Application of TEAD family transcription factor gene or carrier containing TEAD family transcription factor gene in preparation of medicine for treating skin injury

Abstract

The invention relates to application of a TEAD family transcription factor gene or a carrier containing the TEAD family transcription factor gene in preparation of a medicine for treating skin injury. The vector containing the TEAD family transcription factor gene is transfected into the damaged skin tissue, so that the yield of exosomes in the skin tissue can be remarkably increased, and the skin injury repair process is promoted. The method is not only suitable for multiple types of skin injury repair processes, but also has important potential value in biomedical research and clinical application such as nerve injury repair, metabolic regulation and tumor microenvironment regulation and control.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the use of a TEAD family transcription factor gene or a vector containing the same in the preparation of a drug for treating skin injuries. Background Art

[0002] Skin injury repair is an important research area in skin regenerative medicine. With the acceleration of the aging process and the frequent occurrence of skin diseases and injuries, the demand for rapid and effective skin repair methods is gradually increasing. Traditional skin injury repair methods, such as drug therapy, cell therapy, and bioengineered material therapy, have promoted skin healing to a certain extent, but have limitations. Traditional drug therapy uses a combination of various drugs, including antibiotics, anti-inflammatory drugs, painkillers, and topical drugs for promoting healing (such as silver ion dressings). The drugs usually only act on the surface and have poor effects on deep tissue injuries or chronic wounds. Long-term use of antibiotics may cause adverse reactions such as drug resistance or local irritation. Cell therapy mainly refers to the use of autologous or allogeneic fibroblasts, keratinocytes, mesenchymal stem cells, etc., directly transplanted to the injury site to promote healing. This method is technically complex, costly, and the processes of cell isolation, expansion, and transplantation are cumbersome, with high requirements for the qualifications and technical levels of medical institutions. Allogeneic cell therapy also has the risk of immune rejection. Bioengineered materials (such as skin substitutes) can be synthetic or bio-derived materials, including collagen, hyaluronic acid-based materials, artificial dermis, acellular matrix, etc. Some materials are degraded before wound healing, affecting the treatment effect, and the materials need to be selected according to the wound condition, with high costs.

[0003] TEAD (TEA Domain) family transcription factors are a group of proteins that play key roles in various biological processes. Members of this family have been found in humans and other mammals and are highly conserved. The TEAD family contains four main members, namely TEAD1 to TEAD4, which have high structural and functional similarities and are involved in various important physiological processes such as cell proliferation and growth, tissue development, cell differentiation, metabolic regulation, and tissue regeneration and repair. Currently, it is commonly used in anti-tumor research, but there is no report on promoting skin injury repair. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a new use of a TEAD family transcription factor gene or a vector containing the same, that is, its use in the preparation of a drug for treating skin injuries.

[0005] The present invention directly introduces genes of the TEAD family into cells at the injury site, and achieves long-term gene expression through viral vectors (such as adeno-associated virus (AAV), lentivirus, etc.), enhances tissue repair function and inhibits inflammation, providing a non-invasive and efficient gene therapy method for skin injury repair. Further, the inventors of the present invention found a biotherapy method for promoting skin injury repair by overexpressing TEAD family transcription factors, which increases the production and secretion of exosomes; for the first time in the field of treating skin injuries, the molecular mechanism of TEAD1 regulating exosome synthesis and secretion was confirmed. Compared with the prior art, the present invention has the advantages of using less virus, low production, transportation and storage costs, simple operation, strong targeting, extremely low immunogenicity, no change in the genome, long duration of a single treatment, etc. and fewer side effects (for example: it will not cause immune reactions and out-of-control gene expression), and is applicable to the treatment of a wide range of skin injuries.

[0006] Therefore, in the first aspect of the present invention, there is provided the use of a TEAD family transcription factor gene or a vector containing the same in the preparation of a drug for treating skin injuries.

[0007] In some embodiments, the vector includes a recombinant vector that expresses or overexpresses a TEAD family transcription factor gene.

[0008] In some preferred embodiments, the backbone of the recombinant vector is selected from any one of pAV-CMV-P2A-GFP, pAAV-U6-sgRNA-CMV-GFP, pAAV-CMV-nls-CasRx-P2A-GFP, pCMV-PE2-P2A-GFP, pAAV-EF1a-GFP, and / or pAAV-CMV-GFP-P2A-Puro.

[0009] The above backbone vectors are all conventional existing vectors that can be used for gene expression, and can be selected according to different gene recombination expression methods. For example, if gene editing tools need to be combined, pAAV-U6-sgRNA-CMV-GFP or pAAV-CMV-nls-CasRx-P2A-GFP can be selected; if long-term stable expression is required, pAAV-EF1a-GFP or pAAV-CMV-GFP-P2A-Puro can be selected. Obviously, GFP used for protein labeling is for detection, and it is not required in the drug or can be replaced with other labeling proteins.

[0010] In some embodiments, the vector further includes a delivery vector, which is preferably selected from viral vectors such as lentiviral vectors, adenoviral vectors, retroviral vectors, or adeno-associated viral vectors, etc. The viral vector is used for packaging and delivering genes, such as the recombinant expression vector containing the TEAD gene described above in the present invention.

[0011] In some preferred embodiments, the viral vector is an adeno-associated viral vector.

[0012] In some more preferred embodiments, the adeno-associated viral vector is AAV9, which is used for packaging and delivering the vector. The present invention has strictly screened different serotypes of adeno-associated virus (AAV). The serotype of AAV has a significant impact on its tissue tropism. The inventors of the present invention found that the AAV9 serotype exhibits excellent transduction efficiency and safety in skin tissues, thereby ensuring that the overexpression vector constructed in the present invention can be effectively delivered to skin cells to achieve stable expression of the target gene.

[0013] In some embodiments, the TEAD family transcription factor gene includes the TEAD family transcription factor gene derived from mammals.

[0014] In some preferred embodiments, the TEAD family transcription factor gene derived from mammals is the mouse-derived TEAD family transcription factor gene.

[0015] In some embodiments, the mouse-derived TEAD family transcription factor gene includes TEAD1, TEAD2, TEAD3, and TEAD4; TEAD1, TEAD2, TEAD3, and TEAD4 have a high degree of homology and are functionally consistent.

[0016] In one embodiment of the present invention, TEAD1 is taken as an example of the TEAD family transcription factor gene.

[0017] In one preferred embodiment of the present invention, the amino acid sequence of TEAD1 is as shown in SEQ ID NO: 1; the recombinant overexpression vector is selected as the common and relatively low-cost pAV-CMV-P2A-GFP; and the viral vector is selected as AAV9.

[0018] The "skin injury" as used in the present invention refers to the impairment of the skin barrier function, which may involve the epidermis, dermis, or subcutaneous tissue, with or without inflammation, necrosis, infection, etc.; including but not limited to the commonly mentioned wounds, traumas, and skin lesions, etc. According to the etiology, the skin injury can include traumatic injuries and secondary injuries.

[0019] Among them, the trauma includes primary injuries caused by physical factors such as common mechanical trauma and burns, as well as chemical factors; the secondary injuries include skin injury complications in diabetic patients, and skin injuries caused by skin diseases such as impetigo and eczema.

[0020] In a more preferred embodiment, the present invention takes an animal model of mechanical trauma by surgical cutting as an example for illustration; among them, common mechanical traumas include contusions, abrasions, lacerations, avulsions and / or puncture wounds.

[0021] "Treatment" as used in the present invention means promoting the repair of skin injuries and / or promoting wound healing.

[0022] In some embodiments, the carrier can promote the secretion of exosomes by endogenous cells.

[0023] In some embodiments, the cells are at least one of keratinocytes, fibroblasts, endothelial cells and mesenchymal stem cells.

[0024] In some preferred embodiments, the stem cells are mesenchymal stem cells.

[0025] In some more preferred embodiments, the mesenchymal stem cells are adipose mesenchymal stem cells.

[0026] In some embodiments, the drug is a drug for local use such as injection.

[0027] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0028] The reagents and raw materials used in the present invention are all commercially available.

[0029] The positive and progressive effects of the present invention are as follows:

[0030] The method of the present invention has the advantages of using less virus, low production, transportation and storage costs, simple operation, strong targeting, extremely low immunogenicity, no change in the genome, long duration of a single treatment, and fewer side effects (for example: it will not cause immune reactions and out-of-control gene expression), and is applicable to the treatment of a wide range of skin injuries. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 For the establishment of a mouse skin injury repair model.

[0032] Figure 2 For the comparison of the mouse skin injury repair process; A: Measurement results of the healing area of the mouse skin wound; B: Statistical chart of the healing rate of the mouse skin wound.

[0033] Figure 3For the immunofluorescence of mouse skin (A) and WB quantitative results (B).

[0034] Figure 4 For the immunohistochemical results of mouse skin; A: The immunohistochemical staining results of mouse skin; B: The expression of TEAD1, exosome marker CD63, angiogenesis marker protein CD31, inflammatory factor IL-6 and cell viability marker Ki67 in the control group and the experimental group injected with TEAD1-AAV virus.

[0035] Figure 5 For TEAD1 promoting adipose mesenchymal stem cells (ADSCs) to secrete exosomes; A: Detecting the mRNA and protein levels after different treatments (knockout / overexpression of TEAD1) of ADSC cells; B: Electron microscopy images of exosomes in different groups; C: The particle size and secretion amount of exosomes in the control group and the TEAD1 overexpression group; D: The expression of exosome marker proteins in different groups of ADSCs.

[0036] Figure 6 For the cell phenotypes of keratinocytes Hacat treated with exosomes produced by different groups; A: Detecting the cell proliferation of different groups by CCK8; B: Detecting the cell migration of different groups by Transwell; C: Detecting the cell migration of different groups by scratch assay.

[0037] Figure 7 For the phenotypes of vascular endothelial cells HUVEC treated with exosomes produced by different groups; A: Detecting the angiogenesis ability of different groups by angiogenesis assay; B: Detecting the cell proliferation of different groups by CCK8. Detailed implementation mode

[0038] The following examples are used for the disclosure of the present invention, but do not limit the scope of the present invention. For those without specific techniques or conditions noted in the examples, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The reagents or instruments involved are all conventional products that can be purchased through regular channels.

[0039] Example 1 Promoting the repair of mouse skin injury by overexpressing TEAD1 using adeno-associated virus AAV9

[0040] (1) Construction of overexpression vector and packaging of adeno-associated virus type AAV9

[0041] Taking the adeno-associated virus method as an example, the steps of packaging mouse-derived TEAD1 gene adeno-associated virus type AAV9 are described.

[0042] Gene name: TEAD1; Species: Mouse-derived; Gene size: 1356 bp; NM number: NM_001166584.2.

[0043] Furthermore, the protein amino acid sequence of TEAD1 is SEQ ID NO: 1:

[0044] MEPSSWSGSESPAENMERMSDSADKPIDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKSRDFHSKLKDQTAKDKALQHMAAMSSAQIVSATAIHNKLGLPGIPRPTFPGAPGFWPGMIQTGQPGSSQDVKPFVQQAYPIQPAVTAPIPGFEPASAPAPSVPAWQGRSIGTTKLRLVEFSAFLEQQRDPDSYNKHLFVHIGHANHSYSDPLLESVDIRQIYDKFPEKKGGLKELFGKGPQNAFFLVKFWADLNCNIQDDAGAFYGVTSQYESSENMTVTCSTKVCSFGKQVVEKVETEYARFENGRFVYRINRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILLVVTNRDTQETLLCMACVFEVSNSEHGAQHHIYRLVKD

[0045] Furthermore, the protein amino acid sequence of TEAD2 is SEQ ID NO: 2:

[0046] MGEPRAGAALDDGSGWTGSEEGSEEGTGGSEGAGGDGGPDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKSREIQSKLKDQVSKDKAFQTMATMSSAQLISAPSLQAKLGPTGPQASELFQFWSGGSGPPWNVPDVKPFSQTPFTLSLTPPSTDLPGYEPPQALSPLPPPTPSPPAWQARGLGTARLQLVEFSAFVEPPDAVDSYQRHLFVHISQHCPSPGAPPLESVDVRQIYDKFPEKKGGLRELYDRGPPHAFFLVKFWADLNWGPSGEEAGAGGSISSGGFYGVSSQYESLEHMTLTCSSKVCSFGKQVVEKVETERAQLEDGRFVYRLLRSPMCEYLVNFLHKLRQLPERYMMNSVLENFTILQVVTNRDTQELLLCTAYVFEVSTSERGAQHHIYRLVRD

[0047] Furthermore, the protein amino acid sequence of TEAD3 is SEQ ID NO: 3:

[0048] MASNSWNASSSPGEAREDGPEGLDKGLDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARKKVREYQVGIKAMNLDQVSKDKALQSMASMSSAQIVSASVLQNKFSPPSPLPQAVFSTSSRFWSSPPLLGQQPGPSQDIKPFAQPAYPIQPPLPPTLSSYEPLAPLPSAAASVPVWQDRTIASSRLRLLEYSAFMEVQRDPDTYSKHLFVHIGQTNPAFSDPPLEAVDVRQIYDKFPEKKGGLKELYEKGPPNAFFLVKFWADLNSTIQEGPGAFYGVSSQYSSADSMTISVSTKVCSFGKQVVEKVETEYARLENGRFVYRIHRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTSRDSQETLLVIAFVFEVSTSEHGAQHHVYKLVKD

[0049] Furthermore, the protein amino acid sequence of TEAD4 is SEQ ID NO: 4:

[0050] MEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKAREIQAKLKDQAAKDKALQSMAAMSSAQIISATAFHSSMALARGPGRPAVSGFWQGALPGQAGTSHDVKPFSQQTYAVQPPLPLPGFESPAGPAPSPSAPPAPPWQGRSVASSKLWMLEFSAFLEQQQDPDTYNKHLFVHIGQSSPSYSDPYLEAVDIRQIYDKFPEKKGGLKDLFERGPSNAFFLVKFWADLNTNIEDEGSSFYGVSSQYESPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHGAQHHIYRLVKE

[0051] In the description of this application, the protein sequences corresponding to murine genes are used. The TEAD family of transcription factors is conserved among different species. The protein sequences corresponding to any homologous genes of the TEAD family fall within the scope of this patent claim.

[0052] 1. Synthesize the target gene by total gene synthesis. Vector selection: pAV-CMV-P2A-GFP (Vigenebio, pAV100004-OE). Add the kozak sequence GCCACC before the ATG of the target gene, and add a small Flag protein tag to the C-terminus of the target gene. The vector carries GFP, and the target gene and GFP are non-fusion expressed through P2A. The restriction enzyme sites at both ends are ASISI / MluI. Use T4 ligase to ligate the target gene with the vector. Transform using DH5α competent cells. Then pick colonies and verify by restriction enzyme digestion. Select the vector with correct restriction enzyme digestion for sequencing. The sequencing primers are CMV-seq-F: 5'-CGCAAATGGGCGGTAGGCGTG-3' (SEQ ID NO: 5), EGFP-SEQ-R: 5'-CTCGTCGTCTTGTAGTTCCCGT-3' (SEQ ID NO: 6). Extract the correct plasmid using an endotoxin removal kit (Tiangen, DP117) to obtain the overexpression vector pAV-CMV-TEAD1 of the TEAD1 gene, and then package the adenovirus.

[0053] 2. Culture HEK 293T cells one day in advance. When packaging, the cell density is 85%-90% and the cells are evenly distributed with good condition. Change the cell medium to serum-free DMEM medium (1% HEPES and 1% P / S) one to two hours before transfection. Prepare the packaging mix: transfection reagent Lipo3000 (Thermo, L3000015): packaging plasmid pAAV-Rep-Cap (Addgene, #197565): vector plasmid pAV-CMV-TEAD1-P2A-GFP (Vigenebio, pAV100004-OE): helper plasmid helper (Addgene, #127694) = 15:2:2:1. Let it stand at room temperature for 30 min. Add the above standing liquid to HEK293T cells and shake well. Culture the cells in an incubator at 37°C and 5% CO2 for 72 h, then collect the virus.

[0054] 3. Resuspend the cells and collect them together with the medium into a 50 ml centrifuge tube. Centrifuge to separate the cell pellet and the supernatant. Transfer the medium supernatant to a new tube, precipitate PGE8000 overnight (add 2.33 g NaCl and 8.5 g PEG8000 per 100 mL), centrifuge at 3500 g and 4°C for 30 min the next day, discard the supernatant, and resuspend with PBS + 0.001% PF68. Resuspend the cell pellet with PBS + 0.001% PF68, freeze-thaw once, then add 5M NaCl and vortex well. Mix the two resuspended liquids, shake well and then sonicate. Centrifuge at 3500 g for 30 min and collect the supernatant.

[0055] 4. Prepare iodixanol with different concentrations (5%, 15%, 25%, 40%). Take an ultracentrifugation tube and layer different concentrations of iodixanol (40%, 25%, 15%, 5%) into it. Add the higher concentration first and then the lower concentration. Add the treated virus solution to the top layer. Ultracentrifuge to purify the virus. After centrifugation, collect the virus. Place the collected liquid in an ultrafiltration tube to concentrate the virus. Pipette the remaining liquid in the ultrafiltration tube repeatedly and aspirate it into a virus storage tube. Aspirate 10 μl of the virus solution for titer detection and specificity detection. Aspirate the virus solution and infect HEK293 cells with a 96-well plate. Observe the fluorescence 24 h and 48 h after infecting the cells.

[0056] (2) Construction of mouse skin wound healing model

[0057] Animal experiments were carried out in accordance with the guiding principles of the Animal Experiment Committee of Fudan University. The establishment process of the mouse skin injury repair model is as Figure 1 shown.

[0058] 1. Select 10-week-old male BALB / C mice, with 5 mice in each of the control group and the experimental group. Fast the mice for 8 - 12 hours before surgery, but allow free access to water. Weigh the mice and record the data. Anesthetize the mice using an intraperitoneal injection of an anesthetic (10 mg / mL tribromoethanol).

[0059] 2. Shave the hair on the back of the mice and disinfect the surgical area with 75% alcohol.

[0060] 3. Use a scalpel to cut a square wound with a diameter of 1 cm on the back of the mice. Pay attention to controlling the depth of the wound to avoid damaging important organs or tissues.

[0061] 4. Put the mice back into the cage and provide a suitable environment and diet. Regularly check the healing of the wound and record the wound healing process.

[0062] (3)Gene overexpression

[0063] According to the aforementioned experimental steps, construct a mouse skin injury repair model, and divide the mice into an experimental group (T1 - T5) and a control group (C1 - C5). Experimental group: Through subcutaneous injection at the damaged site, introduce 15 μl of AAV9-TEAD1 (5.38*10E13 vg / ml) virus solution into the skin tissue at the damaged site to achieve local high expression of TEAD1; Control group: Inject an equal volume of AAV9 virus solution without TEAD1. Measure the healing area of the mouse skin wound at intervals within two weeks. Within two weeks, we selected the 1st, 3rd, 7th, 10th, and 13th days for measurement respectively. The measurement results are as Figure 2 shown in A. The healing rate of the mouse skin wound in the experimental group (TEAD1-AAV) is significantly faster than that in the control group. According to the measurement data, on the 7th day, the wound healing rate of the experimental group (AAV9-TEAD1) is 58%, and that of the control group is 33%. The wound healing rate of the experimental group is 1.76 times higher than that of the control group. On the 10th day, the wound healing rate of the experimental group (AAV9-TEAD1) is 80%, and that of the control group is 61%. The wound healing rate of the experimental group is 1.3 times higher than that of the control group ( Figure 2 shown in B). In the first three days, the healing areas of the two groups of mice are basically the same. Starting from the 7th day, the healing area of the mouse skin wound in the TEAD1-AAV group is significantly higher than that in the control group. At 13 days, that is, at the end of wound healing, there is no significant difference in the healing area of the skin wounds of the two groups of mice (ns P>0.05; * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001). Use methods such as WB to compare the expression level of TEAD1 in the skin tissue of the mice. The above results indicate that overexpressing the TEAD1 gene based on the adeno-associated virus method can effectively promote the repair process of skin injury.

[0064] (4) Exosome characterization

[0065] CD63 is an exosome marker protein. To verify the effective secretion of exosomes and compare the secretion amount of exosomes, we characterized the expression of TEAD1 and CD63 in mouse skin wound healing tissue using immunofluorescence staining and IHC staining. First, we performed multiple immunofluorescence staining on the mouse skin tissue ( Figure 3 ), including TEAD1 antibody (abclonal, A6768) (green), CD63 antibody (proteintech, 25682-1-AP) (red) and DAPI (Biyuntian, C1002) (blue).

[0066] The skin tissue was collected and fixed with 4% paraformaldehyde to maintain the morphology and structure of the tissue. The fixed tissue was dehydrated and transparentized. The tissue was sliced ​​into thin slices with a thickness of 4 microns using a slicer and placed on a slide. Permeabilization was performed using a permeabilization solution (0.5% Triton X-100). Blocking was performed at room temperature using a blocking solution (1% BSA) to block nonspecific antigen epitopes and reduce background interference.

[0067] For immunofluorescence staining, drop appropriately diluted specific primary antibodies (TEAD1 antibody, abclonal, A6768, green; CD63, proteintech, 25682-1-AP, red) onto tissue sections and incubate overnight at 4°C. Wash sections with PBS to remove unbound primary antibodies. Drop appropriately diluted fluorescently labeled secondary antibodies (CD63 antibody, proteintech, SA00013-4) onto sections and incubate at 37°C in the dark for a period of time. Use DAPI staining solution (Biyuntian, C1002) to stain the sections, and wash the sections with PBS to remove excess DAPI staining solution. Add anti-fluorescence quencher onto the sections, and then cover with coverslips for sealing. Try to avoid bubbles when sealing. The results of immunofluorescence staining are as follows: Figure 3 As shown in A, in the immunofluorescence results of mouse skin, it can be seen that in the experimental group samples injected with TEAD1-AAV virus, TEAD1 (green) and exosome marker CD63 (red) were significantly higher than those in the control group. This shows that the use of adeno-associated virus method can overexpress the TEAD1 gene in damaged skin tissue of mice and promote the secretion of exosomes.

[0068] Furthermore, we extracted proteins from the healed mouse skin and performed WB verification of TEAD1 protein. The results showed that ( Figure 3In the skin tissues of 5 mice injected with TEAD1-AAV, the protein expression of TEAD1 was significantly upregulated (ns P>0.05; * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001).

[0069] (5) Evaluation of skin injury repair ability

[0070] Record the wound healing area at different time points, analyze the data using the t-test, compare the differences between different treatment groups, and evaluate the wound healing effect.

[0071] After the wound had basically healed, immunohistochemistry was used to detect the generation of new tissue and the level of inflammatory response to verify the promoting effect of TEAD1 overexpression on skin injury repair. The immunohistochemistry method refers to the description in (4) Exosome characterization. Among them, the expression of TEAD1 was characterized using the antibody TEAD1 antibody (abclonal, A6768), the skin endothelial differentiation marker was characterized using CD31 (proteintech, 28083-1-AP); the inflammation in skin tissues was characterized by the protein expression level of IL-6 (abclonal, A21264); the activity degree of cells in new tissue was expressed using the Ki67 index (proteintech, 28074-1-AP).

[0072] Finally, the healed skin tissues of mice were extracted and immunohistochemically characterized. In addition to the expression of TEAD1 protein and the exosome marker protein CD63, we also characterized the expression of the skin endothelial differentiation marker CD31, the inflammatory marker IL-6 (abclonal, A21264) in skin tissues, and the marker Ki67 (proteintech, 28074-1-AP) of cell activity in the newly formed tissues. During IHC staining, the diluted primary antibodies (TEAD1 antibody, abclonal, A6768; CD63, proteintech, 25682-1-AP; CD31, proteintech, 28083-1-AP; IL6, abclonal, A21264; KI67, proteintech, 28074-1-AP;) were dropped onto the sections, and the sections were placed in a wet box and incubated overnight at 4°C. The sections were rinsed 3 times with PBS for 5 minutes each time. The secondary antibody was added and incubated at room temperature for 60 minutes. The sections were rinsed 3 times with PBS for 5 minutes each time. The sections were immersed in DAB solution (Beyotime, P0203), and the progress of the color reaction was observed, usually for 1-5 minutes. At the end of the reaction, the reaction was stopped by rinsing with distilled water. The sections were immersed in hematoxylin solution and stained for 30 seconds to 1 minute to counterstain the cell nuclei. Rinsed with tap water and blued. Dehydrated with gradient alcohols of 70%, 95%, and 100%. Cleared with xylene for 5 minutes each time. A neutral gum was added and sealed with a coverslip. After the sealed slides were dried, the staining results were observed under a microscope. The positive signals should appear brown (DAB color development), and the background staining was blue (hematoxylin). The immunohistochemical staining results are as Figure 4 shown in A of, which respectively show the expression of TEAD1, the exosome marker CD63, the angiogenesis marker protein CD31, the inflammatory factor IL-6, and the cell viability marker Ki67 in the control group and the experimental group injected with TEAD1-AAV virus. From Figure 4 B of, it can be seen that in the skin tissues of mice injected with TEAD1-AAV, the expression of all 4 protein markers was significantly up-regulated, and only the IL-6 protein was down-regulated (ns P>0.05; * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001). These results indicate that after overexpressing TEAD1, not only the exosome secretion in skin tissues increases, the skin tissue cells become more active, the angiogenesis ability is enhanced, but the inflammation situation is reduced. This creates a good tissue microenvironment for skin injury repair.

[0073] The experimental results of the above embodiments showed that by achieving overexpression of TEAD1, multiple endogenous (the injured person himself) cells related to skin healing, such as keratinocytes, fibroblasts, endothelial cells, and mesenchymal stem cells, were significantly activated and / or promoted. To explore the effect of overexpression of TEAD1 on promoting exosome secretion on wound healing, primary mouse ADSCs (PMID: 20483444) were isolated, and ADSCs cells were transfected with TEAD1 knockout or overexpression vectors ( Figure 5 A). Subsequently, the supernatants of ADSCs in different treatment groups were collected, and exosomes in the control group, TEAD1 knockout, and overexpression groups were isolated respectively by a cell supernatant exosome extraction kit. By evaluating TEM images, it was found that exosomes in the control group, TEAD1 knockout, and overexpression groups contained typical exosome structures, including homogeneous, spherical, and membrane vesicles ( Figure 5 B). In addition, the size distribution of exosomes in different treatment groups was measured by NTA, and it was found that they were mainly between 50 and 120 nm. After knocking down TEAD1, the exosome secretion of ADSCs was significantly reduced; while when TEAD1 was overexpressed, the exosome secretion was significantly increased ( Figure 5 C). The surface markers of exosomes were detected by Western blotting. The results showed that exosomes in different treatment groups expressed exosome markers such as CD63, HSP70, ALIX, and Calnexin ( Figure 5 D). These data indicated that the morphology and particle size of exosomes in different treatment groups were similar, and overexpression of TEAD1 had no effect on the morphology and particle size of exosomes of ADSCs, but knocking out or overexpressing TEAD1 would have a significant impact on the yield of exosomes (ns P>0.05; * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001).

[0074] To study the effects of ADSCs and exosomes derived from ADSCs overexpressing TEAD1 on skin healing and angiogenesis, ADSCs and exosomes derived from ADSCs overexpressing TEAD1 were co-cultured with HaCaT (Cell Bank of the Chinese Academy of Sciences, SCSP-5091) and HUVEC (ATCC, PCS-100-013) respectively. CCK-8 assays were performed to evaluate the effects of exosomes derived from ADSCs with different treatments on the viability of HaCaT and HUVEC. The results showed that exosomes derived from ADSCs overexpressing TEAD1 could promote the proliferation of HaCaT and HUVEC. On the contrary, after knocking out TEAD1, the proliferation of HaCaT and HUVEC was inhibited ( Figure 6 A and Figure 6B). The effects of exosomes derived from different treatment groups on the migration of HaCaT were further evaluated. The results showed that, compared with the control group, exosomes derived from ADSCs overexpressing TEAD1 could promote the migration of HaCaT, while the migration of Hacat was inhibited by exosomes derived from ADSCs with TEAD1 knocked out ( Figure 6 C). Subsequently, the capillary network formation ability and cell proliferation ability of HUVECs treated differently were evaluated on Matrigel. The results showed that exosomes derived from ADSCs overexpressing TEAD1 enhanced the tube formation ability and cell proliferation ability of HUVECs ( Figure 7 A and Figure 7 B). It can be seen from Figure 6 and Figure 7 that under the regulation of TEAD1, a large amount of exosomes were secreted, and their synergistic effect accelerated the proliferation and migration ability of the keratinocyte Hacat, and promoted the angiogenesis of the vascular endothelial cell HUVEC, achieving an unexpectedly high-efficient tissue repair effect (ns P>0.05; * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001).

[0075] In the present invention, viral vectors such as AAV are used to deliver the TEAD1 gene for overexpression. Different from conventional gene therapy, it targets the skin tissue, does not involve genetic cells, has no genetic risk, and viral vectors such as AAV will not insert gene sequences into the genome, and will be gradually lost during the long-term proliferation of cells, without long-term risk, that is, it will not be expressed continuously. In addition, the use of a low-concentration virus solution in the present invention not only improves the safety of treatment, but also reduces side effects.

[0076] Compared with the existing exogenous mesenchymal stem cell exosome therapy for skin injury, the present invention does not require the preparation, preservation, transportation and transplantation of in vitro exosomes and / or mesenchymal stem cells, reducing the harsh conditions and high costs in the above processes; avoiding that exogenous exosomes may contain immunogenic proteins and RNAs, with a single source and composition; and does not require precise selection of cell types. It also solves the problems of high cell dosage (about 2.8 times that of the present invention) during the preparation of exogenous exosomes, limited action time, and difficulty in the directional migration and retention of exosomes.

[0077] It can be seen that the gene therapy of the present invention has strong pertinence, can directly repair or regulate specific TEAD genes, promote skin injury repair, and through a precise regulation tool, can precisely control the time and space of gene expression, provide personalized treatment plans, and meet the current demand of "precision medicine".

Claims

1. Use of a TEAD family transcription factor gene or a vector containing the same in the preparation of a drug for treating skin damage.

2. The use according to claim 1, characterized in that The vector includes a recombinant vector, and the backbone of the recombinant vector is preferably selected from any one of pAV-CMV-P2A-GFP, pAAV-U6-sgRNA-CMV-GFP, pAAV-CMV-nls-CasRx-P2A-GFP, pCMV-PE2-P2A-GFP, pAAV-EF1a-GFP and pAAV-CMV-GFP-P2A-Puro.

3. The use according to claim 1 or 2, characterized in that The vector also includes a delivery vector, which is preferably selected from a viral vector such as a lentiviral vector, an adenoviral vector, a retroviral vector or an adeno-associated viral vector; Preferably, the viral vector is an adeno-associated viral vector; More preferably, the adeno-associated virus vector is AAV9.

4. The use according to any one of claims 1 to 3, characterized in that The TEAD family transcription factor gene comprises a TEAD family transcription factor gene of mammalian origin; Preferably, the mammalian-derived TEAD family transcription factor gene is a mouse-derived TEAD family transcription factor gene.

5. The use according to claim 4, characterized in that The mouse TEAD family transcription factor genes include TEAD1, TEAD2, TEAD3 and TEAD4; For example, the TEAD family transcription factor gene is TEAD1.

6. The use according to claim 1, characterized in that The TEAD family transcription factor gene is TEAD1, and the amino acid sequence of TEAD1 is shown in SEQ ID NO: 1; the vector includes pAV-CMV-P2A-GFP as an expression vector skeleton, and AAV9 as a viral vector.

7. The use according to any one of claims 1 to 6, characterized in that The skin damage includes trauma and secondary damage; Preferably, the trauma includes mechanical trauma and burns; the secondary injury includes complications of skin injury in diabetic patients; More preferably, the skin injury is mechanical trauma.

8. The use according to any one of claims 1 to 7, characterized in that The vector promotes the secretion of exosomes from endogenous cells.

9. The use according to claim 8, characterized in that The endogenous cells are at least one of keratinocytes, fibroblasts, endothelial cells and mesenchymal stem cells; Preferably, the stem cells are mesenchymal stem cells; More preferably, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

10. The use according to any one of claims 1 to 9, characterized in that: The drug is a drug for local use.

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