Capsid protein mutant MutC capable of improving AAV virus packaging capacity and its application

Abstract

The present invention discloses a capsid protein mutant, MutC, that can improve the packaging capacity of AAV viruses and its applications. The inventors discovered that the capsid protein mutant, MutC, obtained by replacing amino acids 561-588 of the wild-type AAV2 capsid protein with QAEEIATTNPVATEQWGCTNNQANMNGVDTATANIDEEWP, can effectively improve the packaging capacity of AAV viruses, fundamentally addressing the issues of AAV2 production throughput and production costs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a capsid protein mutant MutC capable of improving the packaging ability of AAV viruses and an application thereof. Background Art

[0002] Adeno-associated virus (AAV) is a delivery vector widely used in gene therapy. Its principle is to replace the sequence between the ITRs of the AAV genome with the target gene sequence through genetic engineering methods, and then deliver it to the target cells through cell infection to achieve the purpose of gene therapy. Based on the characteristics of recombinant AAV such as safety, efficiency, stability, sustainability, specificity, and low integration, AAV has become one of the main delivery methods in the field of gene therapy. In the application process of gene therapy, living animals often need to be injected with high-dose, high-purity AAV viruses, and the high cost of AAV production has become one of the bottlenecks in the gene therapy process.

[0003] As downstream gene therapy R&D technologies mature, the limitations of upstream AAV production throughput are becoming increasingly apparent. To address this issue, existing strategies focus on the following two aspects: one is to optimize the existing AAV production process; the other is to find mutant viruses with stronger packaging capabilities. The AAV production process is more about optimizing its external packaging conditions and does not involve the modification of the AAV itself. Based on the structural characteristics of the AAV virus, its viral characteristics such as tissue targeting, immunogenicity, and packaging yield are mainly determined by its surface capsid protein. How to modify the AAV capsid protein to improve the packaging yield of the AAV virus is an urgent problem to be solved. Summary of the Invention

[0004] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide a capsid protein mutant MutC and its application that can improve the AAV virus packaging ability.

[0005] The technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides:

[0007] An AAV virus capsid protein mutant MutC, compared with the wild-type AAV2 virus capsid protein, the amino acids 561-588 of the wild-type AAV2 virus capsid protein are replaced by the polypeptide sequence QAEEIATTNPVATEQWGCTNNQANMNGVDTATANIDEEWP.

[0008] In some examples of AAV viral capsid protein mutant MutC, its amino acid sequence is shown in SEQ ID NO.: 2.

[0009] The second aspect of the present invention provides:

[0010] A nucleic acid sequence molecule expressing the AAV virus capsid protein mutant MutC according to the first aspect of the present invention.

[0011] The third aspect of the present invention provides:

[0012] An expression system, which can express the AAV virus capsid protein mutant MutC described in the first aspect of the present invention, or contains the nucleic acid sequence molecule described in the second aspect of the present invention.

[0013] In some examples of expression systems, the expression system is a recombinant AAV vector, and the recombinant AAV vector is obtained by modifying a wild-type AAV vector.

[0014] In some examples of expression systems, the wild-type AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10.

[0015] In some examples of expression systems, the expression system further comprises a nucleic acid molecule encoding a functional gene product.

[0016] A fourth aspect of the present invention provides:

[0017] A composition comprises the expression system described in the third aspect of the present invention and an acceptable carrier.

[0018] A fifth aspect of the present invention provides:

[0019] The fourth aspect of the present invention is the use of the composition in the preparation of gene therapy preparations or transgenic preparations.

[0020] A sixth aspect of the present invention provides:

[0021] A method for constructing a transgenic animal model comprises introducing the composition described in the fourth aspect of the present invention into the animal body.

[0022] The beneficial effects of the present invention are:

[0023] The capsid protein mutant MutC of some examples of the present invention can effectively improve the AAV virus packaging capacity and fundamentally solve the problems of AAV2 production flux and production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0025] Figure 1 This is an agarose gel electrophoresis identification image of the mutant plasmid constructed in the test example of this invention;

[0026] Figure 2 This is the pAAV2-Rep2 / Cap2 packaging plasmid map in the examples of the present invention;

[0027] Figure 3 This is the pAAV2-MutC-Rep2 / Cap2 packaging plasmid map constructed in the examples of the present invention;

[0028] Figure 4 This is a map of the pAAV2-CMV-EGFP-hGH expression plasmid in the three-plasmid virus packaging system of the embodiment of the present invention;

[0029] Figure 5 This is a map of the pHelper helper plasmid in the three-plasmid virus packaging system in the embodiment of the present invention;

[0030] Figure 6 This is a graph showing the detection results of the total amount of viral particles of the adeno-associated virus variant prepared by the solution of the present invention and the wild-type adeno-associated virus in the control group in this test example;

[0031] Figure 7 1 is a sequence alignment diagram of the wild-type Cap561-588 and MutC sequences of the examples of the present invention. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the concept of the present invention and the technical effects produced in conjunction with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are 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 all be reagents and materials obtained from commercial channels.

[0033] Example 1

[0034] This example provides an adeno-associated virus variant (AAV2-MutC), the preparation of which includes the following steps:

[0035] 1. Plasmid construction

[0036] The nucleic acid sequences corresponding to the amino acid sequences of MutC and Mut101-Mut120 (Table 1) were synthesized (Sangong Biotechnology Co., Ltd.) and cloned into the pAAV2-Rep2 / Cap2 plasmid using existing methods. The amino acids Cap2561-588 were replaced with the mutant sequence to construct the pAAV2-mutant plasmid vector. The ligation product was transformed into Escherichia coli competent DH5α, and single colonies were picked for enzyme digestion verification and sequencing verification. The results are as follows: Figure 1 Agarose gel electrophoresis confirmed that the mutant plasmid was successfully constructed and had good stability.

[0037] Table 1

[0038]

[0039] 2. Packaging of mutant viruses such as AAV2-WT and AAV2-MutC

[0040] AAV2-WT plasmid and AAV2-mutant plasmid were transfected into HEK293 cells using a viral packaging three-plasmid system. The plasmid maps are shown in Figure 2 . Figures 2 to 5 As shown, the following steps are included:

[0041] A cell transfection

[0042] (1) One day before transfection, trypsinize HEK293 cells and count them. Plate the cells in T175 cell culture flasks so that the confluence reaches 80%-90% on the day of transfection.

[0043] (2) For each flask of cells, dilute 42 μg of packaging plasmid, pHelper, and CMV-EGFP-hGM with 3 mL of CaCl2;

[0044] (3) Add 3 mL of 2x HBSS to the plasmid dilution solution and mix, then let it stand at room temperature for 10 minutes.

[0045] (4) Add the above mixture directly to the cell culture flask, shake the flask and mix gently;

[0046] (5) Incubate at 37°C, 5% CO2 for 72 hours.

[0047] B virus purification

[0048] (1) Viral lysis

[0049] 1) Repeatedly pipette to detach the cells, centrifuge at 3000g for 10 minutes, and separate the supernatant and cells;

[0050] 2) Add 0.245 mL of PEG 8000 per mL of supernatant, mix thoroughly, and refrigerate at 4°C overnight. Resuspend the cells in 2.5 mL of Trislysis buffer, freeze-thaw five times, and incubate at 37°C overnight.

[0051] 3) After 12 hours, centrifuge the supernatant after PEG 8000 concentration at 10°C and 3000g for 15 minutes. Remove the supernatant and resuspend the pellet in 1.5 mL of Tris lysis buffer. Add DNase to the cell suspension that has been stored at 37°C overnight and pipette evenly. Incubate in a 37°C water bath for 30 minutes. Pipet the suspension again and centrifuge at 10°C and 2000g for 15 minutes. Combine the two supernatants into one centrifuge tube.

[0052] (2) Ultracentrifugation

[0053] 1) Take an Ultra-Clear centrifuge tube and add 0.5 mL of 60% iodixanol, 2 mL of 40% iodixanol, 1.5 mL of 25% iodixanol, and 1.5 mL of 15% iodixanol to the bottom of the collected virus suspension, and finally balance with Trislysis buffer;

[0054] 2) Ultracentrifuge at 10°C, 230,000 g, 8 ascent, 9 ascent for 18 h.

[0055] (3) Virus purification

[0056] 1) Take an ultrafiltration tube and soak the filter membrane with 1 mL of Tris lysis buffer;

[0057] 2) Carefully aspirate the 40% iodixanol layer from the ultracentrifuge tube using a pipette, avoiding protein, and transfer to an ultrafiltration tube.

[0058] 3) Add an appropriate amount of Tris lysis buffer and pipette evenly. Centrifuge at 4500g for 20 min. Repeat this step until the iodixanol is removed.

[0059] 4) Add 1 mL of Tris lysis buffer and pipette 40-50 times to form a virus suspension. Transfer to an EP tube.

[0060] 5) Use a 5 mL syringe to draw the virus suspension from the EP tube and filter it through a 0.22 μm filter. Collect 20 μL of the virus liquid as a test sample and aliquot it into 102 μL tubes to obtain the adeno-associated virus variant.

[0061] Comparative Example 1

[0062] This comparative example provides a wild-type adeno-associated virus (AAV2-WT), which differs from the preparation method of Example 1 only in that the sequence of the adeno-associated virus capsid protein adopts the wild-type AAV2 capsid protein sequence.

[0063] Test Case

[0064] 1. Virus titer detection

[0065] (1) Viral lysis

[0066] 1) Take 20 μL of the virus samples of Example 1 and the comparative example respectively;

[0067] 2) Add 1 μL each of 10% SDS, 0.5 mol / L EDTA, and proteinase K and mix well;

[0068] 3) Incubate at 56°C in a thermomixer for 1 hour, then at 90°C for 10 minutes;

[0069] 4) Take 2 μL of lysis buffer, dilute 500-fold, and set aside.

[0070] (2) Preparation of standard samples for standard curve

[0071] Take 2×10 12 The plasmid of 100 copies / mL was diluted with ultrapure water ddH2O in proportion to form 6 gradients as the template of the standard 2×10 7 copies / mL, 2×10 6 copies / mL, 2×10 5 copies / mL, 2×10 4 copies / mL, 2×10 3 copies / mL, 2×10 2 copies / mL.

[0072] (3) Absolute quantitative qPCR

[0073] 1) Prepare the following reaction mixture in 0.2 mL PCR tubes, using three tubes for each viral lysate dilution: 10 μL of 2× qPCR Mix; 0.4 μL each of forward and reverse primers; 5 μL of viral lysate dilution; and 4.2 μL of ddH2O.

[0074] Amplification primers:

[0075] Forward primer 5′- TCATATGCCAAGTACGCCCC-3′ (SEQ ID NO. 4);

[0076] Reverse primer 5′-CCCGTGAGTCAAACCGCTAT-3′ (SEQ ID NO. 5).

[0077] 2) PCR amplification

[0078] Pre-denaturation: 95°C, 2 min;

[0079] 40× cycles: 95°C, 15 s; 60°C, 60 s.

[0080] 3) qPCR data processing: Virus titer = dilution factor * viral gene array copy number.

[0081] The experimental results are as follows Figure 6 As shown in the figure, it can be seen that the present invention scheme transfects the AAV2-WT virus packaging three-plasmid system (including pAAV2-Rep2 / Cap2, pAAV2-CMV-EGFP-hGH, pHelper three plasmids) and the AAV2-mutant virus packaging three-plasmid system (including pAAV2-mutant plasmid, pAAV2 -CMV-EGFP-hGH, pHelper three plasmids) into HEK293 cells in equal amounts, among which the total amount of AAV2-MutC virus particles is increased by 145% compared with AAV2-WT, proving that the adeno-associated virus variant prepared by the present application scheme can significantly improve the AAV2 virus packaging capacity. The sequence alignment result of the adeno-associated virus variant capsid protein sequence MutC and the wild-type AAV2 capsid protein (SEQ ID NO.: 1) is shown in the figure. Figure 7 The sequence of the variant adeno-associated virus capsid protein differs from that of the wild-type AAV2 capsid protein in that the wild-type AAV2 capsid protein sequence is replaced by four amino acids, Q561, A562, A566, and W576, and a subsequent amino acid sequence is replaced. The amino acid sequence of the wild-type adeno-associated virus capsid protein Cap2 is shown in SEQ ID NO.1, and the amino acid sequence of the variant adeno-associated virus capsid protein Cap2 is shown in SEQ ID NO.2.

[0082] Amino acid sequence of MutC sequence:

[0083] QAEEIATTNPVATEQWGCTNNQANMNGVDTATANIDEEWP (as shown in SEQ ID NO. 3).

[0084] In summary, the adeno-associated virus variant prepared by the scheme of the present invention can significantly enhance the AAV2 virus packaging ability compared with the wild type.

[0085] The amino acid sequence of wild-type adeno-associated virus capsid protein Cap2 is SEQ ID NO.1: (protein ID: YP_680426.1).

[0086] Amino acid sequence of adeno-associated virus variant capsid protein Cap2, SEQ ID NO.2:

[0087] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAP GKKRPVEHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWG YFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMIT QAEEIATTNPVATEQWGCTNNQANMNGVDTATANIDEEWP QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL.

[0088] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. An AAV virus capsid protein mutant MutC, characterized in that Compared with the wild-type AAV2 viral capsid protein shown in SEQ ID NO.1, amino acids 561-588 of the wild-type AAV2 viral capsid protein are replaced by the polypeptide sequence QAEEIATTNPVATEQWGCTNNQANMNGVDTATANIDEEWP.

2. The AAV virus capsid protein mutant MutC according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

3. A nucleic acid sequence molecule expressing the AAV virus capsid protein mutant MutC according to claim 1 or 2.

4. An expression system, characterized in that It can express the AAV virus capsid protein mutant MutC described in claim 1 or 2, or contain the nucleic acid sequence molecule described in claim 3.

5. The expression system according to claim 4, characterized in that The expression system is a recombinant AAV vector, and the recombinant AAV vector is obtained by transforming a wild-type AAV vector.

6. The expression system according to claim 5, characterized in that The wild-type AAV is AAV2.

7. The expression system according to any one of claims 4 to 6, characterized in that The expression system also includes a nucleic acid molecule encoding a functional gene product.

8. A composition comprising the expression system according to any one of claims 4 to 7 and an acceptable carrier.

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.

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