Codon-optimized TWNK gene, adeno-associated virus vector and application thereof

Abstract

The invention discloses a codon-optimized TWNK gene, an adeno-associated virus vector and application of the codon-optimized TWNK gene, a TWNK-OPT gene obtained by performing codon optimization on a TWNK-WT gene is recombined with an adeno-associated virus expression vector to obtain pAAV-smCBA-TWNK-OPT recombinant plasmid and rAAV-smCBA-TWNK-OPT recombinant virus, so that the expression quantity of the TWNK gene and protein is increased, and the expression quantity of the TWNK gene and the protein is increased; the cost of gene therapy drugs and adverse reactions such as immune reaction caused by high-dose virus injection can be reduced, and the method can be used for treating Perrault syndrome.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a codon-optimized TWNK gene, an adeno-associated virus vector and their applications. Background Art

[0002] The TWNK gene is located on chromosome 10, in the second reading frame, and is thus also called C10orf2; the protein encoded by it is scattered in the cytoplasm, like twinkling stars, so it is called the TWNK protein; it was first discovered by Spelbrink and his colleagues in 2001, and was identified as a causal mutation for autosomal dominant progressive external ophthalmoplegia (adPEO), also known as the PEO1 gene. The TWNK protein encoded by it is an mtDNA replication helicase, which is essential for maintaining the integrity and replication process of mitochondrial DNA.

[0003] Perrault syndrome (PRLTS) is mainly characterized by progressive sensorineural hearing loss (SNHL) and ovarian dysfunction in women, and some patients may be accompanied by neurological symptoms. PRLTS is an autosomal recessive genetic disease. At present, PRLTS has been identified as being related to eight gene mutations, namely TWNK, CLPP, ERAL1, GGPS1, HARS2, HSD17B4, LARS2, RMND1, and TWNK; clinically, PRLTS is divided into type I (static and without neurological involvement) and type II (accompanied by progressive neurological diseases); studies on the genotype-phenotype correlation of the reported cases so far have shown that patients with Perrault syndrome caused by TWNK mutations will present neurological signs in adulthood, with progressive sensory and motor peripheral neuropathy, limited eye movement, nystagmus, cerebellar ataxia and mild intellectual disability.

[0004] At present, the treatment methods for PRLTS are still very limited: for the symptoms of ovarian dysplasia in women, hormone replacement therapy is mainly used for personalized treatment; for the hearing-impaired, special educational resources, hearing aids and tactile sensing devices are mainly adopted; cochlear implantation is performed on children over 12 months old with severe and profound hearing loss. With the continuous development and improvement of gene therapy technology, the autosomal recessive genetic disease PRLTS can be alleviated and treated by replacing normal genes or compensating for mutant genes.

[0005] Chinese Patent CN118703570A discloses an rAAV vector for treating TWNK-mutated PRLTS and its application. It uses the TWNK gene to prepare an rAAV vector, and then transfects the vector containing the TWNK gene into cells to compensate and correct the TWNK gene mutation sites in brain-derived organs, so that it can be used to prepare drugs for treating Perrault syndrome, solving the problem of the lack of precise and effective gene therapy methods for Perrault syndrome caused by TWNK gene mutations currently.

[0006] Recently, we further found that there is still a large room for improvement in the expression efficiency of the wild-type TWNK gene. Therefore, it results in high costs of gene therapy drugs and high risks of side effects such as immune reactions. In addition, due to codon bias and each host cell having its own codon usage frequency table, when using AAV virus to deliver the functional TWNK gene for heterologous expression, there is often a deviation between the codons of the heterologous mRNA and the optimal codon usage frequency of the host cell. It is possible that the abundance of tRNA corresponding to the codons in the heterologous protein mRNA sequence is very low in the host cell, resulting in ribosome stalling on the mRNA and even causing mRNA degradation, thus affecting protein translation efficiency. Therefore, in the process of treating diseases with gene therapy, not only the targeting and expression efficiency of the delivered viral vector need to be improved, but also the yield and quality of the translation products of the functional gene need to be carefully considered. It can be seen that finding an mRNA sequence with a sufficiently stable secondary structure, high translation efficiency, and optimized codons is crucial for the treatment of Perrault syndrome. Summary of the Invention

[0007] Aiming at the above technical problems, the present invention provides a codon-optimized TWNK gene, an adeno-associated virus vector and its application, which improve the expression levels of the TWNK gene and protein. After preparing the adeno-associated virus vector or adeno-associated virus therefrom, it can reduce the cost of Perrault syndrome gene therapy drugs and reduce adverse reactions such as immune reactions caused by high-dose virus injection.

[0008] To achieve the above object, the present invention provides a codon-optimized TWNK gene, and the codon-optimized TWNK gene is TWNK-OPT, with a nucleotide sequence of SEQ ID NO:2 and an amino acid sequence of SEQ ID NO:3.

[0009] Preferably, the TWNK gene sequence has at least 78.1% homology with the wild-type TWNK gene sequence.

[0010] The present invention also provides a recombinant adeno-associated virus vector containing a codon-optimized TWNK gene. The adeno-associated virus vector comprises an adeno-associated virus genome containing the smCBA promoter and a codon-optimized TWNK gene sequence; the codon-optimized TWNK gene sequence is the nucleotide sequence shown in SEQ ID NO: 2.

[0011] The present invention also provides a method for constructing a recombinant adeno-associated virus vector containing a codon-optimized TWNK gene, which comprises the following steps: (1) Optimize the codons of the wild-type TWNK gene, and add protective bases and double digestion sites to the 5' end and 3' end of the sequence respectively, synthesize the TWNK-OPT gene fragment, and perform PCR amplification and recovery; (2) Double digest the pAAV-smCBA-luc plasmid and the TWNK-OPT gene, and ligate them; (3) Transform the ligation product to obtain a plasmid, and perform double digestion detection and sequencing to obtain the recombinant adeno-associated virus expression vector pAAV-smCBA-TWNK-OPT.

[0012] Preferably, the double digestion sites in step (1) are HindⅢ and XhoI.

[0013] Preferably, the enzyme used for ligation in step (2) is T4 DNA ligase, and the ligation condition is ligation at 16°C for 16 h.

[0014] The present invention also provides a method for preparing a recombinant adeno-associated virus containing a codon-optimized TWNK gene. Co-transfect the recombinant adeno-associated virus expression vector pAAV-smCBA-TWNK-OPT, pHelper, and pAAV2 into cells, collect the supernatant and cells, and after purification, obtain the recombinant adeno-associated virus rAAV-smCBA-TWNK-OPT.

[0015] Preferably, the cells are any one of HEK293T cells, HEK293 cells, HeLa cells, Sf9 / Sf21 insect cells, and BHK cells.

[0016] The present invention also provides an application of a recombinant adeno-associated virus containing a codon-optimized TWNK gene, and the application is for the preparation of a drug for treating Perrault syndrome.

[0017] Preferably, the Perrault syndrome is caused by a TWNK gene mutation.

[0018] The beneficial effects of the present invention are as follows: 1. Provided is a codon-optimized TWNK-OPT gene that has at least 78.1% homology with the wild-type TWNK gene, which improves the expression levels of the TWNK gene and the TWNK protein.

[0019] 2. Recombinant the TWNK-OPT with an adeno-associated virus vector to obtain the recombinant plasmid pAAV-smCBA-TWNK-OPT, and then package and purify to obtain the recombinant virus rAAV2-smCBA-TWNK-OPT. This recombinant virus vector expresses the TWNK protein in cells, and its expression level is significantly higher than that of rAAV2-smCBA-TWNK-WT. It can be used to prepare gene therapy drugs for the treatment of Perrault syndrome, which is beneficial to reducing the cost of drugs and reducing adverse reactions such as immune responses caused by high-dose virus injection. Description of the Drawings

[0020] Figure 1 Sequencing result of the recombinant plasmid pAAV-smCBA-TWNK-OPT in Example 1.

[0021] Figure 2 Schematic diagram of the construction of the pAAV-CBA-TWNK-OPT vector in Example 1.

[0022] Figure 3 Protein expression gel electrophoresis diagram after transfection of the recombinant virus vector into cells in Example 2.

[0023] Figure 4 Immunofluorescence detection diagram of neural stem cells in Example 5.

[0024] Figure 5 Sequencing result diagram of the TWNK gene mutation site in Example 6. In the figure, A is c.794G>A P.Arg265His; B is c.1181G>A P.Arg394His.

[0025] Figure 6 Histogram of mitochondrial genome copy number in neural stem cells after treatment with the recombinant virus in Example 7.

[0026] Figure 7 Protein in vitro expression gel electrophoresis diagram after infection of cells with the recombinant virus in Example 8. Detailed Embodiments

[0027] The technical solution of the present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the following embodiments are only the preferred embodiments of the present invention and should not be construed as a limitation of the present invention. The protection scope of the present invention shall be subject to the content recorded in the claims. Any modifications and substitutions made by those skilled in the art to the technical solution of the present invention without creative work fall within the protection scope of the present invention.

[0028] Plasmids and cells: HindⅢ, xholⅠ: Takara; vector1212, pAAV-smCBA-Luciferase plasmid, AAV2 capsid plasmid, pHelper helper virus plasmid, HEK293T cells and iPS cells: provided by Hubei Key Laboratory of Tumor Immunology and Microenvironment; Reagents and culture media: T4 ligase: Takara Plasmid miniprep kit, protein Marker: Nanjing Novoprotein Biological Technology Co., Ltd.; DMEM culture medium, PVDF membrane: Zechuan Biotechnology Co., Ltd.; Cytokey transfection reagent: Zhongji Biotechnology Co., Ltd.; PBS: Weigh 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO 4 , 0.24 g of KH 2 PO 4 Dissolve in 800 mL of distilled water, adjust the solution to pH 7.4 with HCl, and finally make up the volume to 1 L with distilled water. Store at 4 °C after autoclaving; Trypsin digestion solution: Zhongke Maichen Technology Co., Ltd. Fetal bovine serum: Biological Industries; Protease inhibitor, flag primary antibody: Bioworld Technology Co., Ltd. RIPA lysis buffer: Beyotime Biotechnology Co., Ltd.; 30% acrylamide gel solution: Dissolve 29 g of acrylamide and 1 g of methylenebisacrylamide in a total volume of 60 mL of water, heat to 37 °C to dissolve, and make up the volume to 100 mL with water; filter and sterilize with a 0.45 μm filter, and store in a brown bottle at room temperature; 1M Tris-HCl pH 6.8: Weigh 121.1 g of Tris and place it in a 1000 mL beaker. Add about 800 mL of deionized water, stir well to dissolve, add concentrated HCl to adjust the pH value to 6.8, make up the volume of the solution to 1000 mL, autoclave at high temperature and high pressure, and store at room temperature; 10% SDS: Dissolve 100 g of SDS (Sodium dodecyl sulfate crystals) in 900 mL of purified water, heat to 68 °C to dissolve the SDS crystals, add purified water to a volume of 1 L, dispense and store at room temperature; 10% APS: Dissolve 0.5 g of APS in 5 mL of deionized water and store at 4 °C in the dark; TEMED: Thermo Fisher Scientific; Electrophoresis buffer: Dissolve 3.03 g of Tris, 14.4 g of Glycine, and 1 g of SDS in 1000 ml of deionized water and stir well to dissolve; 1×TBST buffer solution: 50 mL of Tris-HCL (1 M, pH 7.5), 8 g of NaCl, 0.2 g of KCl, 0.5 mL of Tween, make up to 1 L with distilled water; 1% BSA: Dissolve 1 g of BSA in 100 ml of 5% TBST solution and stir well to dissolve; Secondary antibody: Sevier (goat anti-mouse) Universal nuclease: YEASEN Biotech Co., Ltd. Iodixanol, iPS cell digestion solution, iPS cell culture medium, neural stem cell induction medium, neural stem cell culture medium: Stem cell; Y-27632: Sigma-Aldrich; BlasTaq™ 2X qPCR Mix: Applied Biological Materials Example 1 Construction of pAAV-smCBA-TWNK-OPT vector (1) Obtain the CDS sequence of the TWNK gene from NCBI, named TWNK-WT, and the sequence is SEQ ID NO:1; (2)Codon optimization was performed on TWNK-WT, and the factors for adjustment included the following: adjusting codon usage preferences to adapt to the highest expression profile of the target host; upgrading the CAI (Codon Adaptation Index) from 0.79 to 0.97; adjusting the average GC content from 55% to 62%, and local high-GC or low-GC regions were removed; adjusting the mRNA secondary structure and modifying RNA unstable motifs to regulate the efficiency of gene expression; adding 6 protective bases to the 5' end of the optimized TWNK gene, then adding a HindIII restriction site, and adding an XhoI restriction site to the 3' end of the TWNK gene to obtain the codon-optimized nucleotide sequence SEQ ID NO: 2, named TWNK-OPT, and its amino acid sequence is SEQ ID NO: 3; (3)TWNK-OPT and the pAAV-smCBA-luc vector plasmid were respectively double-digested with HindⅢ and XhoI at 37 °C for 2 h to obtain digestion products, and the digestion system is shown in Table 1; (4)The digestion products were subjected to agarose gel electrophoresis at 120 V for 30 min to separate DNA fragments, and then purified using a gel extraction kit to obtain the purified TWNK-OPT fragment and the purified linearized pAAV-smCBA-luc vector; (5)The concentration of the purified fragment was detected using a UV spectrophotometer. The TWNK-OPT fragment and the purified AAV vector plasmid were mixed at a ratio of 3:1, and ligated with T4 DNA ligase at 16 °C for 16 h to obtain a ligation product, and the ligation system is shown in Table 2; Table 1 Double digestion reaction system

[0029] Table 2 Ligation system

[0030] (6)All the ligation products were transferred into competent cells Trans1-T1. After culturing at 37 °C for 1 h, the bacterial solution was inoculated on a solid culture plate containing ampicillin and cultured overnight in a 37 °C bacterial incubator. Then, single colonies were picked for amplification, and small-scale plasmid extraction was performed for sanger sequencing verification; (7)Refer to steps (1)-(6) to construct the pAAV-smCBA-TWNK-WT recombinant vector.

[0031] The results are as Figure 1 shown: The sequencing results of the recombinant plasmid pAAV-smCBA-TWNK-OPT indicate that the recombinant adeno-associated virus vector plasmid containing TWNK-OPT was successfully constructed. The schematic diagram of the construction of pAAV-smCBA-TWNK-OPT is as Figure 2 shown.

[0032] Example 2 Western Blot Detection The pAAV-smCBA-TWNK-OPT recombinant vector and pAAV-smCBA-TWNK-WT recombinant vector constructed in Example 1 were transfected into cells, and then proteins were extracted for Western Blot detection. The specific method is as follows: (1) The pAAV-smCBA-TWNK-OPT recombinant vector and pAAV-smCBA-TWNK-WT recombinant vector were transfected into HEK293T cells through transfection reagents respectively. After culturing for 48 hours, the supernatant was discarded, and RIPA lysis buffer was added to extract cell proteins, and the proteins were quantified by the BCA method; (2) Take 30 μg of protein samples, separate them by 10% SDS-PAGE, transfer them to a PVDF membrane at a constant current of 300 mA, and block the PVDF membrane with 5% skim milk at room temperature for 1 h; (3) Incubate the primary antibodies (anti-TWNK, anti-β-actin) overnight at 4°C, incubate the secondary antibody goat anti-rabbit IgG at room temperature for 1 h, wash it 3 times with TBST, add the substrate reaction solution for 2 min, and then develop the color by the ECL method. The protein expression level was judged according to the color development result.

[0033] The results are as Figure 3 shown. The pAAV-CBA-TWNK-OPT recombinant vector and pAAV-smCBA-TWNK-WT recombinant vector can normally express anti-TWNK protein in cells, and the bands of anti-TWNK protein are both at 77 kD; in addition, the anti-TWNK protein band of the pAAV-CBA-TWNK-OPT recombinant vector is thicker, indicating that the TWNK sequence after codon optimization can express TWNK protein better.

[0034] Example 3 Virus Packaging The three-plasmid (vector plasmid, packaging plasmid, and helper plasmid) system was used to transfect 293T cells for rAAV virus packaging. The specific steps are as follows: Cell transfection: Culture HEK 293T cells to a density of about 90%, and change the medium 2 hours before transfection; (2) Prepare the solutions required for transfection: Solution A: 8 μg of pAAV2 plasmid, 6 μg of pAAV-smCBA-TWNK-OPT recombinant plasmid and 10 μg of pHelper plasmid were mixed in serum-free and antibiotic-free DMEM medium according to the three-plasmid system; Solution B: 15 μL of the transfection reagent Cytokey was diluted in serum-free and antibiotic-free DMEM medium; After standing at room temperature for 5 min, solution A was added dropwise to solution B and gently mixed. After standing at room temperature for 20 min, the AB mixture was slowly and evenly dropped into well-cultured cells. The medium was changed 6 h after transfection, and the cells were cultured for another 72 h. (4) Virus collection: The cell pellet and supernatant producing the virus were collected in a 50 ml centrifuge tube. After centrifugation, the culture medium supernatant and cell pellet were harvested separately. After adding PEG8000 to precipitate the supernatant, it was centrifuged at 3000 r for 2 h, and the supernatant was discarded. The precipitate was resuspended in PBS. After the cell pellet was repeatedly frozen and thawed three times, 50 U / mL universal nuclease was added and digested at 37 °C for 1 h. After centrifugation at 12000 rpm for 10 min, the supernatant was taken, and rAAV2-smCBA-TWNK-OPT obtained from the cell pellet and supernatant was combined. (5) Virus purification: Iodixanol (iodix) density gradient centrifugation was used to purify adeno-associated virus. 9 mL of 15% iodix / 1M NaCl, 6 mL of 25% iodix, 6 mL of 40% iodix, and 3 mL of 60% iodix were sequentially added to an ultracentrifuge tube. Finally, the virus was added to the top layer, and it was made up with PBS. (6) The ultracentrifuge tube obtained in step (5) was centrifuged at 60000 r and 4 °C for 2.5 h using an ultra-high freezing speed centrifuge. After centrifugation, it could be seen that the iodixanol of different concentrations was clearly separated in color. AAV was located in the colorless 40% iodixanol layer. After being aspirated with a syringe, the virus was ultrafiltered and concentrated using an ultrafiltration centrifuge tube with a molecular weight cut-off of 100 KD and collected in an EP tube and stored at -80 °C. (7) Refer to steps (1)-(6) to obtain purified rAAV2-smCBA-TWNK-WT.

[0035] Example 4 qPCR detection of virus titer Using ssAAV-eGFP as the standard quality plasmid, it was serially diluted, and then qPCR reactions were carried out separately to draw a curve. By comparing with the Ct value, the number of virus copies contained in the recombinant virus was obtained, and thus the virus titer was calculated. The specific steps are as follows: (1) The standard quality plasmid ssAAV-eGFP was serially diluted to 10 µg / µL, 1 µg / µL, 0.1 µg / µL, 0.01 µg / µL, and 0.001 µg / µL respectively. (2) Universal primers were designed for the ITR of the rAAV virus. The sequence of the upstream primer was SEQ ID NO:4, and the sequence of the downstream primer was SEQ ID NO:5. (3) Perform qPCR experiments on the recombinant virus and standard quality plasmids of different concentrations. The qPCR reaction system is shown in Table 3, and the reaction procedure is as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and end-point reading of the plate, for a total of 39 cycles; the melting curve is between 65°C and 95°C; end; Table 3 qPCR reaction system

[0036] (4) Calculate the copy number of the standard quality plasmid as 2×10 8 / ng. The calculation formula is copy number (copies / µL) = concentration (ng / µL)×10^ -9 ×6.02×10 23 (Avogadro's constant) / molecular weight (base pair number bp×648).

[0037] Example 5 Construction of a neural stem cell model with TWNK mutations Establish a neural stem cell model using iPS cells induced from fibroblasts of a Perrault syndrome patient with heterozygous mutations in the TWNK gene. The mutation sites are c.794G>A (P.Arg65His) and c.1181G>A (P.Arg394His). The specific steps are as follows: (1) Seed the iPS cells in a six-well plate. The next day is recorded as the first day of neural stem cell induction. Observe the cells. At this time, the cell density is approximately 15 - 25%. Replace the iPS cell medium with neural stem cell induction medium (490 mL of Neurobasal medium, 10 mL of Gibco neural induction culture additive), and place the cells back into the CO 2 incubator for continued culture; (2) On the second day of induction, replace the neural stem cell induction medium, and then replace the medium every other day. Cell fusion may occur around the fourth day of induction. If the cell density is high, the number of medium replacements for the cells can be appropriately increased to ensure cell nutrition until the seventh day of cell induction; (3) On the seventh day of neural stem cell induction, neural stem cells can be collected at this time. Wash the cells once with DPBS, add 1 mL of pre-warmed StemPro Accutase to digest the cells, incubate at 37°C for 3 min, discard the StemPro Accutase digestion solution, add pre-warmed neural expansion medium (49 mL of Neurobasal medium, 49 mL of Advanced DMEM / F-12, 2 mL of Gibco neural induction culture additive), gently pipette the cells, and collect them; (4)Centrifuge at 300×g for 4 min, discard the supernatant, resuspend the cells in pre-warmed neural expansion medium, and take a small amount of cells for counting; (5)Add Y27632 to the above cell suspension to a final concentration of 5 μM; (6)According to 1×10 5 cells / cm 2 Inoculate the cell suspension into a six-well plate coated with Geltrex at a density of; (7)Shake the six-well plate to disperse the cells evenly, and place the cells in an incubator overnight; (8)The next day, replace the neural expansion medium without Y27632. After that, replace the cell medium every other day until the cell density reaches 80%, and the cells can be cryopreserved or passaged; (9)Immunofluorescence staining was used to analyze the expression of neural stem cell-specific molecular markers.

[0038] In this experiment, we selected Nestin and Sox2 as molecular markers to identify neural stem cells. Nestin belongs to the intermediate filament protein family and is specifically expressed in embryonic and adult neural stem cells but not in mature nerve cells. Currently, it is widely used as a marker molecule for neural stem cells; Sox2, as an important transcription factor, plays an important role in maintaining the stemness of stem cells. Neural stem cells still have the potential to differentiate into various mature nerve cells. Therefore, the expression of Sox2 is positive in neural stem cells; The results are shown in Figure 4 , In neural stem cells induced from iPS cells of patients with Perrault syndrome, the expression of Nestin and Sox2 was detected, proving that this method successfully induced the differentiation of iPS cells of patients with Perrault syndrome into neural stem cells.

[0039] Example 6 Verification of TWNK Mutation Sites in Neural Stem Cell Model Extract the genomic DNA of neural stem cells at the mature stage obtained in Example 5, and use PCR to amplify the gene of the TWNK mutation site to verify whether the neural stem cell model is successfully constructed. The specific steps are as follows: (1)Take the neural stem cells at the mature stage obtained in Example 5 and extract the genomic DNA using a tissue and cell DNA extraction kit; (2)According to the DNA fragment sequence (735 bp) where the TWNK mutation site is located, design PCR primers. The forward primer is TWNK-F with the sequence SEQ ID NO:4, and the reverse primer is TWNK-R with the sequence SEQ ID NO:5; (3)Use DNA polymerase for PCR amplification, and then detect the amplified product by agarose gel electrophoresis; (4) Recover the correct part of the electrophoresis band by gel extraction, and then send it to Genewiz (Suzhou) Co., Ltd. for sequencing.

[0040] Table 4 PCR Amplification System

[0041] Table 5 PCR Amplification Program

[0042] The electrophoresis results showed that the size of the electrophoresis band was consistent with the size of the DNA fragment of the designed primer, indicating that the fragment containing the TWNK mutation site was successfully amplified; the sequencing results were as Figure 5 shown. The 794th base of the TWNK gene was indeed mutated to A, and the 1181st base was indeed mutated to A, indicating that the c.794G>A(p.Arg265His) and c.1181G>A(p.Arg394His) mutation sites were still retained in neural stem cells, demonstrating the successful construction of the neural stem cell model with TWNK mutation.

[0043] Example 7 rAAV-mediated Restoration of Mitochondrial Genome Copy Number in Neural Stem Cells with Perrault Syndrome by TWNK Add the rAAV2-smCBA-TWNK-OPT virus and rAAV2-smCBA-TWNK-WT virus prepared in Example 3 to the well-cultured neural stem cells, and observe the restoration level of the copy number of the TWNK gene. The specific steps are as follows: (1) Experimental group 1: Add the rAAV2-smCBA-TWNK-OPT virus with MOI = 2.0E+05 to the well-cultured neural stem cells obtained in Example 5; Experimental group 2: Add the rAAV2-smCBA-TWNK-WT virus with MOI = 2.0E+05 to the well-cultured neural stem cells obtained in Example 5; Control group: Add sterile ultrapure water without virus particles to the neural stem cells obtained in Example 5; (2) Gently shake evenly and then put it back into the 37°C incubator for continued culture for 72 hours. Collect the neural stem cells, extract the whole genome using a cell / tissue DNA extraction kit, and detect the mtDNA copy number by qPCR. The system of the qPCR reaction is shown in Table 6, and the reaction program is pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing and extension at 55°C for 30 s, for a total of 40 cycles; the qPCR primer sequences for mtDNA are shown in Table 7.

[0044] Table 6 qPCR Reaction System

[0045] Table 7 qPCR primers for mtDNA

[0046] The results were as Figure 6 shown. After 72 h of infection with rAAV-CBA-TWNK-OPT virus and rAAV-CBA-TWNK-WT virus, the copy number of mtDNA in neural stem cells increased, indicating that both rAAV-CBA-TWNK-OPT virus and rAAV-CBA-TWNK-WT virus could increase the copy number of mitochondrial genome in neural stem cells. Among them, at the same dose, rAAV-CBA-TWNK-OPT virus had the best effect on increasing the copy number of mitochondrial genome in neural stem cells. Therefore, if rAAV-CBA-TWNK-OPT is used as a drug to treat Perrault syndrome caused by TWNK mutation, it can theoretically better increase the copy number of mitochondrial genome in cells.

[0047] Example 8 rAAV2-smCBA-TWNK-OPT virus has higher efficiency in expressing TWNK protein (1) The neural stem cells obtained in Example 5 were seeded into a 6-well plate at a density of 1×10 6 cells / well. rAAV-smCBA-TWNK-WT and rAAV-smCBA-TWNK-OPT with the same virus titer were used to infect neural stem cells at an MOI of 1000. At 72 h after infection, the cells were collected and lysed to obtain TWNK-WT lysate and TWNK-OPT lysate respectively; (2) Prepare a 10% SDS-PAGE gel. The protein concentrations of TWNK-WT lysate and TWNK-OPT lysate were measured by the BCA method. The cell lysates with the same protein amount were added to the 8% SDS-PAGE gel, and the gel was run at a voltage of 80 V until it reached the junction of the stacking gel and the separating gel, and then run at a voltage of 120 V until the bottom of the gel; (3) Transfer the SDS-PAGE gel containing TWNK-WT protein and TWNK-OPT protein to a PVDF membrane with a constant current of 300 mA; (4) Incubate the PVDF membrane transferred with TWNK-WT protein and TWNK-OPT protein with 5% skim milk at room temperature for 1 h, and wash the membrane 3 times with TBST, 10 min each time; (5) Incubate with the primary antibody of TWNK at 4 °C for 14 h; (6) After 14 h, wash the membrane 3 times with TBST, 10 min each time, incubate with the secondary antibody of goat anti-rabbit containing HRP at room temperature for 1 h, and wash the membrane 3 times with TBST, 10 min each time; (7)Develop the film using the ECL method to obtain the comparison results of the expression levels of neural stem cells of TWNK-WT and TWNK-OPT.

[0048] The results are as Figure 7 shown. Both the rAAV-CBA-TWNK-OPT virus and the rAAV-CBA-TWNK-WT virus can infect Perrault syndrome neural stem cells in vitro and normally express the TWNK protein. Among them, the expression level of the TWNK protein in the rAAV-CBA-TWNK-OPT virus group is higher. The expression of the TWNK protein in rAAV-CBA-TWNK-OPT is twice that of the rAAV-CBA-TWNK-WT virus. If rAAV-CBA-TWNK-OPT is used as a drug to treat Perrault syndrome caused by TWNK mutations, theoretically, the dosage and cost of rAAV virus can be saved, and adverse reactions such as immune responses caused by high-dose virus injection can be reduced.

Claims

1. A codon-optimized TWNK gene, characterized in that: The codon-optimized TWNK gene is TWNK-OPT, the nucleotide sequence is SEQ ID NO: 2, and the amino acid sequence is SEQ ID NO:

3.

2. A codon-optimized TWNK gene according to claim 1, characterized in that: The TWNK gene sequence has at least 78.1% homology with the wild-type TWNK gene sequence.

3. A recombinant adeno-associated virus vector containing a codon-optimized TWNK gene, characterized in that: The adeno-associated virus vector comprises an adeno-associated virus genome containing a smCBA promoter and a codon-optimized TWNK gene sequence; the codon-optimized TWNK gene sequence is the nucleotide sequence shown in SEQ ID NO: 2 described in claim 1.

4. A method for constructing a recombinant adeno-associated virus vector containing a codon-optimized TWNK gene as claimed in claim 3, characterized in that: The steps include: (1) The wild-type TWNK gene was codon optimized, and protective bases and double restriction sites were added to the 5' and 3' ends of the sequence, respectively. The TWNK-OPT gene fragment was synthesized and amplified by PCR and recovered; (2) Double-digest the pAAV-smCBA-luc plasmid and TWNK-OPT gene and connect them; (3) The ligation product was transformed to obtain a plasmid, and the recombinant adeno-associated virus expression vector pAAV-smCBA-TWNK-OPT was obtained through double enzyme digestion detection and sequencing.

5. The construction method according to claim 4, characterized in that: The double restriction enzyme cutting sites in step (1) are HindⅢ and XhoI.

6. The construction method according to claim 4, characterized in that: The enzyme used in the ligation in step (2) is T4 DNA ligase, and the ligation conditions are 16° C. for 16 h.

7. A method for preparing a recombinant adeno-associated virus containing a codon-optimized TWNK gene, characterized in that: The recombinant adeno-associated virus expression vector pAAV-smCBA-TWNK-OPT constructed according to claim 4 is co-transfected into cells with pHelper and pAAV2, and the supernatant and cells are collected to purify the recombinant adeno-associated virus rAAV-smCBA-TWNK-OPT.

8. The preparation method according to claim 7, characterized in that: The cell is any one of HEK293T cells, HEK293 cells, HeLa cells, Sf9 / Sf21 insect cells and BHK cells.

9. Use of a recombinant adeno-associated virus containing a codon-optimized TWNK gene, characterized in that: The application is application in preparing medicine for treating Perrault syndrome.

10. The use according to claim 9, characterized in that: The Perrault syndrome is caused by mutations in the TWNK gene.

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