Capsid protein mutant capable of improving AAV (adeno-associated virus) yield and full-eye infection ability and application of capsid protein mutant
By performing peptide replacement in the amino acid sequence of the specific amino acid at the AAV2 virus capsid protein, the whole eye infection capacity and yield of the AAV virus are improved, the problem of low efficiency of AAV virus in eye infection is solved, and more efficient gene therapy effects are achieved.
Patent Information
- Application Number
- CN202510300718.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-16
- Publication Date
- 2025-06-13
AI Technical Summary
The AAV virus is inefficient in the eyes, especially the infectious ability to infect cells and central nervous cells of the posterior retinal layer, resulting in poor gene therapy.
By performing polypeptide replacement in the amino acid sequences of positions 561 to 588 of the AAV2 viral capsid protein, a series of capsid protein mutants were obtained, which improved the whole-eye infection capacity and yield of the AAV virus.
It significantly improves the infection capacity and yield of AAV2 virus on the whole eye, can more effectively infect posterior retinal cells and other target cells, and improves the effect of gene therapy.
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Figure CN120136981A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and specifically relates to a capsid protein mutant that improves the production of AAV virus and the ability of whole-eye infection, and its application. Background Art
[0002] Adeno-associated virus (AAV) has become the preferred vector for in vivo treatment in the field of gene therapy due to its characteristics of low immunogenicity, tissue targeting, low integrativity, and long-term stable expression, and is widely used in the research of various diseases such as genetic diseases, cancers, and some chronic diseases. However, it still faces multiple challenges: First, the production process is complex, the cost is expensive, and the production capacity is limited; Second, high-dose administration may exhibit neurotoxicity and hepatotoxicity, causing safety problems. Third, the infection ability for some tissue cells is weak. For example, intravitreal injection is difficult to infect the posterior layer cells of the retina, and intravenous injection is difficult to infect central nerve cells. Modifying and screening the AAV capsid protein to improve its targeting and transfection efficiency for specific tissues, with the expectation of achieving lower and safer dosing, is an effective way to solve this problem.
[0003] Due to the existence of the blood-eye barrier, the eye has a certain immune privilege, and local administration is relatively safe. Moreover, most fundus diseases are monogenic genetic diseases. Therefore, ophthalmology has become a hot field in gene therapy. Different fundus diseases have different pathogenesis. When using AAV virus for gene therapy, the cell layers to be infected are different. The commonly used injection methods in clinical practice are intravitreal injection and subretinal injection. The subretinal injection has a better infection effect, especially for the RPE layer and the outer nuclear layer. However, this method is highly invasive, prone to retinal detachment, and may lead to photoreceptor degeneration, visual function damage, and gliosis. Intravitreal injection has a lower surgical risk, but conventional serotypes such as AAV2 and AAV5 can only infect the RGC layer and have a poor infection effect on cone and rod cells and RPE. Therefore, developing AAV mutants that can improve the whole-eye expression ability or can penetrate the RGC layer and the inner nuclear layer to infect the outer nuclear layer cells and RPE cells is of great significance for solving the problem of gene therapy delivery vectors in ophthalmology.
[0004] Previous studies have shown that mutating the amino acid sites in the 561-588 region of Cap2 can optimize its infection ability for mouse eye tissues. However, the specific replacement method to improve the whole-eye infection ability of AAV virus is still unclear. Summary of the Invention
[0005] The present invention aims to modify the AAV capsid protein to obtain AAV virus mutants with higher production and better whole-eye expression ability, and specifically provides a capsid protein mutant that improves the production of AAV virus and the ability of whole-eye infection, and its application.
[0006] The technical solution adopted by the present invention is: In a first aspect of the present invention, there is provided: a capsid protein mutant that enhances the ability of AAV virus to infect the whole eye, obtained by replacing the amino acids at positions 561 to 588 of the wild-type AAV2 virus capsid protein with any one of the following polypeptides: 1) DEEEIATVNPVATEQYGSVSTNLQRGNR 2) DENEIATTNPVATEQYGWVSTNLQRGNR 3) DEEEIVATNPVATEMYGVVSTNLQRGNR 4) DEQEIAATNPVATEQYGSVSYNLQRINR 5) DESEIATTNPVATEQYGVVSTNLQRGNR 6) DEEEIRTTNPVATEQYGSVSTNLQRGNAAAQGYPPKPPAR 7) DEEEIRTTNPVATEQYGSVSTNLQRGNTGTSQLGARGLSR 8) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSARGGYTGLSR 9) DEEEIRTTNPVATEQYGSVSTNLQRGNTGTSTFGASRGLSR 10) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSVTFSGTQRGLSR 11) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSALPSHLSGRGLSR 12) DEEEIRTTNPVATEQYGSVSTNLQRGNTGLQATMGRGLSR 13) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSLGTNPTRGLSR 14) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSYVGQPPRGLSR 15) DEEEIRTTNPVATEQYGSVSTNLQRGNTGRDKVGIQGLSR.
[0007] In some examples of the capsid protein mutant, the GenBank number of the amino acid sequence of the wild-type AAV2 virus capsid protein is YP_680426.1.
[0008] The second aspect of the present invention provides: A gene encoding the capsid protein mutant described in the first aspect of the present invention.
[0009] In some examples of the gene, codon optimization is performed according to the expression system.
[0010] The third aspect of the present invention provides: An expression system into which the gene described in the second aspect of the present invention is inserted.
[0011] In some examples of the expression system, the expression system is a recombinant AAV vector.
[0012] In some examples of the expression system, the AAV is any one selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 and its variants.
[0013] The fourth aspect of the present invention provides: A composition comprising the expression system described in the third aspect of the present invention.
[0014] The fifth aspect of the present invention provides: The use of the composition described in the fourth aspect of the present invention in the preparation of a gene therapy preparation or a transgenic preparation.
[0015] In some examples of the use, the gene therapy preparation is used for treating eye diseases.
[0016] In some examples of the use, the transgenic preparation is used for treating eye diseases or for constructing a disease animal model.
[0017] In some examples of the use, the animal is selected from mice, rats, cynomolgus monkeys, rhesus monkeys, etc.
[0018] In some examples of the use, the disease is an eye disease, especially an eye genetic disease.
[0019] The sixth aspect of the present invention provides: A method for constructing a transgenic animal model, comprising introducing the composition described in the fourth aspect of the present invention into an animal.
[0020] In some examples of the construction method, the animal is selected from mice, rats, cynomolgus monkeys, rhesus monkeys, etc.
[0021] In some examples of the construction method, the transgenic animal model is a transgenic animal model for eye diseases.
[0022] In some examples of the construction method, the eye disease is an eye genetic disease.
[0023] The beneficial effects of the present invention are: The capsid protein mutants of some examples of the present invention can effectively improve the whole-eye infection ability of AAV2, fundamentally solving the problem of the eye infection efficiency of AAV2.
[0024] The capsid protein mutants of some examples of the present invention have better yields.
[0025] The capsid protein mutants of some examples of the present invention can be well applied to gene therapy of the eye or construction of an eye disease model. Description of the Drawings
[0026] Figure 1 It is the identification diagram of the agarose gel electrophoresis of the mutant plasmid constructed in the test example of the present invention.
[0027] Figure 2 It is the plasmid map of pAAV2-Rep2 / Cap2 packaging plasmid in the embodiment of the present invention.
[0028] Figure 3 It is the plasmid map of pAAV2-7M8-Rep2 / Cap2-7M8 packaging plasmid in the embodiment of the present invention.
[0029] Figure 4 It is the plasmid map of the pAAV2-Mut-Rep2 / Cap2-Mut packaging plasmid constructed in the embodiment of the present invention.
[0030] Figure 5 It is the plasmid map of the pAAV-CAG-Luciferase-HGH expression plasmid of the virus packaging three-plasmid system in the embodiment of the present invention.
[0031] Figure 6 It is the plasmid map of the pHelper helper plasmid of the virus packaging three-plasmid system in the embodiment of the present invention.
[0032] Figure 7 It is the yield results of AAV2 wild type, 7M8 and AAV2 mutants prepared by the method in the test example of the present invention.
[0033] Figure 8 It is the in vivo imaging results of the mouse eye infection effects of AAV2 wild type, 7M8 and some AAV2 mutants (AAV2-EYE-08 to EYE-22) prepared by the method in the test example of the present invention.
[0034] Figure 9 It is the chemiluminescence detection results of the mouse eye infection effects of AAV2 wild type, 7M8 and AAV2 mutants (AAV2-EYE-08 to EYE-22) prepared by the method in the test example of the present invention.
[0035] Figure 10Amino acid sequence alignment diagram of wild-type AAV2, 7M8 and AAV2 mutants (AAV2-EYE-08 to EYE-22) of the embodiments of the present invention. Detailed implementation manners
[0036] The following will clearly and completely describe the concept and technical effects generated by the present invention in combination with embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources. Embodiment
[0037] This embodiment provides a variety of adeno-associated virus variants (AAV2-EYE-08 to AAV2-EYE-22, AAV2-Mut201 to AAV2-Mut213) based on AI design and past research. The preparation of the adeno-associated virus variants includes the following steps: Table 1 Amino acid sequence information of AAV2 mutants replaced at the Cap2 D561 to R588 sites Sequence Name Amino Acid Sequence Information Number AAV2-WT DEEEIRTTNPVATEQYGSVSTNLQRGNR 3 7M8 DEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRPAR 4 AAV2-EYE-08 DEEEIATVNPVATEQYGSVSTNLQRGNR 5 AAV2-EYE-09 DENEIATTNPVATEQYGWVSTNLQRGNR 6 AAV2-EYE-10 DEEEIVATNPVATEMYGVVSTNLQRGNR 7 AAV2-EYE-11 DEQEIAATNPVATEQYGSVSYNLQRINR 8 AAV2-EYE-12 DESEIATTNPVATEQYGVVSTNLQRGNR 9 AAV2-EYE-13 DEEEIRTTNPVATEQYGSVSTNLQRGNAAAQGYPPKPPAR 10 AAV2-EYE-14 DEEEIRTTNPVATEQYGSVSTNLQRGNTGTSQLGARGLSR 11 AAV2-EYE-15 DEEEIRTTNPVATEQYGSVSTNLQRGNTGSARGGYTGLSR 12 AAV2-EYE-16 DEEEIRTTNPVATEQYGSVSTNLQRGNTGTSTFGASRGLSR 13 AAV2-EYE-17 DEEEIRTTNPVATEQYGSVSTNLQRGNTGSVTFSGTQRGLSR 14 AAV2-EYE-18 DEEEIRTTNPVATEQYGSVSTNLQRGNTGSALPSHLSGRGLSR 15 AAV2-EYE-19 DEEEIRTTNPVATEQYGSVSTNLQRGNTGLQATMGRGLSR 16 AAV2-EYE-20 DEEEIRTTNPVATEQYGSVSTNLQRGNTGSLGTNPTRGLSR 17 AAV2-EYE-21 DEEEIRTTNPVATEQYGSVSTNLQRGNTGSYVGQPPRGLSR 18 AAV2-EYE-22 DEEEIRTTNPVATEQYGSVSTNLQRGNTGRDKVGIQGLSR 19 AAV2-Mut201 DEQEIAATNPVATEIYGEVSTNLQRGDR 20 AAV2-Mut202 CEEEISTTNPVATEQYGDVSENLQRGNA 21 AAV2-Mut203 DENEIATTNPVATEQYGSVGENLQRGNR 22 AAV2-Mut204 DEEEIRTTNPVATEQYGSVSTNLQRGNTGARTPVQLKPGLSR 23 AAV2-Mut205 DEEEIRTTNPVATEQYGSVSTNLQRGNTGPVGSRFVPGLSR 24 AAV2-Mut206 DEEEIRTTNPVATEQYGSVSTNLQRGNTGPPLQLIRGLSR 25 AAV2-Mut207 DEEEIRTTNPVATEQYGSVSTNLQRGNTGTGDSPLPGLSR 26 AAV2-Mut208 DEEEIRTTNAVATEGYGSVSENLQRGGR 27 AAV2-Mut209 DEEEIRTTNPVSTEQYGWVSDNLQRGNR 28 AAV2-Mut210 AEEEIRTTNPVATEQYGSVGENLQRGNR 29 AAV2-Mut211 DEEEIRTTNPVATEQYGSVSTNLQRGNTGRDLRLSLGLSR 30 AAV2-Mut212 DEEEIRTTNPVATEQYGSVSTNLQRGNTGRLSTGRSHGLSR 31 AAV2-Mut213 DEEEIRTTNPVATEQYGSVSTNLQRGNTGSCAVEGHEPGLSR 32 1. Plasmid construction The nucleic acid sequences corresponding to the amino acid sequences of AAV2-EYE-08 to EYE-22 and AAV2-Mut201 to AAV2-Mut213 (as shown in Table 1) were synthesized by gene synthesis (GenScript Biotech), and the synthesized sequences were cloned onto the pAAV2-Rep2 / Cap2 plasmid by seamless method. The Cap2 561 to 588 sequence of wild-type AAV2 was replaced with the mutant sequence to construct the pAAV-mutant plasmid vector; the ligation product was transformed into Escherichia coli competent Stbl3, single colonies were picked for amplification culture, and plasmids were extracted for sequencing verification and restriction enzyme digestion verification. The sequencing verification was consistent with the theoretical sequence. Identified by agarose gel electrophoresis, the results are as Figure 1 shown, and the mutant plasmid construction was successful.
[0038] 2. AAV2-WT, 7M8 and AAV mutant virus packaging The packaging plasmid of AAV2-WT, 7M8 or AAV2-mutant, the pAAV-CAG-Luciferase-hGH expression plasmid and the pHelper helper plasmid were co-transfected into suspension HEK293T cells.
[0039] Virus packaging includes the following steps: A. Cell culture (1) Take out a vial of suspended 293T cells from the liquid nitrogen tank, place it in a 37°C water bath, shake it quickly to thaw the cells, add them to a centrifuge tube containing 5 ml of Wayne 293 serum-free medium, centrifuge at 200×g for 5 min, discard the supernatant, resuspend in 10 ml of Wayne 293 serum-free medium, place in a 125 ml Erlenmeyer flask, supplement with Wayne 293 serum-free medium to 20 ml, and place in an orbital shaker incubator at 120 rpm, 37°C, and 5% CO 2 for culturing; (2) After culturing for 48 - 72 hours, when the cell density reaches 2 - 3E+6 cell / ml, the cells can be passaged; (3) Take out the seed cells to the laminar flow hood, shake well and aspirate about 500 μl of the sample. Take 20 μl of the sample and add 20 μl of trypan blue to mix well. Aspirate 20 μl of the mixture and add it to the cell counting chamber. Read the data 3 times on an automatic cell counter, and take the average value as the cell density; (4) Take an appropriate amount of cells, dilute them with fresh Wayne 293 serum-free medium to 0.6 - 0.65E+6 cell / ml, place in a 500 ml Erlenmeyer flask for culturing, and the culture volume per flask is 25 ml; culture in an orbital CO 2 cell incubator, and the culture conditions are: 120 rpm, 37°C, and 5% CO 2 .
[0040] B. Cell Transfection (1) Culture the above cells for 48 h. When the cell density reaches 3 - 3.8E+6 cell / ml, the cells can be transfected; (2) For each flask of cells, add a total of 100 μg of packaging plasmid, pHelper, and pAAV-CAG-Luciferase--hGH to 4 mL of DMEM medium in sequence, and mix well to obtain the "plasmid dilution solution"; (3) Take another 1 mL of DMEM medium, add 300 μg of PEI and mix well to obtain the "PEI dilution solution"; (4) Pour the "PEI dilution solution" into the "plasmid dilution solution", mix well quickly, and let it stand at room temperature for 25 minutes to form the transfection complex; add the transfection complex dropwise to the cell suspension while gently shaking; (5) Place the culture flask in an orbital CO 2 cell incubator for culturing, and the culture conditions are: 120 rpm, 37°C, and 5% CO 2 .
[0041] C. Virus Harvest (1) After transfection, continue to culture the cells for 72 hours and start virus harvest; (2) Collect the cell suspensions in each bottle into a centrifuge bottle, centrifuge at 1000 g for 5 min, collect the supernatant into a new centrifuge bottle, add 0.245 mL of 50% PEG8000 solution (containing 0.5 mol / L NaCl) to each mL of the supernatant, mix well, and let stand overnight at 2 - 8 °C; (3) Add an appropriate amount of 0.5% TritonX - 100 cell lysis buffer (containing universal nuclease) to the cell pellet, incubate at 37 °C for 1 - 2 h, then add 1 / 10 volume of 5 mol / L NaCl, mix well, centrifuge at 4 °C and 3000 g for 15 min, collect the supernatant, which is the "crude virus extract" and store it temporarily at 2 - 8 °C; (4) Centrifuge the supernatant after PEG8000 precipitation at 4 °C and 3000 g for 15 min, discard the supernatant, and resuspend it with the "crude virus extract" in step (3) for standby.
[0042] D. Virus purification 1) Take an Ultra - Clear centrifuge tube, successively add 0.5 mL of 60% Iodixanol, 2 mL of 40% Iodixanol, 1.5 mL of 25% Iodixanol, 1.5 mL of 15% Iodixanol, the collected virus suspension, and finally balance with cell lysate; 2) Ultra - centrifuge at 10 °C and 230000 g, with a ramp rate of 8 and a deceleration rate of 9 for 18 h; 3) Take an ultrafiltration tube and moisten the filter membrane with 1 mL of PBS buffer; 4) Carefully aspirate the middle layer of 40% - 60% Iodixanol in the ultra - centrifuge tube with a pipette, avoiding sucking up the protein, and transfer it to the ultrafiltration tube; 5) Add an appropriate amount of PBS buffer, pipette to mix evenly, centrifuge at 4500 g for 3 - 5 min, and repeat this step 5 - 7 times until Iodixanol is removed; 6) Add 1 mL of PBS buffer, pipette 40 - 50 times to form a virus suspension, and transfer it to an EP tube; 7) Aspirate the virus suspension in the EP tube with a 5 mL syringe and filter it through a 0.22 μm filter. After collecting 20 μL of the virus solution for testing samples, aliquot it into 100 μL per tube to obtain the adeno - associated virus variant.
[0043] Comparative Example 1 This comparative example provides a wild - type adeno - associated virus (AAV2 - WT), which is prepared by the method of Example 1 with AAV2 - mutant. The difference is only that the sequence of the adeno - associated virus capsid protein uses the wild - type AAV2 capsid protein sequence (SEQ ID NO.1).
[0044] Comparative Example 2 This comparative example provides an AAV2 adeno-associated virus mutant (AAV2-7M8) with strong whole-eye expression ability that has been used in ophthalmic clinical experiments. It was prepared according to the method of Example 1 with AAV2-mutant, and the only difference is that the adeno-associated virus capsid protein sequence uses the capsid protein sequence of mutant AAV2-7M8 (SEQ ID NO.2).
[0045] Test Example 1. Virus titer detection (1) Virus lysis 1) Take 20 μL of the virus samples from the examples and comparative examples respectively; 2) Add 1 μL of 10% SDS, 0.5 mol / L EDTA, and proteinase K respectively, and mix well; 3) Incubate at 56 °C for 1 hour in a thermostatic mixer, and then incubate at 90 °C for 10 min; 4) Take 10 μL of the virus lysate and dilute it 10-fold serially to 10,000-fold to obtain the virus diluent for standby.
[0046] (2) Preparation of the standard product for the standard curve Take the plasmid standard product with a concentration of 2×10 12 copies / mL, and dilute it 10-fold with ddH 2 O to 6 gradients as the standard curve template 2×10 11 copies / mL, 2×10 10 copies / mL, 2×10 9 copies / mL, 2×10 8 copies / mL, 2×10 7 copies / mL, 2×10 6 copies / mL.
[0047] (3) Standard curve quantitative qPCR 1) Take a 0.2 mL PCR tube and prepare the following reaction system, with 3 replicates for each virus diluent; 2× qPCR Mix 10 μL; 0.2 μL of forward and reverse primers each; 5 μL of virus diluent; 4.2 μL of ddH 2 O; Amplification primers: Forward primer 5’- GGAACCCCTAGTGATGGAGTT-3’ (SEQ ID NO.33); Reverse primer 5’- CGGCCTCAGTGAGCGA-3’ (SEQ ID NO.34); 2) PCR amplification Pre-denaturation: 95°C, 2 min; 40× cycles: 95°C, 15 s; 60°C, 60 s; 3) qPCR data processing: Virus titer = dilution factor * copy number of viral gene array.
[0048] The AAV2-WT virus packaging three-plasmid system (including three plasmids: pAAV2, pAAV-CAG-Luciferase, and pHelper) and the AAV2-mutant virus packaging three-plasmid system (including three plasmids: pAAV2-mutant, pAAV-CAG-Luciferase, and pHelper) in the present invention were respectively transfected into HEK293T cells in equal amounts. The cells were lysed to harvest the virus solution, which was ultracentrifuged and purified with iodixanol, and the virus titer was detected by QPCR to evaluate its yield. Among them, Figure 2 This is the plasmid map of pAAV2-Rep2 / Cap2 packaging plasmid in the embodiment of the present invention. Figure 3 This is the plasmid map of pAAV2-7M8-Rep2 / Cap2-7M8 packaging plasmid in the embodiment of the present invention. Figure 4 This is the plasmid map of the constructed pAAV2-Mut-Rep2 / Cap2-Mut packaging plasmid in the embodiment of the present invention. Figure 5 This is the plasmid map of the pAAV-CAG-Luciferase-HGH expression plasmid of the virus packaging three-plasmid system in the embodiment of the present invention. Figure 6 This is the plasmid map of the pHelper auxiliary plasmid of the virus packaging three-plasmid system in the embodiment of the present invention. The experimental results are as Figure 7 shown. It can be seen from the figure that the yields of 6 mutants are less than 0.5 times that of AAV2-WT and are identified as low-yield variants and not verified in vitro; for the remaining 22 variants, only the yields of 12 AAV2-EYE variants are less than that of AAV2-WT, which is 0.84 times its yield. The virus yields of 21 (accounting for 75%) mutants are higher than that of AAV2-WT, which is 1.26 to 4.36 times its yield, and the AAV2-EYE-20 variant has the highest yield.
[0049] 2. Mouse whole-eye expression ability test Samples from the examples and comparative examples were respectively diluted to 2E+9 vg / µl, and were injected into the vitreous cavity of Bab / c mice, with 1.5 µl injected into each eye (the total virus amount was 3E+9 vg), and 3 mice were injected with each virus. 3-4 weeks after injection, the Luciferase expression was detected by in vivo imaging and chemiluminescence methods.
[0050] (1) In vivo imaging detection Three weeks after virus injection, potassium D-luciferin solution was intraperitoneally injected. Two minutes after the injection, the mice were placed in the induction box of an anesthesia machine for isoflurane-induced anesthesia. When the mice's breathing became stable and there were no more large movements, they were transferred to the breathing mask of the imager to maintain anesthesia. Imaging parameters were set, and imaging and photographing were performed.
[0051] In the protocol of the present invention, AAV2-WT, AAV2-7M8, and variant viruses were intravitreally injected into mice. Three weeks later, the in vivo imaging results showed that for all mice injected with variant viruses, the Luciferase signal in the eyes was stronger than that of AAV2-WT and AAV2-7M8. Some of the best-performing examples and comparative examples are as Figure 8 shown. It shows that the adeno-associated virus variants prepared by the protocol of the present invention can improve the infectivity of AAV2 virus to the whole eye.
[0052] (2)Chemiluminescence detection 1)Sample collection The mice were sacrificed by cervical dislocation. The eyelids were opened, and the eyeballs were removed with forceps. The muscles and connective tissues around the eyeballs were removed. The eyeballs were placed in 2 ml grinding tubes, one tube for each eyeball, and stored temporarily in an ice bath.
[0053] 2)Sample pretreatment 500 μl of 1xCell Culture Lysis Reagent was added to the homogenization tube containing the mouse eyeballs (single eyeball) and homogenized in a grinder pre-cooled to -65 °C in advance. After homogenization, it was incubated at 4 °C for 1 - 2 h, centrifuged at 15000 r / min at 4 °C for 3 min, and the supernatant was aspirated.
[0054] 3)Color development A black opaque 96-well plate was taken. 20 μl of the sample was added to each well, with 2 - 3 replicates for each sample. The substrate chromogenic solution that had been taken out and restored to room temperature in the dark was added, 100 μl to each well. Immediately after adding the chromogenic solution, it was put into a fluorescence microplate reader for detection, and the fluorescence signal value was read.
[0055] In the protocol of the present invention, AAV2-WT, AAV2-7M8, and mutant viruses were intravitreally injected into mice. To more precisely quantify the expression effect, three weeks later, the eyeballs were removed and homogenized for chemiluminescence detection. The results are as Figure 9 shown. For all 16 groups (accounting for 76.2%) of the mice injected with mutant viruses, the Luciferase expression level in the whole eye was higher than that of AAV2-WT and AAV2-7M8, 1.23 - 8.4 times that of AAV2-WT. Among them, the expression level of 15 groups was more than 5 times that of AAV2-WT, and it was considered that the whole-eye expression ability was significantly improved. Among them, AAV2-EYE-09 had the highest expression level. It shows that the adeno-associated virus variants prepared by the protocol of the present invention can improve the whole-eye expression ability of AAV2 virus.
[0056] The sequence alignment result diagram of the adeno-associated virus variant capsid protein is as follows Figure 10 shown. The difference between the capsid protein sequences of the adeno-associated virus variants EYE-08 to EYE-22 and the wild-type AAV2 capsid protein is that the sequence of the wild-type capsid protein from D561 to R588 (shown in SEQ ID NO.3) is replaced with a polypeptide sequence of 28 to 42 amino acids. Among them, EYE-08 to EYE-12 are partial amino acid substitutions, and EYE-13 to EYE-13 are multiple amino acid insertions.
[0057] The amino acid sequence of the wild-type adeno-associated virus capsid protein Cap2 is shown in SEQ ID NO.1, and the adeno-associated virus variant 7M8 sequence is shown in SEQ ID NO.2.
[0058] In summary, the adeno-associated virus variant prepared by the solution of the present invention can significantly enhance the whole-eye infection ability of the AAV2 virus compared with the wild type.
[0059] Amino acid sequence of wild-type adeno-associated virus capsid protein Cap2, SEQ ID NO.1: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (Protein ID: YP_680426.1).
[0060] Amino acid sequence of AAV2 adeno-associated virus mutant capsid protein Cap2-7M8, SEQ ID NO.2: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRV T TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LALGETTRPA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKS I NVDFTVDTNGVYSEPRPIGTRYLTRNL。
[0061] The above is a further detailed description of the present invention and should not be regarded as a limitation on the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention pertains, any simple deduction or substitution without departing from the concept of the present invention falls within the protection scope of the present invention.
Claims
1. A capsid protein mutant that improves the ability of AAV virus to infect the entire eye, characterized in that: The peptides are obtained by replacing amino acids 561 to 588 of the wild-type AAV2 viral capsid protein with any of the following: 1) DEEEIRTTNPVATEQYGSVSTNLQRGNAAAQGYPPKPPAR 2) DEEEIRTTNPVATEQYGSVSTNLQRGNTGTSQLGARGLSR 3) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSARGGYTGLSR 4) DEEEIRTTNPVATEQYGSVSTNLQRGNTGTSTFGASRGLSR 5) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSVTFSGTQRGLSR 6) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSALPSHLSGRGLSR 7) DEEEIRTTNPVATEQYGSVSTNLQRGNTGLQATMGRGLSR 8) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSLGTNPTRGLSR 9) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSYVGQPPRGLSR 10) DEEEIRTTNPVATEQYGSVSTNLQRGNTGRDKVGIQGLSR.
2. The capsid protein mutant according to claim 1, characterized in that The GenBank number of the wild-type AAV2 virus capsid protein amino acid sequence is YP_680426.
1.
3. A gene encoding the capsid protein mutant according to claim 1 or 2.
4. The gene according to claim 3, characterized in that Codon optimization was performed according to the expression system.
5. An expression system, into which the gene according to claim 3 or 4 is inserted.
6. The expression system according to claim 5, characterized in that The expression system is a recombinant AAV vector.
7. The expression system according to claim 6, characterized in that The AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 and variants thereof.
8. A composition, characterized in that The invention comprises the expression system according to any one of claims 5 to 7.
9. Use of the composition according to claim 8 in the preparation of gene therapy preparations or transgenic preparations.
10. A method for constructing a transgenic animal model, comprising introducing the composition of claim 8 into an animal.