A codon-optimized twink gene, an adeno-associated viral vector and application thereof

Abstract

The application discloses a codon-optimized TWNK gene, an adeno-associated virus vector and application thereof, a TWNK-OPT gene is obtained by codon optimization on a TWNK-WT gene, and then the TWNK-OPT gene is recombined with an adeno-associated virus expression vector to obtain a pAAV-smCBA-TWNK-OPT recombinant plasmid and rAAV-smCBA-TWNK-OPT recombinant virus, the expression amount of the TWNK gene and protein is improved, and this is favorable for reducing the cost of a gene therapy drug and adverse reactions such as an immune response caused by high-dose virus injection, and can be used for treating Perrault syndrome.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a codon-optimized TWNK gene, an adeno-associated viral vector and application thereof. BACKGROUND

[0002] TWNK gene is located on chromosome 10, the second reading frame, thus also known as C10orf2; the protein encoded by the gene is scattered in the cytoplasm, like a twinkling star, so it is called TWNK protein; in 2001, Spelbrink and his colleagues first discovered and identified that TWNK gene is a causal mutation of autosomal dominant progressive external ophthalmoplegia (adPEO), also known as PEO1 gene; the TWNK protein encoded by the gene is a 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 females, and some patients can be accompanied by neurological symptoms. PRLTS is an autosomal recessive disease, and currently eight kinds of gene mutations related to PRLTS have been identified, including 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 (with progressive neurological disease); the study on the genotype-phenotype correlation of the cases reported so far shows that patients with Perrault syndrome caused by TWNK mutation will have neurological signs in adulthood, with progressive sensory and motor peripheral neuropathy, limited eye movement, nystagmus, cerebellar ataxia and mild mental retardation.

[0004] Currently, the treatment methods for PRLTS are still very limited: for female ovarian hypoplasia, mainly individualized treatment with hormone replacement therapy; for hearing impaired persons, mainly special education resources and use of hearing aids, tactile sensing devices; for children over 12 months with severe and severe hearing loss, cochlear implantation. With the continuous development and improvement of gene therapy technology, normal gene replacement or compensation of mutant genes can be used to alleviate and treat autosomal recessive diseases of PRLTS.

[0005] Chinese patent CN118703570A discloses a rAAV vector for treating TWNK mutant PRLTS and its application, which utilizes the TWNK gene to prepare a rAAV vector, and then transfects the vector containing the TWNK gene into cells to compensate and correct the TWNK gene mutation site in the brain accumulator organ, so that it can be used to prepare a drug for treating Perrault syndrome, solving the problem of lack of precise and effective gene therapy method for Perrault syndrome caused by TWNK gene mutation.

[0006] Recently, we have further found that the expression efficiency of wild-type TWNK gene still has a lot of room for improvement, so that the cost of gene therapy drugs is high, and the risk of side effects such as immune response is large; in addition, due to the existence of codon bias and each host cell has its own codon usage frequency table, when using AAV virus to deliver functional TWNK gene for heterologous expression, there is often a deviation between the codon of heterologous mRNA and the optimal codon usage frequency of the host cell, and the tRNA abundance corresponding to the codon in the heterologous protein mRNA sequence in the host cell is very low, which causes the ribosome to stall on the mRNA, and even causes mRNA degradation, thereby affecting the protein translation efficiency. Therefore, in the process of using gene therapy to treat diseases, 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 product of the functional gene need to be considered, so it can be seen that finding an mRNA sequence with a stable secondary structure, high translation efficiency and optimized codon is crucial for the treatment of Perrault syndrome. SUMMARY

[0007] In view of the above technical problems, the present application provides a codon-optimized TWNK gene, an adeno-associated virus vector and its application, which improves the expression level of TWNK gene and protein, and after preparing an adeno-associated virus vector or an adeno-associated virus, it can reduce the cost of Perrault syndrome gene therapy drugs and reduce the adverse reactions such as immune response caused by high-dose virus injection.

[0008] In order to achieve the above purpose, the present application provides a codon-optimized TWNK gene, which is TWNK-OPT, the nucleotide sequence is SEQ ID NO: 2, and the amino acid sequence is 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 application further provides a recombinant adeno-associated virus vector containing a codon-optimized TWNK gene, wherein 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 a nucleotide sequence shown in SEQ ID NO: 2.

[0011] The application further provides a construction method of a recombinant adeno-associated virus vector containing a codon-optimized TWNK gene, comprising the following steps:

[0012] (1) codon-optimizing a wild-type TWNK gene, adding a protection base and a double enzyme digestion site at the 5' end and the 3' end of the sequence respectively, synthesizing a TWNK-OPT gene fragment and performing PCR amplification and recovery;

[0013] (2) double enzyme digestion of a pAAV-smCBA-luc plasmid and the TWNK-OPT gene, and ligation;

[0014] (3) transformation of the ligation product to obtain a plasmid, double enzyme digestion detection and sequencing to obtain a recombinant adeno-associated virus expression vector pAAV-smCBA-TWNK-OPT.

[0015] Preferably, the double enzyme digestion site in step (1) is HindIII and XhoI.

[0016] Preferably, the enzyme used in the ligation in step (2) is T4 DNA ligase, and the ligation condition is 16 ℃ for 16 h.

[0017] The application further provides a preparation method of a recombinant adeno-associated virus containing a codon-optimized TWNK gene, wherein the recombinant adeno-associated virus rAAV-smCBA-TWNK-OPT is obtained by co-transfecting a recombinant adeno-associated virus expression vector pAAV-smCBA-TWNK-OPT, pHelper and pAAV2 into cells, collecting supernatant and cells, and purifying the supernatant and the cells.

[0018] Preferably, the cells are any one of HEK293T cells, HEK293 cells, HeLa cells, Sf9 / Sf21 insect cells and BHK cells.

[0019] The application further provides an application of a recombinant adeno-associated virus containing a codon-optimized TWNK gene, and the application is an application in preparation of a medicine for treating Perrault syndrome.

[0020] Preferably, the Perrault syndrome is caused by a TWNK gene mutation.

[0021] The application has the following beneficial effects:

[0022] 1. A codon-optimized TWNK-OPT gene having at least 78.1% homology with the wild-type TWNK gene, which improves the expression level of the TWNK gene and TWNK protein.

[0023] 2. The TWNK-OPT is recombined with an adeno-associated virus vector to obtain a pAAV-smCBA-TWNK-OPT recombinant plasmid, which is then packaged and purified to obtain a rAAV2-smCBA-TWNK-OPT recombinant virus. The recombinant virus vector expresses TWNK protein in cells, and the expression level is significantly higher than that of rAAV2-smCBA-TWNK-WT. The recombinant virus vector can be used to prepare a gene therapy drug for treating Perrault syndrome, which is beneficial to reduce the cost of the drug and reduce the adverse reactions such as immune response caused by high-dose virus injection. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Sequencing results of the recombinant plasmid pAAV-smCBA-TWNK-OPT in Example 1.

[0025] Figure 2 A schematic diagram of the construction of the pAAV-CBA-TWNK-OPT vector in Example 1.

[0026] Figure 3 A protein expression gel electrophoresis map of the recombinant virus vector transfected cells in Example 2.

[0027] Figure 4 A graph of immunofluorescence detection of neural stem cells in Example 5.

[0028] Figure 5 A graph of sequencing results of the TWNK gene mutation site in Example 6, where A is c.794G>AP.Arg265His, and B is c.1181G>AP.Arg394His.

[0029] Figure 6 A column chart of the copy number of mitochondrial genome in neural stem cells after treatment with the recombinant virus in Example 7.

[0030] Figure 7 A gel electrophoresis map of in vitro protein expression of cells infected with the recombinant virus in Example 8. DETAILED DESCRIPTION

[0031] The technical solutions of the present application are further explained and described below in combination with the drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present application and should not be understood as limiting the present application. The protection scope of the present application should be subject to the content recited in the claims. The modifications and replacements of the technical solutions of the present application made by those skilled in the art without creative labor fall within the protection scope of the present application.

[0032] Plasmid and cell:

[0033] Hind III, xhol I: Takara;

[0034] vector1212, pAAV-smCBA-Luciferase plasmid, AAV2 capsid plasmid, pHelper helper virus plasmid, HEK293T cell and iPS cell: provided by Tumor Immunity and Microenvironment Hubei Key Laboratory;

[0035] Reagents and medium:

[0036] T4 ligase: Takara

[0037] Plasmid extraction kit, protein marker: Nanjing Novizen Biological Technology Co., Ltd.;

[0038] DMEM medium, PVDF membrane: Zekuan Biological Technology Co., Ltd.;

[0039] cytokey transfection reagent: Zhongji Biological Technology Co., Ltd.;

[0040] PBS: weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4, dissolve in 800mL distilled water, adjust the solution to 7.4 with HCl, and finally add distilled water to 1L, sterilize with high pressure, and store at 4℃;

[0041] Trypsin digestion solution: Zhongkemaichen Technology Co., Ltd.

[0042] Fetal bovine serum: Biological Industries;

[0043] Protease inhibitor, flag primary antibody: Bao Biological Technology Co., Ltd.

[0044] RIPA lysis buffer: Biyun Tian Biological Technology Co., Ltd.;

[0045] 30% acrylamide gel solution: 29 g acrylamide and 1 g methylene bisacrylamide were dissolved in a total volume of 60 mL of water, heated to 37°C to dissolve, and water was added to a final volume of 100 mL; after filtering and sterilizing with a 0.45 μm filter, it was stored in a brown bottle at room temperature;

[0046] 1M Tris-HCl pH 6.8: 121.1 g of Tris was weighed into a 1000 mL beaker, about 800 mL of deionized water was added, and it was stirred to dissolve, and concentrated HCl was added to adjust the pH to 6.8. The solution was brought to 1000 mL, autoclaved at high temperature and high pressure, and stored at room temperature;

[0047] 10% SDS: 100 g of SDS (sodium dodecyl sulfate crystals) was dissolved in 900 mL of purified water, heated to 68°C to dissolve the SDS crystals, and purified water was added to a volume of 1 L, aliquoted and stored at room temperature;

[0048] 10% APS: 0.5 g of APS was dissolved in 5 mL of deionized water, and stored at 4°C in the dark;

[0049] TEMED: Thermo Fisher Scientific;

[0050] Electrophoresis buffer: Tris 3.03 g, glycine 14.4 g, SDS 1 g were dissolved in 1000 ml of deionized water, and stirred to dissolve;

[0051] 1x TBST buffer solution: Tris-HCL (1M, pH 7.5) 50 mL, NaCl 8 g, KCL 0.2 g, Tween 0.5 mL, add distilled water to 1 L;

[0052] 1% BSA: 1 g of BSA was dissolved in 100 ml of 5% TBST solution, and stirred to dissolve;

[0053] Secondary antibody: Sevier (goat anti-mouse)

[0054] Benzonase: Yisen Biotechnology Co., Ltd.

[0055] Iodixanol, iPS cell digestion solution, iPS cell culture medium, neural stem cell induction culture medium, neural stem cell culture medium: Stem cell;

[0056] Y-27632: Sigma-Aldrich;

[0057] BlasTaq™ 2X qPCR Mix: Applied Biological Materials

[0058] Example 1 Construction of pAAV-smCBA-TWNK-OPT vector

[0059] (1) Obtain the CDS sequence of TWNK gene from NCBI, named TWNK-WT, the sequence is SEQ ID NO: 1;

[0060] (2) Codon optimization is performed on TWNK-WT, the adjustment factors include the following contents: adjust the codon usage bias to adapt to the highest expression profile of the target host; the CAI (codon adaptation index) is upgraded from 0.79 to 0.97; the average GC content is adjusted from 55% to 62%, and local high GC or low GC has been removed; adjust the mRNA secondary structure, modify the RNA unstable motif, etc. to adjust the efficiency of gene expression; add 6 protection bases at the 5' end of the optimized TWNK gene, then add a HindIII restriction site, and add an XhoI restriction site at 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;

[0061] (3) TWNK-OPT and pAAV-smCBA-luc vector plasmid are respectively digested with HindIII and XhoI at 37°C for 2h to obtain the digestion products, and the enzyme digestion system is shown in Table 1;

[0062] (4) The enzyme digestion products are separated by 120V agarose gel electrophoresis for 30min, and then purified using a gel recovery kit to obtain purified TWNK-OPT fragments and purified linearized pAAV-smCBA-luc vector;

[0063] (5) The concentration of the purified fragments is detected using a UV spectrophotometer, and the TWNK-OPT fragments and the purified AAV vector plasmid are mixed in a ratio of 3:1, and T4 DNA ligase is used to connect at 16°C for 16h to obtain the ligation product, and the ligation system is shown in Table 2;

[0064] Table 1 Double enzyme digestion reaction system

[0065]

[0066] Table 2 Ligation system

[0067]

[0068] (6) The ligation product was all transferred into competent cell Trans1-T1, which was cultured at 37°C for 1 h, then the bacterial liquid was inoculated on solid culture plate containing ampicillin, and after overnight culture at 37°C in bacterial incubator, single colony was picked for amplification, plasmid was extracted and subjected to sanger sequencing verification;

[0069] (7) The pAAV-smCBA-TWNK-WT recombinant vector was constructed according to steps (1)-(6).

[0070] Results are shown in Figure 1 The sequencing results of the recombinant plasmid pAAV-smCBA-TWNK-OPT showed that the recombinant adeno-associated virus vector plasmid containing TWNK-OPT was successfully constructed, and the schematic diagram of the construction of pAAV-smCBA-TWNK-OPT is shown in Figure 2 .

[0071] Example 2 Western Blot detection

[0072] The pAAV-smCBA-TWNK-OPT recombinant vector and the pAAV-smCBA-TWNK-WT recombinant vector constructed in Example 1 were subjected to cell transfection, and then the protein was extracted for Western Blot detection, and the specific method was as follows:

[0073] (1) The pAAV-smCBA-TWNK-OPT recombinant vector and the pAAV-smCBA-TWNK-WT recombinant vector were transfected into HEK293T cells by transfection reagent, and after 48 hours of culture, the supernatant was discarded, RIPA lysis buffer was added to extract cell protein, and BCA method was used for protein quantification;

[0074] (2) 30 μg of protein sample was separated by 10% SDS-PAGE, and then transferred to PVDF membrane at a constant current of 300 mA, and the PVDF membrane was blocked with 5% skim milk at room temperature for 1 h;

[0075] (3) The primary antibody (anti-TWNK, anti-β-actin) was incubated at 4°C overnight, and the secondary antibody (goat anti-rabbit IgG) was incubated at room temperature for 1 h, then TBST was washed for 3 times, substrate reaction solution was added and reacted for 2 min, and then ECL method was used for color development. The protein expression level was determined according to the color development results.

[0076] Results are shown in Figure 3As shown, the pAAV-CBA-TWNK-OPT recombinant vector and the pAAV-smCBA-TWNK-WT recombinant vector can normally express anti-TWNK protein in cells, and the bands of anti-TWNK protein are all 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 better express TWNK protein.

[0077] Virus packaging of Example 3

[0078] The rAAV virus packaging was performed by transfecting 293T cells using a three-plasmid (vector plasmid, packaging plasmid, and helper plasmid) system, and the specific steps were as follows:

[0079] Cell transfection: HEK 293T cells were cultured to a density of about 90%, and the medium was changed 2 hours before transfection;

[0080] (2) Prepare the solution required for transfection: A solution: 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 double-antibody-free DMEM medium according to the three-plasmid system; B solution: 15 uL of transfection reagent Cytokey was diluted in serum-free and double-antibody-free DMEM medium;

[0081] (3) After 5 min at room temperature, add A solution dropwise to B solution and mix gently, and after 20 min at room temperature, slowly drop the AB mixture into the well-cultured cells. Replace the culture medium 6 h after transfection and continue to culture for 72 h;

[0082] (4) Virus collection: collect the cell pellet and supernatant producing virus in a 50 ml centrifuge tube, and after centrifugation, collect the culture medium supernatant and cell pellet; after adding PEG8000 to precipitate the supernatant, centrifuge at 3000 r for 2 h, discard the supernatant, and resuspend in PBS; after repeated freezing and thawing of the cell pellet three times, add 50 U / mL of omnipotent nuclease, digest at 37°C for 1 h, centrifuge at 12000 rpm for 10 min, and take the supernatant. Combine the rAAV2-smCBA-TWNK-OPT obtained from the cell pellet and the supernatant;

[0083] (5) Virus purification: use iodixanol (iodix) density gradient centrifugation method to purify the adeno-associated virus, and sequentially add 9 mL of 15% iodix / 1M NaCl, 6 mL of 25% iodix, 6 mL of 40% iodix, and 3 mL of 60% iodix in an ultracentrifuge tube, and finally add the virus on the uppermost layer, and fill with PBS;

[0084] (6) Using an ultra-high-speed freezing centrifuge, the ultracentrifuge tube obtained in step (5) was centrifuged at 60000 r, 4°C for 2.5 h. After centrifugation, different concentrations of iodixanol could be seen, and AAV was located in the colorless 40% iodixanol layer. After suction, the virus was concentrated by ultrafiltration centrifuge tube with a molecular weight cutoff of 100 KD, collected in an EP tube, and stored at -80°C.

[0085] (7) The purified rAAV2-smCBA-TWNK-WT was obtained according to steps (1)-(6).

[0086] Example 4 qPCR detection of virus titer

[0087] The ssAAV-eGFP standard plasmid was gradient diluted, and then qPCR reaction was performed to draw a curve. By comparing the Ct value, the number of virus copies contained in the recombinant virus was obtained, and the virus titer was calculated. The specific steps are as follows:

[0088] (1) The standard plasmid ssAAV-eGFP was gradient diluted to 10 µg / µL, 1 µg / µL, 0.1 µg / µL, 0.01 µg / µL, and 0.001 µg / µL, respectively.

[0089] (2) The universal primer was designed for the ITR of rAAV virus, wherein the sequence of the upstream primer was SEQ ID NO: 4, and the sequence of the downstream primer was SEQ ID NO: 5.

[0090] (3) qPCR experiments were performed on the recombinant virus and the standard plasmid with different concentrations, respectively. The qPCR reaction system is shown in Table 3, and the reaction program is as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 10 s, 60°C annealing for 30 s, and end-point plate reading, a total of 39 cycles; the melting curve was between 65°C and 95°C; and the end.

[0091] Table 3 qPCR reaction system

[0092]

[0093] (4) The copy number of the standard plasmid was calculated as 2*10 8 copies / µL, and the calculation formula was copy number (copies / µL) = concentration (ng / µL)*10 -9 *6.02*10 23 (Avogadro's number) / molecular weight (base pair number bp*648).

[0094] Example 5 Construction of TWNK mutant neural stem cell model

[0095] The iPS cells obtained by inducing differentiation of fibroblasts of a patient with Perrault syndrome using a TWNK gene hybrid mutation, wherein the mutation sites are c.794G>A (P.Arg65His) and c.1181G>A (P.Arg394His), are used to establish a neural stem cell model, and the specific steps are as follows:

[0096] (1) The iPS cells are inoculated in a six-well plate, and the next day is recorded as the first day of neural stem cell induction. At this time, the cell density is about 15-25%, and the iPS cell culture medium is replaced with neural stem cell induction medium (Neurobasal medium 490 mL, Gibco neural induction culture additive 10 mL). The cells are placed back into the CO2 incubator for continuous culture;

[0097] (2) The neural stem cell induction medium is replaced on the second day, and the medium is replaced every other day thereafter. Cell fusion can occur around the fourth day of induction. If the cell density is high, the cell medium replacement frequency can be appropriately increased to ensure cell nutrition until the seventh day of cell induction;

[0098] (3) On the seventh day of neural stem cell induction, the neural stem cells can be collected. The cells are washed once with DPBS, 1 mL of preheated StemPro Accutase is added to digest the cells, and the cells are incubated at 37°C for 3 min. The StemPro Accutase digestion solution is discarded, and preheated neural expansion medium (Neurobasal medium 49 mL, Advanced DMEM⁄F-12 49 mL, Gibco neural induction culture additive 2 mL) is added to gently blow the cells and collect them;

[0099] (4) Centrifuge at 300×g for 4 min, discard the supernatant, and resuspend the cells in preheated neural expansion medium. Count a small amount of cells;

[0100] (5) Add Y27632 to the above cell suspension to a final concentration of 5 μM;

[0101] (6) Seed the cell suspension into a Geltrex-coated six-well plate at a density of 1×10 5 cells / cm 2 ;

[0102] (7) Shake the six-well plate to evenly distribute the cells, and place the cells in the cell culture incubator overnight;

[0103] (8) Replace the neural expansion medium without Y27632 the next day, and then replace the cell culture medium every other day until the cell density reaches 80%. The cells can be frozen or passaged;

[0104] (9) Immunofluorescence staining was used to analyze the expression of neural stem cell specific molecular markers.

[0105] In this experiment, we selected Nestin and Sox2 as molecular markers for identifying 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 neural cells. It is currently widely used as a marker molecule for neural stem cells. Sox2 is an important transcription factor that plays an important role in stem cell stemness maintenance. Neural stem cells still have the potential to differentiate into various mature neural cells, so Sox2 expression in neural stem cells is positive. The results are shown in Figure 4 In the iPS cell-induced neural stem cells of the Perrault syndrome patient, the expression of Nestin and Sox2 was detected, proving that the method successfully induced the iPS cells of the Perrault syndrome patient to differentiate into neural stem cells.

[0106] Example 6 Verification of TWNK mutation site in neural stem cell model

[0107] The genome of the mature stage neural stem cells obtained in Example 5 was extracted, and the gene of the TWNK mutation site was amplified by PCR to verify whether the neural stem cell model was successfully constructed. The specific steps are as follows:

[0108] (1) The mature stage neural stem cells obtained in Example 5 were used to extract the genome using a tissue and cell DNA extraction kit;

[0109] (2) According to the sequence of the DNA fragment (735 bp) where the TWNK mutation site is located, PCR primers were designed, wherein the forward primer is TWNK-F with the sequence of SEQ ID NO: 4, and the reverse primer is TWNK-R with the sequence of SEQ ID NO: 5;

[0110] (3) DNA polymerase was used for PCR amplification, and then the amplification product was detected by agarose gel electrophoresis;

[0111] (4) The correct part of the electrophoresis band was recovered, and then sent to Suzhou Jinweizhi Biological Technology Co., Ltd. for sequencing.

[0112] Table 4 PCR amplification system

[0113]

[0114] Table 5 PCR amplification program

[0115]

[0116] The electrophoresis result shows that the electrophoresis band size is consistent with the DNA fragment size of the designed primer, indicating that the fragment at the TWNK mutation site is successfully amplified; the sequencing result is shown in Figure 5 Figure 1, the base at position 794 of the TWNK gene is indeed mutated to A, and the base at position 1181 is indeed mutated to A, indicating that the c.794G>A (p.Arg965His) and c.1181G>A (p.Arg9394His) mutation sites are still retained in the neural stem cells, indicating that the neural stem cell model of TWNK mutation is successfully constructed.

[0117] Example 7 rAAV-mediated TWNK restores mitochondrial genome copy number in neural stem cells of Perrault syndrome

[0118] The rAAV2-smCBA-TWNK-OPT virus and the rAAV2-smCBA-TWNK-WT virus prepared in Example 3 are added to the well-cultured neural stem cells, and the recovery level of the copy number of the TWNK gene is observed, and the specific steps are as follows:

[0119] (1) Experimental group 1: Take the rAAV2-smCBA-TWNK-OPT virus with MOI = 2.0E+05 and add it to the well-cultured neural stem cells obtained in Example 5;

[0120] Experimental group 2: Take the rAAV2-smCBA-TWNK-WT virus with MOI = 2.0E+05 and add it to the well-cultured neural stem cells obtained in Example 5;

[0121] Control group: Take sterile ultrapure water without virus particles and add it to the neural stem cells obtained in Example 5;

[0122] (2) After gently shaking and uniformity, return to the 37°C incubator for further culture for 72 hours, collect the neural stem cells, extract the whole genome using the cell / tissue DNA extraction kit, and detect the mtDNA copy number by qPCR, wherein the qPCR reaction system is shown in Table 6, and the reaction program is 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 55°C annealing and extension for 30 s, a total of 40 cycles; the qPCR primer sequence of mtDNA is shown in Table 7.

[0123] Table 6 qPCR reaction system

[0124]

[0125] Table 7 qPCR primer of mtDNA

[0126]

[0127] The results are shown in Figure 6As shown, the mtDNA copy number in neural stem cells was increased after 72h of infection with rAAV-CBA-TWNK-OPT virus and rAAV-CBA-TWNK-WT virus, indicating that both rAAV-CBA-TWNK-OPT virus and rAAV-CBA-TWNK-WT virus can increase the mitochondrial genome copy number in neural stem cells, and under the same dose, rAAV-CBA-TWNK-OPT virus has the best effect on increasing the mitochondrial genome copy number in neural stem cells, so theoretically, rAAV-CBA-TWNK-OPT can better increase the mitochondrial genome copy number in cells if it is used as a drug to treat Perrault syndrome caused by TWNK mutation.

[0128] Example 8 rAAV2-smCBA-TWNK-OPT virus has higher expression efficiency of TWNK protein

[0129] (1) The neural stem cells obtained in Example 5 were inoculated into a 6-well plate at a density of 1x10 6 cells / well, and rAAV-smCBA-TWNK-WT and rAAV-smCBA-TWNK-OPT were used to infect neural stem cells at a MOI of 1000 with the same virus titer. 72h after infection, the cell lysate was collected to obtain TWNK-WT lysate and TWNK-OPT lysate, respectively;

[0130] (2) Prepare 10% SDS-PAGE gel, and determine the protein concentration of TWNK-WT lysate and TWNK-OPT lysate by BCA method. Add the same amount of cell lysate to the 8% SDS-PAGE gel, and run to the junction of the compression gel and the separation gel at a voltage of 80V, and run to the bottom of the gel at a voltage of 120V;

[0131] (3) Transfer the SDS-PAGE gel containing TWNK-WT protein and TWNK-OPT protein to the PVDF membrane with a constant current of 300mA;

[0132] (4) Incubate the PVDF membrane with TWNK-WT protein and TWNK-OPT protein with 5% skim milk at room temperature for 1h, and wash the membrane with TBST for 3 times, 10min each time;

[0133] (5) Incubate the primary antibody of TWNK at 4°C for 14h;

[0134] (6) After 14h, wash the membrane with TBST for 3 times, 10min each time, and incubate the secondary antibody of goat anti-rabbit with HRP at room temperature for 1h, and wash the membrane with TBST for 3 times, 10min each time;

[0135] (7) The ECL method was used for development to obtain the comparison results of the neural stem cell expression levels of TWNK-WT and TWNK-OPT.

[0136] The results are shown in Figure 7 As shown in the results, both the rAAV-CBA-TWNK-OPT virus and the rAAV-CBA-TWNK-WT virus can infect the Perrault syndrome neural stem cells in vitro and express the TWNK protein normally, and the expression amount of the TWNK protein in the rAAV-CBA-TWNK-OPT virus group is higher, the expression of the TWNK protein in the rAAV-CBA-TWNK-OPT is 2 times of the rAAV-CBA-TWNK-WT virus, and if the rAAV-CBA-TWNK-OPT is used as a drug for treating the Perrault syndrome caused by the TWNK mutation, theoretically, the use amount and cost of the rAAV virus can be saved, and the adverse reactions such as the immune response caused by the 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, with the nucleotide sequence SEQ ID NO:2 and the amino acid sequence SEQ ID NO:

3.

2. 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 an 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 as described in claim 1.

3. A method for constructing a recombinant adeno-associated virus vector containing a codon-optimized TWNK gene as described in claim 2, characterized in that: Includes the following steps: (1) Codon optimization was performed on the wild-type TWNK gene, 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) The pAAV-smCBA-luc plasmid and TWNK-OPT gene were double-digested with enzymes and then ligated; (3) The ligation product was transformed to obtain a plasmid, which was then subjected to double enzyme digestion and sequencing to obtain the recombinant adeno-associated virus expression vector pAAV-smCBA-TWNK-OPT; The codon-optimized TWNK gene sequence is the nucleotide sequence shown in SEQ ID NO: 2 as described in claim 1.

4. The construction method according to claim 3, characterized in that: The double restriction sites mentioned in step (1) are HindⅢ and XhoI.

5. The construction method according to claim 3, characterized in that: The enzyme used for ligation in step (2) is T4 DNA ligase, and the ligation conditions are 16℃ for 16h.

6. 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 3 was co-transfected into cells with pHelper and pAAV2. The supernatant and cells were collected, and the recombinant adeno-associated virus rAAV-smCBA-TWNK-OPT was obtained after purification. The codon-optimized TWNK gene sequence is the nucleotide sequence shown in SEQ ID NO: 2 according to claim 1.

7. The preparation method according to claim 6, characterized in that: The cells are any one of HEK293T cells, HEK293 cells, HeLa cells, Sf9 / Sf21 insect cells, and BHK cells.

8. The application of a recombinant adeno-associated virus containing a codon-optimized TWNK gene, characterized in that: The application is in the preparation of a drug for treating Perrault syndrome; the codon-optimized TWNK gene sequence is the nucleotide sequence shown in SEQ ID NO: 2 as described in claim 1.

9. The application according to claim 8, characterized in that: The Perrault syndrome is caused by a mutation in the TWNK gene.

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