Application of TEAD family transcription factor gene or vector containing TEAD family transcription factor gene in preparation of medicine for treating nerve injury

Abstract

The invention relates to application of a TEAD family transcription factor gene or a vector containing the TEAD family transcription factor gene in preparation of a medicine for treating nerve injury. The vector containing the TEAD family transcription factor gene is transfected to the spinal cord injury part, and the expression of the TEAD family transcription factor gene is induced under the proper culture condition, so that the yield of the cell exosome can be remarkably improved, and the spinal cord nerve injury repair is promoted. The method is not only suitable for various cell types, but also has important potential value in biomedical research and clinical application of nerve injury repair, metabolic regulation and the like.

Description

Technical Field

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

[0002] Spinal cord injury (SCI) is a serious neurological disease mainly caused by physical trauma to the spinal cord. According to the pathophysiological process, spinal cord injury can be divided into primary injury and secondary injury. In the initial stage, physical trauma to the spinal cord causes disruption of the blood-spinal cord barrier, resulting in hemorrhage and edema. Ischemia and reperfusion lead to necrosis and apoptosis of neurons and glial cells, axonal degeneration and demyelination in the lesion center, and trigger secondary injury including inflammatory responses. Excessive inflammatory responses and extravasation of infiltrating leukocytes spread from the lesion center to adjacent areas, resulting in apoptosis and aggravating tissue damage. The neuroinflammatory response mediated by microglia at the injury site may also amplify the secondary injury of spinal cord injury. Necrosis and apoptosis of neurons and axonal demyelination and loss at the lesion site lead to permanent neurological deficits. Therefore, axonal regeneration and formation of new connections in the injury area will contribute to promoting functional recovery after spinal cord injury.

[0003] The TEAD (Transcriptional Enhanced Associate Domain) transcription factor family is a transcription factor family composed of four members, including TEAD1, TEAD2, TEAD3, and TEAD4, which have high structural and functional similarities. These transcription factors regulate the expression of a series of genes by interacting with other proteins (especially YAP / TAZ), and thus affect processes such as cell proliferation, differentiation, and migration. Currently, it is commonly used in anti-tumor research, but there is no report on promoting nerve 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, the use in the preparation of a medicament for treating nerve injury.

[0005] The present invention directly introduces genes of the TEAD family into damaged site cells, and realizes long-term gene expression through viral vectors (such as adeno-associated virus (AAV), lentivirus, etc.), enhances axon regeneration and nerve repair functions, and provides a non-invasive and highly efficient gene therapy approach for nerve injury repair. Compared with the prior art, this method has significant advantages such as less virus usage, low production, transportation and storage costs, simple operation, strong targeting, extremely low immunogenicity, no change in the genome, long sustainable time for single treatment and small side effects (for example: it will not trigger immune response and out-of-control gene expression), and is expected to be widely applied to the treatment of various nerve injuries, including traumatic brain injury, spinal cord injury and neurodegenerative diseases, etc.

[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 nerve injury.

[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, pCMV-PE2-P2A-GFP, pAAV-CMV-nls-CasRx-P2A-GFP, pAAV-EF1a-GFP and 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, and the delivery vector 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 in the present invention above.

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

[0012] In some more preferred embodiments, the adeno-associated virus 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 neural tissues, thereby ensuring that the overexpression vector constructed in the present invention can be effectively delivered to neural cells and achieve stable expression of the target gene.

[0013] In some embodiments, the TEAD family transcription factor gene comprises a TEAD family transcription factor gene of mammalian origin.

[0014] In some preferred embodiments, the TEAD family transcription factor gene of mammalian origin is a murine TEAD family transcription factor gene.

[0015] In some embodiments, the murine TEAD family transcription factor gene comprises 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 virus vector is selected as AAV9.

[0018] As used in the present invention, "nerve injury" refers to a pathological state in which the nerve structure (such as axons, myelin sheaths, or nerve bundles) or function is damaged due to mechanical, chemical, biological and other factors, resulting in sensory, motor or autonomic nerve dysfunction, which can be caused by trauma (such as laceration, fracture), metabolic abnormalities (such as diabetes), infections (such as meningitis), poisoning (heavy metals or drugs), etc. Nerve injury can be divided into central nerve injury and peripheral nerve injury. The central nerve injury involves the brain and spinal cord, such as traumatic brain injury, stroke or multiple sclerosis, often resulting in irreversible dysfunction; the peripheral nerve injury is, for example, brachial plexus injury or diabetic neuropathy.

[0019] In some embodiments, the nerve injury includes one or more of acute injury, chronic injury, inflammatory injury, and ischemic injury;

[0020] and / or, the nerve injury includes peripheral nerve injury and central nerve injury.

[0021] In some preferred embodiments, the acute injury is traumatic spinal cord compression; the chronic injury is spinal cord damage caused by degenerative spinal diseases; the inflammatory injury is multiple sclerosis or an autoimmune disease; the ischemic injury is an injury caused by insufficient blood supply to the spinal cord;

[0022] and / or, the central nerve injury includes cranial nerve injury and spinal nerve injury.

[0023] In some embodiments, the carrier promotes the secretion of exosomes by endogenous cells.

[0024] In some embodiments, the endogenous cells are at least one of neural stem cells, glial cells, inflammatory cells, and mesenchymal stem cells.

[0025] In some preferred embodiments, the mesenchymal stem cells are spinal cord mesenchymal stem cells.

[0026] In some embodiments, the drug is a topically used drug.

[0027] On the basis of conforming to the common knowledge in the art, the above-mentioned 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 nerve injuries. Description of the Drawings

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

[0032] Figure 2 For the gait analysis at the eighth week of mouse spinal cord injury repair. A: The footprint information of the mouse walking captured by the video acquisition system of the gait analyzer; B: The mouse gait analysis result.

[0033] Figure 3 For the immunofluorescence staining of mouse spinal cord injury repair. A: Comparison of multiplex immunofluorescence staining of spinal cord samples of the control group and the TEAD1 overexpression group of mice with spinal cord injury repair, including TEAD1 (green), CD63 (purple), CD90 (orange), NeuN (red), and DAPI (blue); B: Statistical chart of the proportion of NeuN at the injury site in mice with spinal cord injury.

[0034] Figure 4 Overexpression of the TEAD1 gene promotes the secretion of exosomes by bone marrow mesenchymal stem cells. A: Transmission electron microscopy was used to observe exosomes secreted by BMSCs treated differently; B: Western blotting was used to identify exosome surface markers; C: Results of nanoparticle tracking analysis of exosomes secreted by BMSCs treated differently.

[0035] Figure 5 This shows the effect of exosomes secreted by BMSCs overexpressing TEAD1 on nerve cells HT22. A: Transwell assay was used to detect the effect of exosomes secreted by BMSCs treated differently on the migration of nerve cells HT22; B: Scratch assay was used to detect the effect of exosomes secreted by BMSCs treated differently on the migration of nerve cells HT22. Specific implementation manners

[0036] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. For those not specifying specific techniques or conditions 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 from regular channel merchants.

[0037] Example 1 Use of adeno-associated virus AAV9 to overexpress TEAD1 to promote the repair of spinal cord injury in mice

[0038] (1) Construction of overexpression vector

[0039] Gene name: TEAD1; Species: mouse; Gene size: 1356 bp; NM number: NM_001166584.2.

[0040] The mouse TEAD1 gene was synthesized by total gene synthesis. A kozak sequence GCCACC was added before the target gene, a flag tag was added to the C-terminus of the target gene, and the target gene and GFP were expressed non-fusion through P2A. The target gene and the vector pAV-CMV-P2A-GFP (Vigenebio, pAV100004-OE) were cut with ASISI / MluI, and then the target gene and the vector were recovered by gel extraction. The target gene was ligated to the vector using T4 ligase. Transformation was carried out using DH5α competent cells. Colony picking was performed and enzyme digestion verification was carried out. The vector with correct enzyme digestion was selected for sequencing. Sequencing primers: CMV-seq-F: 5'-CGCAAATGGGCGGTAGGCGTG-3', EGFP-SEQ-R: 5'-CTCGTCGTCTTGTAGTTCCCGT-3'. The correct plasmid was extracted using an endotoxin removal kit (Tiangen, DP117) to obtain the TEAD1 gene overexpression vector pAV-CMV-TEAD1, and then adenovirus packaging was carried out.

[0041] Further, the protein amino acid sequence of TEAD1 is SEQ ID NO: 1:

[0042] MEPSSWSGSESPAENMERMSDSADKPIDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKSRDFHSKLKDQTAKDKALQHMAAMSSAQIVSATAIHNKLGLPGIPRPTFPGAPGFWPGMIQTGQPGSSQDVKPFVQQAYPIQPAVTAPIPGFEPASAPAPSVPAWQGRSIGTTKLRLVEFSAFLEQQRDPDSYNKHLFVHIGHANHSYSDPLLESVDIRQIYDKFPEKKGGLKELFGKGPQNAFFLVKFWADLNCNIQDDAGAFYGVTSQYESSENMTVTCSTKVCSFGKQVVEKVETEYARFENGRFVYRINRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILLVVTNRDTQETLLCMACVFEVSNSEHGAQHHIYRLVKD

[0043] Further, the protein amino acid sequence of TEAD2 is SEQ ID NO: 2:

[0044] MGEPRAGAALDDGSGWTGSEEGSEEGTGGSEGAGGDGGPDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKSREIQSKLKDQVSKDKAFQTMATMSSAQLISAPSLQAKLGPTGPQASELFQFWSGGSGPPWNVPDVKPFSQTPFTLSLTPPSTDLPGYEPPQALSPLPPPTPSPPAWQARGLGTARLQLVEFSAFVEPPDAVDSYQRHLFVHISQHCPSPGAPPLESVDVRQIYDKFPEKKGGLRELYDRGPPHAFFLVKFWADLNWGPSGEEAGAGGSISSGGFYGVSSQYESLEHMTLTCSSKVCSFGKQVVEKVETERAQLEDGRFVYRLLRSPMCEYLVNFLHKLRQLPERYMMNSVLENFTILQVVTNRDTQELLLCTAYVFEVSTSERGAQHHIYRLVRD

[0045] Furthermore, the protein amino acid sequence of TEAD3 is SEQ ID NO: 3:

[0046] MASNSWNASSSPGEAREDGPEGLDKGLDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARKKVREYQVGIKAMNLDQVSKDKALQSMASMSSAQIVSASVLQNKFSPPSPLPQAVFSTSSRFWSSPPLLGQQPGPSQDIKPFAQPAYPIQPPLPPTLSSYEPLAPLPSAAASVPVWQDRTIASSRLRLLEYSAFMEVQRDPDTYSKHLFVHIGQTNPAFSDPPLEAVDVRQIYDKFPEKKGGLKELYEKGPPNAFFLVKFWADLNSTIQEGPGAFYGVSSQYSSADSMTISVSTKVCSFGKQVVEKVETEYARLENGRFVYRIHRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTSRDSQETLLVIAFVFEVSTSEHGAQHHVYKLVKD

[0047] Furthermore, the protein amino acid sequence of TEAD4 is SEQ ID NO: 4:

[0048] MEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKAREIQAKLKDQAAKDKALQSMAAMSSAQIISATAFHSSMALARGPGRPAVSGFWQGALPGQAGTSHDVKPFSQQTYAVQPPLPLPGFESPAGPAPSPSAPPAPPWQGRSVASSKLWMLEFSAFLEQQQDPDTYNKHLFVHIGQSSPSYSDPYLEAVDIRQIYDKFPEKKGGLKDLFERGPSNAFFLVKFWADLNTNIEDEGSSFYGVSSQYESPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHGAQHHIYRLVKE

[0049] In the above description of the present invention, the protein sequence corresponding to the murine gene is used. The TEAD family transcription factors are conserved among different species, and the protein sequences corresponding to any homologous genes of the TEAD family fall within the scope of the claims of this patent.

[0050] (2) Packaging of AAV9-type adeno-associated virus overexpressing the target gene

[0051] 1. Prepare HEK 293T cells: Seed HEK 293T cells one day in advance. When packaging, the cell density is 85%-90% and the cells are evenly distributed with good condition.

[0052] 2. Packaging virus: Replace with serum-free DMEM medium (1% HEPES and 1% P / S) one to two hours before transfection. Prepare the packaging mix according to the ratio of transfection reagent Lipo3000 (Thermo, L3000015): packaging plasmid pAAV-Rep-Cap (Addgene, #197565): vector plasmid pAV-CMV-TEAD1-P2A-GFP (Vigene Biosciences, pAV100004-OE): helper plasmid helper (Addgene, #127694) = 15:2:2:1, and let it stand at room temperature for 30 min. Add the above standing liquid to the HEK293T cell medium and mark it, then shake well after adding. Incubate the cells in an incubator at 37 °C and 5% CO 2 Collect the virus after culturing for 72 h.

[0053] 3. Virus collection: Blow up 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 with PGE8000 overnight (add 2.33 g NaCl + 8.5 g PEG8000 per 100 mL), centrifuge at 3500 g at 4 °C for 30 min the next day, and discard the supernatant. Resuspend the cell pellet with PBS + 0.001% PF68, freeze-thaw once, then add 5 M NaCl and vortex well. Mix the two resuspended liquids, shake well and then sonicate until not viscous. When sonicating, the probe should be washed successively with 84 disinfectant, 75% alcohol, and water between different samples, sonicate for 30 s and stop for 5 - 10 s. Depending on the viscosity of the samples, sonicate the AMPL30% 3 - 4 times and the AMPL20% once. Centrifuge the sonicated liquid at 3500 g for 30 min and collect the supernatant.

[0054] 4. Purification: Density gradient centrifugation with iodixanol. Prepare iodixanol with different concentrations (5%, 15%, 25%, 40%). Take an ultracentrifugation tube and layer by layer add iodixanol with different concentrations (40%, 25%, 15%, 5%). Add the higher concentration first and then the lower concentration, and push the iodixanol with different concentrations slowly to avoid mixing the higher concentration layer with the lower concentration layer. Add the treated virus solution to the top layer. Ultracentrifuge to purify the virus.

[0055] 5. Concentration: After centrifugation, collect the virus. Place the collected liquid in an ultrafiltration tube to concentrate the virus. Repeatedly pipette the remaining liquid in the ultrafiltration tube and aspirate it into a virus storage tube, supplement the volume with virus storage solution, and label the name and date. Aspirate 10 μl of the virus solution for titer detection and specificity detection to obtain AAV9-TEAD1.

[0056] (3) Construction of mouse spinal cord injury model and injection administration

[0057] Animal experiments were conducted in accordance with the guiding principles of the Animal Experiment Committee of Fudan University. After 10-week-old male C57BL / 6 mice were fasted for 6 hours, they were intraperitoneally injected with a ready-to-use 10 mg / mL tribromoethanol anesthetic at a dose of 25 mL / kg. Subsequently, the back of the mice was depilated, the skin was incised, and after the paravertebral muscles were dissected, a T13 laminectomy was performed. A control group (Sham group) and a spinal cord injury group (SCI group) were set up. The paravertebral muscle tissues of mice in each group were isolated. In the spinal cord injury group, the exposed spinal cord at the T13 segment was compressed with a microvascular clamp for 60 seconds, while in the control group, only laminectomy was performed on the experimental mice without spinal cord compression injury. AAV9-TEAD1 was introduced into the damaged spinal cord tissue by in-situ injection to achieve local overexpression of TEAD1. The control group was injected with an equal volume of AAV9 virus solution without TEAD1. Each spinal cord injured mouse was injected with 15 μl of the virus solution (5.38*10E13 vg / ml) ( Figure 1 ). Subsequently, the muscles and skin were sutured, and the operated mice were rewarmed and revived under an electric heating lamp. All revived mice were placed in a cage and allowed to move freely. The loss of lower limb motor ability in the mice was observed and determined as successful modeling. Disinfected food and purified water were provided to restore their strength. The postoperative treatment procedure was intramuscular injection of penicillin solution once a day for 3 consecutive days. For mice that could not urinate autonomously after spinal cord injury, artificial assisted urination was required every day. Squeezing the bladder to passively urinate is the simplest and most feasible means to relieve urinary retention. The bladder of the mice was squeezed to promote urination, and artificial urination was performed twice a day. The mental state, diet, urination and defecation conditions of the mice were closely observed, as well as the presence of limb edema and pressure sores, and the presence of secretions in the urinary system. These nursing measures helped to maintain the basic physiological functions of paralyzed mice, reduce complications, and improve their quality of life.

[0058] (4)Gait analysis of mice to evaluate the ability of motor function recovery

[0059] At the fourth week and the eighth week after modeling, the behavioral function recovery of the mice after drug administration was detected by instruments such as gait analysis instruments ( Figure 2 ). Gait analysis experiment is a method to evaluate the motor function by recording and analyzing parameters such as the posture, speed and stride length of an organism when walking. Experimental mice were selected and trained to ensure that they adapted to the experimental environment and were familiar with the runway. The gait analyzer (Clever sys, Treadmill) was adjusted, including the camera and the runway, to ensure the normal operation of the equipment. The mice were placed in the induction box to naturally enter the runway, and the video acquisition system was started to capture the footprint information of the mice walking ( Figure 2 A). Ensure that the mice walked at a constant speed on the runway until the data collection was completed. The gait analysis software TreadScan was used to process the collected video data to identify and analyze various parameters of the mouse gait. The results of the gait analysis data are asFigure 2 As shown in Figure B of , the imprint lengths of the left and right hindlimbs of the mice in the TEAD1-AAV group at the 8th week were higher. In the experimental group, i.e., the OE-TEAD1 group (mean+sd), the footprint length of the right hindlimb was 4.831±1.045, and the footprint length of the left hindlimb was 4.956±1.266. In the control group (mean+sd), the footprint length of the right hindlimb was 1.346±0.875, and the footprint length of the left hindlimb was 1.363±0.906. The imprint length of the left hindlimb in the experimental group was 3.64 times that of the control group (P=0.0009), and the imprint length of the right hindlimb in the experimental group was 3.59 times that of the control group (P=0.0004). This reflects that the hindlimbs of the mice in the TEAD1-AAV group had a better degree of extension during walking. In addition, there were significant differences in the imprint lengths of the left and right hindlimbs of the mice in the TEAD1-AAV group at the 8th week compared with the control group (ns P>0.05; * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001). This indicates that after treatment with TEAD1-AAV9, the motor function of the mice was well restored. The above results show that overexpressing the TEAD1 gene based on the adeno-associated virus method can effectively promote the repair of spinal cord nerve injury and the recovery process of motor function.

[0060] (5)Immunofluorescence staining of related proteins in spinal cord samples

[0061] At the fourth and eighth weeks, spinal cord samples of 3 mice were taken for multiplex immunofluorescence staining, including TEAD1 antibody (abclonal, A6768) (green), CD63 (proteintech, 25682-1-AP) (purple), CD90 (proteintech, 66766-1-Ig) (orange), NeuN (proteintech, 66836) (red), and DAPI (Beyotime, C1002) (blue).

[0062] After the spinal cord injury was basically healed after 8 weeks, immunofluorescence was used to detect neuronal regeneration and exosome secretion levels to verify the promoting effect of TEAD1 overexpression on spinal cord injury repair. TEAD1 antibody (abclonal, A6768, green) was used to detect endogenous TEAD1 expression in spinal cord tissue to characterize the infection efficiency and expression of TEAD1-AAV9; CD63 (proteintech, 25682-1-AP, purple) was used to characterize exosome secretion markers; CD90 (proteintech, 66766-1-Ig, orange) was used to characterize nerve cells; NeuN (proteintech, 66836, red) was used to evaluate the repair of nerve injury and the effect of nerve regeneration therapy. According to the analysis results, the experimental data were sorted and the experimental conclusions were drawn.

[0063] The steps of immunofluorescence staining are as follows:

[0064] The whole mouse spinal cord tissue sample was peeled off and fixed with 4% paraformaldehyde to maintain the morphology and structure of the tissue. The fixed tissue was dehydrated and transparentized. The tissue was cut 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 solution (1% BSA) was used for room temperature blocking to block nonspecific antigen epitopes and reduce background interference.

[0065] For immunofluorescence staining, appropriately diluted specific primary antibodies (TEAD1, abclonal, A6768; CD63, proteintech, 25682-1-AP; CD90, proteintech, 66766-1-Ig; NeuN, proteintech, 66836) were added to the tissue sections and incubated overnight at 4°C. The sections were washed with PBS to remove unbound primary antibodies. Appropriately diluted fluorescently labeled secondary antibodies (proteintech, SA00013-4) were added to the sections and incubated at 37°C in the dark for a period of time. The sections were stained with DAPI staining solution (Biyuntian, C1002) and washed with PBS to remove excess DAPI staining solution. Anti-fluorescence quencher was added to the sections and then coverslipped for sealing. Avoid bubbles when sealing.

[0066] Immunofluorescence staining results Figure 3As shown in A of , in the spinal cord of the experimental group of mice injected with TEAD1-AAV, TEAD1 (green) and CD63 (purple) were significantly more than those in the control group. The extension distance of NeuN (red) neurons with spinal cord injury in the TEAD1-AAV group at the fourth week was also higher than that in the control group. The neurons in the TEAD1-AAV group at the eighth week had better recovery compared with the fourth week. The statistical chart of the proportion of the NeuN injury site is as shown in Figure 3 B of . As can be seen from the figure, the proportion of NeuN in the injury site in the experimental group at the fourth week and the eighth week was ( Figure 3 The left figure of B of is the data of the eighth week, and the right figure is the data of the fourth week). The proportion of NeuN in the injury site in the control group at the eighth week was 32.7%, and the proportion of NeuN in the injury site in the experimental group (TEAD1 group) was 9.8%, which decreased significantly by 22.9% (P = 0.0005, ns P>0.05; * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001). This indicates that using the adeno-associated virus method to overexpress the TEAD1 gene in the mouse spinal cord and promote the secretion of exosomes can further promote the injury repair and axon regeneration of spinal cord neurons.

[0067] To further verify the effect of overexpressing TEAD1 on the exosome secretion of spinal cord-derived mesenchymal stem cells, exosomes of bone marrow mesenchymal stem cells (BMSCs) (PMCID: PMC9714364) in different treatment groups were collected for evaluation. By evaluating TEM images, it was found that the exosomes of the control group and the exosomes of the TEAD1 knockout and overexpression groups contained typical exosome structures, including homogeneous, spherical and membrane vesicles ( Figure 4 A of ). 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 100 nm. After knocking down TEAD1, the exosome secretion of BMSCs decreased significantly; while when TEAD1 was overexpressed, the exosome secretion increased significantly ( Figure 4 C of ). 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 4 B of ). These data indicate that the morphology and particle size of exosomes in different treatment groups are similar. Overexpressing TEAD1 has no effect on the characterization of exosomes of BMSCs, but knocking out or overexpressing TEAD1 will have a significant impact on the yield of exosomes.

[0068] To further clarify the effect of exosomes secreted by BMSCs overexpressing TEAD1 on nerve cells, we co-cultured BMSCs and exosomes derived from BMSCs with knocked out or overexpressed TEAD1 with HT22 cells (Cell Bank of the Chinese Academy of Sciences, SCSP-5419). We found that compared with the control group, exosomes derived from BMSCs overexpressing TEAD1 could promote the migration of HT22, while the migration of HT22 was inhibited by exosomes derived from BMSCs with knocked out TEAD1 ( Figure 5 A of Figure 5 B of). These results further indicate that exosomes secreted by BMSCs overexpressing TEAD1 may affect spinal cord injury repair by promoting neuron regeneration.

[0069] 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 nerve spinal cord 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 gradually be 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.

[0070] Compared with the existing treatment of nerve injury with exogenous mesenchymal stem cell exosomes, 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 the possible presence of immunogenic proteins and RNAs in exogenous exosomes, with a single source and composition; and not requiring precise selection of cell types. It also solves the problems of high cell usage (about 2.88 times that of the present invention), limited action time, and difficulty in the directional migration and retention of exosomes when preparing exogenous exosomes.

[0071] Thus, it can be seen that the gene therapy of the present invention has strong specificity, can directly repair or regulate specific TEAD genes, promote spinal cord nerve injury repair, and through precise regulation tools, can precisely control the time and space of gene expression, providing personalized treatment plans to meet the current demand for "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 nerve 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, pCMV-PE2-P2A-GFP, pAAV-CMV-nls-CasRx-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, and the delivery vector is preferably 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; For example, 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 backbone; and AAV9 as a viral vector.

7. The use according to any one of claims 1 to 6, characterized in that The nerve injury includes one or more of acute injury, chronic injury, inflammatory injury and ischemic injury; And / or, the nerve damage includes peripheral nerve damage and central nerve damage; Preferably, the acute injury is traumatic spinal cord compression; the chronic injury is spinal cord damage caused by degenerative spinal disease; the inflammatory injury is multiple sclerosis or autoimmune disease; the ischemic injury is injury caused by insufficient blood supply to the spinal cord; And / or, the central nervous system injury includes cranial nerve injury and spinal nerve injury.

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 neural stem cells, glial cells, inflammatory cells and mesenchymal stem cells; Preferably, the mesenchymal stem cells are spinal cord mesenchymal stem cells.

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

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