. Gross cardiac images and tissue sections 7 days after myocardial infarction (F), and the number of lymphatic endothelium cells at the site of myocardial infarction and undamaged distal sites (G), **, P<0.01, Mann-Whitney U test; (H) Differentially expressed genes in the endothelium-specific transcriptome and analysis of downstream target genes of Tbx1 using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq); (I) Functional analysis of Tbx1 target genes.

[0081] Figure 3 The study showed significant alterations in the expression of genes related to lymphatic vessel growth in Tbx1-deficient hearts. (A) Transcriptome analysis revealed a significant decrease in the expression of lymphatic vessel growth-related genes Dtx1, Dtx3, Sema4c, and Foxc2 in cardiac endothelial cells after myocardial infarction. (B, C) Immunofluorescence staining analysis was performed on day 7 after myocardial infarction to determine the distribution and quantification of Dtx1 and Notch1-expressing lymphoendothelial cells in the infarct area.

[0082] Figure 4 This study showed significant alterations in the expression of Tbx1-deficient cardiac lymphoendothelial cells and immune-regulating genes. (A, B) Immunofluorescence staining analysis was performed on day 7 post-myocardial infarction to determine the distribution and quantification of lymphoendothelial cells expressing Ccl21, Ccl28, and Icam1 in the infarct area.

[0083] Figure 5 The results showed a significant increase in autoimmune CD8+ T cells in Tbx1-deficient hearts. (A) Single-cell sequencing results of CD8+ T cells. + T cell number distribution, **p=3x10 -3 ,§§p=5x10 -15 Chi-square test; (B) T cell subset proliferation analysis showed CD8 +T cells exhibit high proliferative capacity; data are presented as mean ± standard deviation, **, p < 5.6 x 10⁻⁶. -14 ,§§p<4.3x10 -11 Student's t-test; (C, D) Flow cytometry analysis of Cd8 in the hearts of endothelial-specific knockout (F-Cko) and lymphoendothelial-specific knockout (P-Cko) mice 7 days after myocardial infarction. + Changes in T cell number were shown as mean ± standard error (n=5), *, p<0.05, **, p<0.01, Mann-Whitney U test; (E, F) Immunofluorescence staining analysis of CD8+ in the heart on day 7 after myocardial infarction. + Distribution and quantification of T cells in the infarct area. Data are presented as mean ± standard error (n=5), *, p<0.05, Mann-Whitney U test; (G) flow cytometry analysis of CD8. + Cytotoxic factors secreted by T cells were shown as mean ± standard error (n=3), ns, which were not statistically significant, according to the Mann-Whitney U test; (H, I) flow cytometry analysis of Myh6 peptide-MHC I tetramer complex staining. Autoreactive CD8+ in the F-Cko group on day 7 post-myocardial infarction. + T cells were significantly increased, as shown in mean ± standard error (n=4), *, p<0.05, Mann-Whitney U test; (J) Flow cytometry analysis of negative control peptide-MHC I tetramer complex staining in post-myocardial infarction samples on CD8 + The positivity rate in T cells was shown as mean ± standard error (n=4), ns, which was not statistically significant according to the Mann-Whitney U test.

[0084] Figure 6 The results showed a decrease in inflammatory M1 macrophages and a significant increase in repair-functional M2 macrophages in Tbx1-deficient hearts. (A, B) Flow cytometry analysis of macrophage number changes in Tbx1Cko heart on day 7 post-myocardial infarction, *, p<0.05, **, p<0.01, Mann-Whitney U test. (C, D) Immunohistochemical staining of decreased M2 macrophage number in the infarcted area of ​​Tbx1Cko heart on day 7 post-myocardial infarction, **, p<0.01, Mann-Whitney U test.

[0085] Figure 7 The mouse Rosa26 was shown. STOP-Tbx1-ZG Schematic diagram of allele construction.

[0086] Figure 8The results showed that, compared with the control group, the mRNA expression levels of genes related to cell proliferation in cardiac endothelial cells were significantly increased (***P<1x10⁻¹). -3

[0087] Figure 9 The results showed that Tbx1 overexpression in cardiac lymphatic endothelial cells improved myocardial repair after myocardial infarction. (A, B) Echocardiography showed ejection fraction (A) and left ventricular end-diastolic volume (B) after myocardial infarction in the control group (n=8) and the Tbx1 OE group (n=10). The statistical method used was the non-parametric Mann-Whitney U-test. ns: no statistical difference, *P<0.05; (C) Massen staining showed cardiac scar tissue (blue) 60 days after myocardial infarction, with the heart shown as a cross-section. (D) The relative area of ​​ischemic tissue in the heart of the Tbx1 OE group (n=5) was significantly smaller than that in the control group (n=4). The statistical method used was the non-parametric Mann-Whitney U-test, *P<0.05.

[0088] Figure 10 The CD8 protein showed that overexpression of Tbx1 in lymphatic endothelium reduced cytotoxicity in the heart. + T cell percentage. (A, B) Flow cytometry analysis of Cd8 in the heart on day 7 after myocardial infarction (Tbx1OE). + Changes in T cell count were shown as mean ± standard error (n=5), *, p<0.05, Mann-Whitney U test; PD-1 + TNFα - Exhaustive CD + The number of 8T cells was significantly increased in the Tbx1OE group, Foxp3 + CD25 + Regulatory CD8 + The number of T cells (Tregs) was significantly increased in the Tbx1OE group. Detailed Implementation

[0089] Through extensive and in-depth research, the inventors have unexpectedly developed an active ingredient for the first time that can be used to treat heart diseases related to cardiac injury. The inventors discovered that overexpression of TBX1 protein in the lymphatic endothelial cells of the cardiac lymphatic vessels promotes lymphatic endothelial cell proliferation. Furthermore, TBX1 can also exert immunomodulatory effects, promoting the establishment of an immunosuppressive microenvironment in the myocardium after myocardial infarction, inhibiting autoimmune responses following cardiac tissue damage, alleviating concurrent cardiac tissue inflammation, and promoting the repair and regeneration of damaged cardiac tissue. Experiments have demonstrated that in a mouse model of myocardial infarction, overexpression of TBX1 in the cardiac lymphatic endothelium significantly improves cardiac function after myocardial infarction and promotes cardiac tissue repair. Based on these findings, this invention was completed.

[0090] the term

[0091] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0092] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. 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.).

[0093] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0094] As used herein, the terms “subject” and “required subject” refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cattle, horses, dogs, cats, pigs, sheep, and goats.

[0095] TBX1

[0096] The present invention provides an active ingredient that can be used to repair cardiac tissue damage, the active ingredient comprising: TBX1 protein, its coding sequence, or a promoter thereof, or a combination thereof.

[0097] The TBX1 gene encodes a T-box transcription factor located in the 22q11.2 region of human chromosome 22 and is the main pathogenic gene for 22q11.2 microdeletion syndrome. The incidence of this disease in newborns is as high as 1 in 2000. 75% of affected infants have congenital heart disease, accompanied by craniofacial abnormalities, thymus agenesis, and other congenital defects. In early embryonic development, Tbx1 is expressed in some cardiac progenitor cells with differentiation potential and is crucial for the development of the cardiac lymphatic system; however, in adulthood, the expression level of Tbx1 in the heart is extremely low.

[0098] The TBX1 gene has a gene ID of 6899 in the NCBI database, and its expressed mRNA sequence has four isoforms: NM_005992.1, NM_080646.2, NM_080647.1, and NM_001379200.1. The mouse Tbx1 gene has a gene ID of 21380 in the NCBI database, and its expressed mRNA sequence has four isoforms: NM_011532.2, NM_001285472.1, NM_001285476.1, and NM_001373938.1. As used herein, the coding sequence for the TBX1 protein can be any of the above sequences or their variants, as long as the sequence effectively expresses a normally functioning or increased TBX1 protein. The function is to promote lymphatic regeneration and the formation of an immunosuppressive microenvironment.

[0099] In another preferred embodiment, the coding sequence of the TBX1 protein is shown in SEQ ID NO.1.

[0100]

[0101] In another preferred embodiment, the coding sequence of the Tbx1 protein is as shown in the CDS sequence used in the examples:

[0102]

[0103] Heart tissue damage

[0104] As used in this article, the term "cardiac tissue injury" refers to damage to cardiac tissue, including degeneration and necrosis of cardiomyocytes and degeneration and necrosis of cardiac blood vessels and lymphatic vessels. Cardiac tissue injury can lead to inflammation and autoimmune responses.

[0105] This invention discovers that TBX1 protein, TBX1 mRNA, or their promoters can be used to repair cardiac tissue damage. Overexpression of TBX1 in cardiac lymphatic vessels not only promotes the proliferation of lymphatic endothelial cells but also regulates autoimmune responses and alleviates cardiac inflammation, thus making it suitable for treating heart diseases associated with cardiac tissue damage.

[0106] The term "cardiac disease associated with cardiac tissue damage" includes acute myocardial injury caused by ischemia and hypoxia, autoimmune heart disease, and other diseases that cause myocardial injury. In another preferred embodiment, the cardiac disease associated with cardiac tissue damage is selected from the group consisting of: myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, cardiac injury caused by COVID-19, or a combination thereof.

[0107] Expression vector and host cell

[0108] The present invention provides an expression vector for expressing the TBX1 protein, which contains the coding sequence of the TBX1 protein of the present invention.

[0109] With the provided sequence information, skilled technicians can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transduction into cells.

[0110] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, plasmids, eukaryotic expression vectors, prokaryotic expression vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, transposons, or combinations thereof.

[0111] Preferably, the nucleic acid sequence encoding the TBX1 protein is provided as a vector, preferably as an expression vector. Preferably, it is provided as a gene therapy vector preferably suitable for transduction and expression in target cells (e.g., cochlear supporting cells). The vector can be viral or non-viral (e.g., plasmid). Viral vectors include those derived from: adenovirus, including mutant forms of adeno-associated virus (AAV), retrovirus, lentivirus, herpesvirus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, poxvirus, and SV40. Preferably, the viral vector is replication-defective, or can be replication-deficient, capable of replication, or conditionally replicating. Viral vectors generally maintain an extrachromosomal state without integrating into the genome of the target cell. A preferred viral vector for introducing the nucleic acid sequence encoding the TBX1 protein into target cells is an AAV vector. Selective targeting can be achieved using specific AAV serotypes (AAV serotypes 2 to 12) or modified versions of any of these serotypes.

[0112] Viral vectors have the ability to enter cells. However, non-viral vectors such as plasmids can be compounded with reagents to facilitate the uptake of viral vectors by target cells. Such reagents include polycationic agents. Alternatively, delivery systems such as liposome-based delivery systems may be used. The vectors used in this invention are preferably adapted for in vivo or in vitro use, and are preferably adapted for human use.

[0113] The vector will preferably contain one or more regulatory sequences to guide the expression of the nucleic acid sequence in target cells. The regulatory sequences may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operatively linked to the nucleic acid sequence. The vector may also include selective markers, for example, to determine the expression of the vector in a growth system (e.g., bacterial cells) or in target cells.

[0114] "Operationally linked" means that nucleic acid sequences are functionally related to their operationally linked sequences such that they are linked in a way that causes them to affect each other's expression or function. For example, a nucleic acid sequence operationally linked to a promoter will have an expression pattern influenced by the promoter.

[0115] This invention also provides a host cell for expressing the TBX1 protein. The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: *Escherichia coli*, *Streptomyces*; bacterial cells of *Salmonella typhimurium*; fungal cells such as yeast; insect cells of *Drosophila* S2 or Sf9; and animal cells such as CHO, COS7, 293 cells, T cells, and NK cells.

[0116] pharmaceutical preparations

[0117] The present invention provides a pharmaceutical formulation or composition comprising (a) the active ingredient described in the second aspect of the present invention, or the carrier described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention, and (b) a pharmaceutically acceptable carrier, excipient, or diluent.

[0118] In another preferred embodiment, the pharmaceutical preparation is used to repair cardiac tissue damage.

[0119] In another preferred embodiment, the pharmaceutical preparation is used to treat heart disease associated with damage to cardiac tissue.

[0120] In another preferred embodiment, the pharmaceutical preparation is used to treat diseases related to lymphatic vessel abnormalities.

[0121] The "active ingredient" in the pharmaceutical formulations described in this invention refers to the TBX1 protein, its coding sequence, its promoter, or a combination thereof, or the expression vector described in this invention, such as a viral vector. The "active ingredient," formulations, and / or compositions described in this invention can be used to treat heart disease associated with cardiac tissue damage and / or lymphatic abnormalities. "Safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. "Pharmaceutically acceptable carrier or excipient" refers to one or more compatible solid or liquid fillers or gel substances suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredient of this invention without significantly reducing the efficacy of the active ingredient.

[0122] The formulations and / or compositions of the present invention may further comprise BMP4, CCL28, anti-CD8 antibody, and other active ingredients with similar functions to TBX1. The formulations and / or compositions may be used in combination with other medicaments known in the art for treating cardiac diseases and / or lymphatic abnormalities associated with cardiac tissue damage.

[0123] The composition can be liquid or solid, such as powder, gel, or paste. Preferably, the composition is liquid, and more preferably, an injectable liquid. Suitable excipients will be known to those skilled in the art.

[0124] In either administration mode, preferably, the carrier is provided as an injectable liquid. Preferably, the injectable liquid is provided as a capsule or syringe.

[0125] Pharmaceutically acceptable examples of carrier components include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0126] The composition may comprise physiologically acceptable sterile aqueous or anhydrous water, dispersion, suspension, or emulsion, and sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0127] Methods for screening promoters of TBX1 protein.

[0128] The present invention also provides a method for screening promoters of TBX1 protein, the method comprising the steps of: administering a candidate drug to in vitro cultured lymphatic endothelial cells and / or experimental animals; if the TBX1 mRNA level, TBX1 protein level, or TBX1 protein function in the in vitro cultured lymphatic endothelial cells or lymphatic tissues of the experimental animals is increased relative to the control group that has not been administered the candidate drug, then the candidate drug can be used as a promoter of TBX1 protein.

[0129] In this invention, the "candidate drug" includes, but is not limited to, small molecule compounds, nucleic acid drugs, or protein drugs. The candidate drug may act directly or indirectly on TBX1, but ultimately increases the function or level of TBX1 protein or TBX1 mRNA in cultured lymphatic endothelial cells or lymphatic tissues of experimental animals.

[0130] In another preferred embodiment, the promoter of the TBX1 protein can be used to treat heart disease associated with cardiac tissue damage.

[0131] The main advantages of this invention are:

[0132] (1) Cardiac injury tends to lead to autoimmune damage, but there are currently no effective means to control this pathological mechanism, resulting in heart failure in a significant number of patients after cardiac injury. This invention proposes for the first time a means to control this pathological mechanism, which is expected to bring new treatment methods to heart diseases caused by this related pathological mechanism.

[0133] (2) This invention proposes a novel endogenous repair and regeneration mechanism for the heart. The active ingredients (or combinations) of this invention enhance the repair and regeneration of the heart after injury from two different levels: inhibiting autoimmune responses and promoting lymphatic vessel proliferation, thus exhibiting excellent effects. This provides a new strategy and solution for regeneration and repair after myocardial infarction in humans.

[0134] (3) In addition, the active ingredients (or combinations) of the present invention can also create favorable conditions for myocardial infarction repair and regeneration by inhibiting the favorable microenvironment created by the autoimmune response.

[0135] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0136] Unless otherwise specified, all materials, reagents, instruments, etc. used in the embodiments are commercially available.

[0137] Experimental methods

[0138] (1) Construction of Rosa26-STOP-Tbx1 / STOP-Tbx1 (Tbx1OE) mouse strain: The LoxP-STOP-LoxP-Tbx1 cDNA-P2A-ZsGreen element was inserted into the Rosa26 gene site, wherein the Tbx1 cDNA is a nucleic acid sequence containing an open reading frame encoding the TBX1 protein, as shown in SEQ ID NO:2. Figure 7 Following Cre-mediated LoxP site recombination, the STOP element was cleaved, allowing Tbx1 expression. The Rosa26-STOP-Tbx1 / STOP-Tbx1 (Tbx1OE) strain of mice survived healthily.

[0139] (2) Transcription sequencing and analysis: using RNA was extracted using LS reagent (Thermo Fisher Science) and sequenced using 1 μg of whole RNA according to the Illumina platform library construction and sequencing method. At least 15 million raw paired-end reads were generated for each sample. The raw reads were aligned to the mouse genome (Gencode, GRCm38, Open Access) using STAR (2.5.2b, MIT license). Gene expression levels were analyzed on a per-gene basis. Differentially expressed genes were identified using the R package DESeq2 (version 1.20.0, LGPL license ≥3) (identification criteria: fold change ≥1.5 and p-value ≤0.05). Functional analysis of the differentially expressed genes was then performed using Metascape (https: / / metascape.org / gp / index.html# / menu / term_service).

[0140] (3) Animal model of myocardial infarction: Mice aged 8-12 weeks were used in the myocardial infarction experiment. The method was based on previous literature descriptions with slight modifications. The brief experimental steps are as follows:

[0141] Mice were intubated and connected to a ventilator (Harvard apparatus). Isoflurane was used to maintain anesthesia at a concentration of 3%-5%, with a ventilation rate of 100 breaths / min and a tidal volume of 350 μL. Myocardial infarction was induced by ligation of the left anterior descending coronary artery. The sham-operated group underwent the same surgical procedure as the experimental group, except that the coronary artery was not ligated. After the animals recovered, buprenorphine (0.1 mg / kg) was rapidly injected subcutaneously, followed by a second injection 24 hours later.

[0142] (4) Measurement of cardiac function: Transthoracic echocardiography was performed 7, 14, and 28 days after ligation of the left anterior descending coronary artery. A Vevo 2100 system (Visual Sonics) was used with a probe frequency of 30 MHz and a frame rate of 30 frames per second. Measurements were performed by acquiring long-axis cross-sections of the left ventricle during the cardiac cycle. The left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), and left ventricular ejection fraction (LVEF) were calculated using the following formulas.

[0143]

[0144]

[0145] Example 1

[0146] Tbx1 is activated and expressed in lymphoendothelial cells after myocardial infarction.

[0147] After inducing myocardial infarction in adult mice by constructing a mouse model, the expression level of Tbx1 in cardiac endothelial cells remained continuously increased, and 90% of the Tbx1-positive cells were lymphatic endothelial cells. Figure 1 A-1C).

[0148] Example 2

[0149] Cardiac lymphatic endothelial cell-specific knockout of Tbx1 leads to impaired post-myocardial infarction repair.

[0150] Tbx1 lymphatic endothelial cell-specific knockout mice were constructed. Figure 2 A), simultaneously inducing myocardial infarction in both the control and knockout groups of mice. Compared to the control group, Tbx1-deficient mice showed poorer repair after myocardial infarction, more severe tissue adhesion, and increased ventricular aneurysm formation. Figure 2 B). Histological examination showed that the inflammation in the heart was resolving slowly. Figure 2 C). Decreased cardiac ejection capacity and increased left ventricular end-diastolic volume ( Figure 2 D-2E). Cytological examination revealed significant impairment of cardiac lymphatic vessel regeneration. Figure 2 F-2G).

[0151] Endothelial transcriptome sequencing ( Figure 3 A) and immunofluorescence staining ( Figure 3 (B) shows that Tbx1 deletion significantly alters the expression of lymphangiogenic genes: one week after myocardial infarction, the expression of genes promoting lymphangiogenic endothelial growth, such as Dtx1, Dtx3, Sema4c, and Foxc2, decreased in cardiac endothelial cells. Simultaneously, the expression of Notch1, a gene inhibiting lymphangiogenic endothelial growth, increased (…). Figure 3 C).

[0152] Example 3

[0153] Tbx1 regulates the autoimmune response after myocardial infarction.

[0154] Immunoprecipitation combined with high-throughput sequencing (ChIP-seq) was used to detect the binding site of Tbx1 as a transcription factor on the genome of cardiac endothelial cells. Genes related to autoimmune tolerance were significantly enriched, indicating that Tbx1 may regulate autoimmune responses after myocardial infarction. Figure 2 H-2I).

[0155] Endothelial-specific transcriptomics revealed that Tbx1 deficiency significantly reduced the levels of immune-regulating mRNAs in endothelial cells, such as Icam1, Ccl21, and Ccl28. Icam1 is a cell surface adhesion protein that plays a crucial role in leukocyte migration and the suppression of leukocyte autoimmune activity. Ccl21 and Ccl28 are cytokines that regulate the immune microenvironment; high concentrations of Ccl21 and Ccl28 can attract immunosuppressive cells, such as regulatory T cells, to inflammatory sites, reducing autoimmune responses. Simultaneously, immunofluorescence experiments showed that 7 days after myocardial infarction modeling in mice, the protein levels of Icam1, Ccl21, and Ccl28 expressed in the lymphatic endothelial cells of Tbx1-deficient mice were significantly reduced. Figure 4 AB).

[0156] Single-cell RNA sequencing analysis revealed CD8 + T cells proliferated actively in the hearts of Tbx1-deficient mice, with a significantly increased cell number. Figure 5 A-5F), and also has the same characteristics as the control group CD8. + T-cell similar toxicity ( Figure 5 G). MHC I tetramer staining further revealed CD8 inhibitors targeting α-cardioglobulin in the F-Cko group. + T cells were significantly elevated ( Figure 5 H-5J) suggests that Tbx1 knockout leads to an increased autoimmune response after myocardial infarction.

[0157] Example 4

[0158] Tbx1 deficiency leads to an increase in the number of inflammatory macrophages after myocardial infarction.

[0159] Coronary artery ligation-induced myocardial infarction was modeled in adult Tbx1-deficient mice. On day 7 post-modeling, flow cytometry analysis was used to analyze macrophage subset numbers. Results showed that M2 macrophages (highly expressing F4 / 80 and lowly expressing Ly6c, which promote regeneration and mediate inflammatory resolution) were significantly reduced in the Tbx1-deficient group; simultaneously, the number of M1 macrophages (lowly expressing F4 / 80 and highly expressing Ly6c, whose enrichment usually indicates an inflammatory phase and long-term accumulation can lead to chronic inflammation and autoimmune responses) was significantly increased in the Tbx1-deficient group. Figure 6 AB).

[0160] Immunohistochemical staining also showed that on day 7 after myocardial infarction, the number of M2 macrophages in the infarct site was reduced in the Tbx1-deficient group. Figure 6 CD).

[0161] Example 5

[0162] Overexpression of the Tbx1 gene in lymphatic endothelial cells activates lymphatic endothelial cell proliferation.

[0163] Lymphatic endothelial cells are more likely to play a crucial role in the repair of myocardial infarction damage. During the transition from the acute inflammatory phase to the repair phase of myocardial infarction, Tbx1 drives transcriptomic changes in lymphatic endothelial cells, which not only promotes the regeneration of cardiac lymphatic vessels but also endows them with additional immunomodulatory functions. This can suppress the autoimmune response after myocardial infarction, preventing autoreactive T cells from continuously attacking the body's own tissues and causing chronic inflammation. Therefore, in this embodiment, using lymphatic endothelial cells as target cells, we investigate the feasibility of treating myocardial infarction by overexpressing Tbx1 and regulating the cardiac immune microenvironment.

[0164] Rosa26 was first constructed. Stop-Tbx1-ZG Allele (Tbx1OE mouse, Figure 7 Lymphatic vessel-specific Prox1Cre mice were crossed with Tbx1 OE mice to obtain double heterozygous mice. Tamoxifen was injected into adult double heterozygous mice to induce Tbx1 overexpression in the lymphatic endothelium. Transcriptome sequencing was used to analyze changes in the gene expression profile of the cardiac lymphatic endothelium, and to detect the molecular biological effects and potential target genes of enhanced Tbx1 expression.

[0165] The results showed that one week after Tbx1 expression was activated in the lymphatic vessels of the heart of adult mice, CD31+ endothelial cells of the heart of Tbx1OE mice were collected for transcriptome analysis.

[0166] The results showed that activation of Tbx1 enhanced the expression of proliferation-related genes in cardiac endothelial cells, such as Top2a, Mki67, Cenpe, and Aurkb, with expression levels increasing more than twofold (n=3, P<1x10). -5 ()( Figure 8 ).

[0167] Example 6

[0168] Effects of Tbx1 overexpression in lymphatic endothelium on post-myocardial infarction repair

[0169] Lymphatic vessel-specific Prox1Cre mice were crossed with Tbx1 OE mice to obtain double heterozygous mice. Tamoxifen was injected into adult double heterozygous mice to induce Tbx1 overexpression in the lymphatic endothelium. One week later, coronary artery ligation was performed to model myocardial infarction. Cardiac function was measured by echocardiography at 7, 14, and 60 days after modeling.

[0170] The results showed that mice overexpressing Tbx1 had significantly better cardiac ejection fraction than the control group two weeks after myocardial infarction. Figure 9 A), and this can continue for up to 2 months after a myocardial infarction, with a corresponding decrease in left ventricular end-diastolic volume. Figure 9B). Meanwhile, histological analysis showed a significant reduction in scar tissue area in Tbx1OE mice 60 days after myocardial infarction. Figure 9 CD).

[0171] Example 7

[0172] Overexpression of Tbx1 in lymphatic endothelium reduces the proportion of cytotoxic CD8+ T cells in the heart.

[0173] Lymphatic vessel-specific Prox1Cre mice were crossed with Tbx1 OE mice to obtain double heterozygous mice. Tamoxifen was infused into adult double heterozygous mice to induce Tbx1 overexpression in the lymphatic endothelium. One week later, coronary artery ligation and myocardial infarction modeling were performed. CD8+ was analyzed by flow cytometry on day 7 post-modeling. + Number of T cell subsets.

[0174] The results showed that depleted CD1 expressing the cell surface antigen PD-1 + The number of CD8 T cells was significantly increased in the Tbx1OE group, and the expression level of the cytokine TNFα in the Tbx1OE group was significantly higher than that in CD8 T cells. + T was significantly reduced ( Figure 10 A). Simultaneously, Foxp3, which has immunosuppressive functions... + CD25 + Regulatory CD8 + The number of T cells was significantly increased in the Tbx1OE group. Figure 10 B).

[0175] discuss

[0176] The occurrence of acute inflammation and damage repair after myocardial infarction is a complex, self-driven process involving various cell types, including immune cells, cardiomyocytes, vascular endothelial cells, and fibroblasts. Most research on post-myocardial infarction repair and regeneration revolves around these cell types.

[0177] The results of this invention suggest that lymphatic endothelial cells are more likely to play a crucial role in the repair of myocardial infarction (MI) damage. During the transition from the acute inflammatory phase to the repair phase of MI, Tbx1 drives transcriptomic changes in lymphatic endothelial cells, not only promoting their proliferation but also endowing them with additional immunomodulatory effects. This suppresses post-MI autoimmune responses, preventing persistent attacks on autoreactive T cells and thus avoiding chronic inflammation. This invention unexpectedly reveals for the first time that Tbx1 plays a role in regulating the regenerative microenvironment and coordinating the promotion of MI repair and regeneration within cardiac lymphatic endothelial cells. Tbx1 can assist in the repair of post-MI damage through lymphatic endothelial cells.

[0178] Another key feature of this invention is the first-ever disclosure of an immune-regulated repair and regeneration mechanism occurring at the initiation stage of myocardial infarction repair. This mechanism exhibits specific stage-specific and cell-type-specific characteristics, distinguishing it from previous studies on immune interventions for myocardial infarction repair and regeneration. Previous research has shown that indiscriminate inflammation suppression is not conducive to myocardial infarction repair, and current clinical studies of immune interventions for myocardial infarction have not demonstrated clear therapeutic benefits. Therefore, this invention will provide a new dimension to the ordered regulatory mechanism of repair and regeneration after myocardial infarction, forming new strategies and approaches to promote regeneration and repair after myocardial infarction by enhancing this novel endogenous repair and regeneration mechanism. Furthermore, the favorable microenvironment created by suppressing autoimmune responses can also create favorable conditions for cell transplantation in myocardial infarction repair and regeneration.

[0179] The heart is an organ prone to autoimmunity. Certain myocardial cell-specific proteins are not typically expressed in the thymus, preventing autoreactive T cells targeting these proteins from being cleared through central tolerance mechanisms and thus releasing them into the peripheral blood. Following cardiac injury, myocardial autoantigens are released, activating autoreactive T cells and triggering an autoimmune response. Besides acute myocardial injury caused by ischemia and hypoxia, other diseases leading to myocardial injury, such as myocarditis, some idiopathic dilated cardiomyopathies, Chagas' cardiomyopathy, and rheumatic heart disease, are all associated with persistent autoimmune responses. Therefore, this study suggests that overexpression of Tbx1 could also be used to treat other autoimmune-related cardiac diseases besides myocardial infarction.

[0180] Furthermore, although the mechanism presented in this invention is primarily based on a cardiac injury model, the genes regulated by Tbx1 and related to lymphatic endothelial growth are universally expressed in lymphatic endothelial cells. These downstream genes regulated by Tbx1 also have similar functions in lymphatic endothelial cells of other tissues. Therefore, similar mechanisms can be used to treat lymphatic abnormality-related diseases in various tissues, such as primary lymphedema caused by lymphatic dysplasia, secondary lymphedema caused by lymphatic vessel rupture or obstruction, and lymphedema present in genetic syndromes such as Turner syndrome, yellow nail syndrome, and Hennekam syndrome.

[0181] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Shanghai Children's Medical Center, affiliated with Shanghai Jiao Tong University School of Medicine <120> Use of enhanced TBX1 expression in repairing cardiac tissue damage <130> P2020‑2413 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1488 <212> DNA <213> Homo sapiens <400> 1 atgcacttca gcaccgtcac cagggacatg gaagccttca cggccagcag cctgagcagc 60 ctgggggccg cggggggctt cccgggcgcc gcgtcgcccg gcgccgaccc gtacggcccg 120 cgcgagcccc cgccgccgcc gccgcgctac gacccgtgcg ccgccgccgc ccccggcgcc 180 ccgggcccgc cgccgccgcc gcacgcctac ccgtttgcgc cggccgccgg ggccgccacc 240 agcgccgccg ccgagcccga gggccccggg gccagctgcg cggccgcagc caaggcgccg 300 gtgaagaaga acgcgaaggt ggccggtgtg agcgtgcagc tagagatgaa ggcgctgtgg 360 gacgagttca accagctggg caccgagatg atcgtcacca aggccggcag gcggatgttt 420 cccaccttcc aagtgaagct cttcggcatg gatcccatgg ccgactatat gctgctcatg 480 gacttcgtgc cggtggacga taagcgctac cggtacgcct tccacagctc ctcctggctg 540 gtggcgggga aggccgaccc tgccacgcca ggccgcgtgc actaccaccc ggactcgcct 600 gccaagggcg cgcagtggat gaagcaaatc gtgtccttcg acaagctcaa gctgaccaac 660 aacctactgg acgacaacgg ccacattatt ctgaattcca tgcacagata ccagccccgc 720 ttccacgtgg tctatgtgga cccacgcaaa gatagcgaga aatatgccga ggagaacttc 780 aaaacctttg tgttcgagga gacacgattc accgcggtca ctgcctacca gaaccatcgg 840 atcacgcagc tcaagattgc cagcaatccc ttcgcgaaag gcttccggga ctgtgaccct 900 gaggactggc cccggaacca ccggcccggc gcactgccgc tcatgagcgc cttcgcgcgc 960 tcgcggaacc ccgtggcttc cccgacgcag cccagcggca cggagaaaga cgcggctgag 1020 gcccggcgag aattccagcg cgacgcgggc gggccagcag tgctcgggga cccggcgcat 1080 cctccgcagc tgctggcccg ggtgctaagc ccctcgctgc ccggggccgg cggcgccggc 1140 ggcttagtcc cgctgcccgg cgcgcccgga ggccggccca gtcccccgaa ccccgagctg 1200 cgcctggagg cgcccggcgc atcggagccg ctgcaccacc acccctacaa atatccggcc 1260 gccgcctacg accactatct cggggccaag agccggccgg cgccctaccc gctgcccggc 1320 ctgcgtggcc acggctacca cccgcacgcg catccgcacc accaccacca ccccgtgagt 1380 ccagccgccg cggccgccgc cgccgctgcc gcagctgccg cggccgccaa catgtactcg 1440 tcggccggag ccgcgccgcc cggctcctac gactattgcc ccagataa 1488 <210> 2 <211> 1467 <212> DNA <213> Mouse (Mus musculus) <400> 2 atgatctccg ccgtgtctag tccgtggctc acgcagctct cgcacttctg cgacgttgca 60 gccttcgcag ccagcagtct gagcggcctg ggatccccgt cgcctggcgc cgacccgttc 120 ggccctcgcg agccgccgcc accgcgctac gatccgtgcg ctgcagtccc cggtgccccg 180 ggcccgccgc cgccgcgcgc ctatcctttc gcgcccgccc ccggggcggc tggcagctcg 240 gcggcggagt ccgagggtcc gggggctagc cgcgcggctg cggtcaaggc tccggtgaag 300 aagaacccga aggtggccag cgtgagcgtg cagctggaga tgaaggcgct gtgggacgag 360 ttcaatcagc tgggcaccga gatgatcgtc accaaggcag gcagacgaat gttccccacg 420 ttccaagtga agctttttgg aatggatccc atggccgact acatgctgct catggacttt 480 gtgcccgtag atgacaagcg ctaccggtat gctttccata gctcctcctg gctggtggcc 540 ggcaaggcag atcctgctac acctggccga gtacactacc acccggactc gccggctaag 660. ggcgcacagt ggatgaaaca gattgtgtct ttcgacaagc tgaaactgac caataacctg ctggatgaca atggccatat tattctcaac tccatgcaca gatatcagcc ccgattccat gttgtctatg tggaccctcg aaaagacagt gagaaatatg cagaggaga cttcaaaact tttgtgtttg aggagacacg cttcactgca gtcactgcct accagaatca ccggatcacg cagcttaaga ttgccagcaa ccccttcgcc aaaggcttcc gggattgcga cccggaggac tggccccgga accaccggcc cggagcgctg ccgctcgtga gtgcctttgc tcgctctcgg 960 aatcccgtgg cttcccccac gcagcccaat ggctcagaca aagacgctgc agaagcccgg 1020 cgcgagttcg accgtgactc cggacccgca gcgctcggcg acgctacgca cccgccgcag 1080 ctgctggcgc gcgtgctgag ccccgcactg cccggccctg gcggcctcgt cccgctaccc 1140 ggcggatccg gaggccgcca cagtcccccg cacgccgatc tgcgcctgga ggcgccgggc 1200 gcgtccgagc cgctgcacca ccatccctac aagtacccgg ccgccgccta cgaccactac 1260 ctcggggcca agagccggcc ggcgccctac ccgctgccag gcctgcgcgg ccacggctac 1320 cacccgcacg cgcacccgca cgcgcacccg caccatcacc accaccccgc ggtgaacccg 1380 gccgccgccg ccgctgctgc cgcagcagcc aacgtgtact cgtcggcggc cgcgccgccc 1440 ggtgcctacg actactgccc cagatag 1467

Claims

1. The use of an active ingredient, characterized in that, Used to prepare a formulation or drug, said formulation or drug being administered to an adult patient for: (i) Suppressing cardiac autoimmune responses; (ii) Promote the transformation of M1 macrophages into M2 macrophages in myocardial tissue; (iii) Repairing heart tissue damage; and (iv) Treatment of myocardial infarction; wherein the active ingredient includes: the TBX1 protein coding sequence.

2. The use as described in claim 1, characterized in that, The TBX1 protein coding sequence includes DNA, cDNA, and mRNA.

3. The use as described in claim 1, characterized in that, The drug or formulation further comprises: additional active ingredients; The additional active ingredients include: BMP4, CCL28, anti-CD8 antibody, or a combination thereof.

4. Use of an expression vector in the preparation of a medicament for use in adult patients to treat myocardial infarction, wherein, The expression vector contains an expression cassette for expressing the TBX1 protein coding sequence.

5. The use as described in claim 4, characterized in that, The expression vector is selected from the group consisting of DNA, RNA, plasmids, eukaryotic expression vectors, prokaryotic expression vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, transposons, or combinations thereof.

6. The use as described in claim 5, characterized in that, The expression vector is a lentiviral vector.

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