Mutant of adeno-associated virus capsid protein and use thereof

Abstract

The application discloses a mutant of an adeno-associated virus capsid protein and application thereof. Compared with an amino acid sequence of a wild-type AAV capsid protein, the mutant has one or more amino acid substitutions at amino acids 561 to 588 of Cap2. The adeno-associated virus variant obtained by the method can significantly improve AAV2 virus packaging capacity and in vivo infectivity, and fundamentally solves the problems of AAV2 production flux and use dosage.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a mutant of an adeno-associated virus capsid protein and its applications. Background Technology

[0002] Adeno-associated virus (AAV) is a widely used delivery vector in gene therapy. Its principle involves replacing the ITR sequences of the AAV genome with the target gene sequence through genetic engineering, then delivering it to target cells via cell infection to achieve gene therapy goals. Recombinant AAV possesses advantages such as safety, high efficiency, stability, persistence, specificity, and low integration, making it one of the main delivery methods in gene therapy. However, in gene therapy applications, live animals often need to be injected with high doses of high-purity AAV virus. Simultaneously, high doses of AAV can lead to toxicity in animals, becoming a major bottleneck for the marketization of AAV gene therapy products. Furthermore, with the maturation of downstream gene therapy research technologies, the limitations of upstream AAV production throughput and dosage are becoming increasingly apparent. To address this issue, existing strategies focus on two aspects: first, optimizing existing AAV production processes to increase virus production throughput; and second, searching for variant viruses with stronger packaging capabilities and greater infectivity in live tissues to increase production throughput while reducing the required virus dosage. Developments in production processes can only increase virus yield, but cannot fundamentally improve the limitations of AAV dosage. This patent modifies the AAV2 capsid protein to obtain an AAV2 mutant with stronger viral packaging and in vivo tissue infection capabilities, thereby fundamentally solving the problems of AAV2 production throughput and dosage. Summary of the Invention

[0003] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a mutant of the adeno-associated virus capsid protein.

[0004] This invention also proposes a nucleic acid molecule.

[0005] The present invention also proposes an expression cassette, recombinant vector, or transgenic cell comprising the above-mentioned variant AAV capsid protein or nucleic acid molecule.

[0006] The present invention also proposes a recombinant AAV variant.

[0007] The present invention also proposes a pharmaceutical composition comprising the above-mentioned recombinant AAV variant, expression cassette, recombinant vector or transgenic cells.

[0008] The present invention also proposes the application of the above-mentioned variant AAV capsid protein, recombinant AAV variant, expression cassette, recombinant vector, transgenic cell or pharmaceutical composition.

[0009] According to a first aspect of the invention, a mutant of the adeno-associated virus capsid protein is provided, which, compared with the amino acid sequence of the wild-type AAV capsid protein, has one or more amino acid substitutions at amino acids 561 to 588 of Cap2 (Seq ID NO.1).

[0010] In some embodiments of the present invention, the one or more amino acid substitutions are located at one or more positions selected from the following:

[0011] (1) Serine at amino acid position 578;

[0012] (2) Threonine at the 581st amino acid position.

[0013] In some embodiments of the present invention, the nucleotide sequence of the mutant Cap2 is shown in Seq ID NO.2.

[0014] In some embodiments of the present invention, the amino acid sequence of Cap2 of the mutant is as shown in SEO ID NO.3.

[0015] According to a second aspect of the invention, a nucleic acid molecule encoding the above-described mutant is proposed.

[0016] According to a third aspect of the present invention, expression cassettes, recombinant vectors, or transgenic cells comprising the aforementioned nucleic acid molecules are proposed.

[0017] A recombinant vector comprising the aforementioned nucleic acid molecules.

[0018] According to a fourth aspect of the invention, a recombinant AAV variant is provided, the recombinant AAV variant comprising the above-described adeno-associated virus variant capsid protein or nucleic acid molecule.

[0019] According to a fifth aspect of the present invention, a pharmaceutical composition is provided comprising at least one of the above-described recombinant AAV variant, nucleic acid molecule, expression cassette, recombinant vector, or transgenic cell.

[0020] In some embodiments of the present invention, the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

[0021] According to a sixth aspect of the invention, the above-described variant AAV capsid protein, recombinant AAV variant, nucleic acid molecule, expression cassette, recombinant vector, transgenic cell, or pharmaceutical composition are proposed for use in the preparation of products that deliver nucleic acid molecules encoding functional gene products into cells and / or tissues.

[0022] A method for generating a somatic cell transgenic animal model includes administering the aforementioned recombinant AAV variant to a non-human animal, wherein the recombinant AAV variant contains at least one transgene, and wherein the recombinant AAV variant infects cells of a target tissue of the non-human animal.

[0023] According to the embodiments of the present invention, the variant AAV capsid protein has at least the following beneficial effects: the adeno-associated virus variant prepared by replacing amino acids 561-588 of the wild-type AAV2 capsid protein Cap2 with a designed amino acid sequence can significantly improve the AAV2 virus packaging capacity and in vivo tissue infection capacity, fundamentally solving the problems of AAV2 production throughput and dosage. Attached Figure Description

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

[0025] Figure 1 This is a sequence alignment diagram of wild-type Cap561-588 and MutB in an embodiment of the present invention;

[0026] Figure 2 This is a plasmid map of the virus packaging three-plasmid system in an embodiment of the present invention, where A is the pAAV2-Rep2 / Cap2 packaging plasmid map; B is the constructed pAAV2-MutB-Rep2 / Cap2 packaging plasmid map; C is the pAAV2-ITR-CMV-Luciferase-BGH pA-ITR expression plasmid map; and D is the pHelper helper plasmid map.

[0027] Figure 3 The graph shows the detection results of the total viral particle count of the adeno-associated virus variant prepared by the present invention in this test example and the wild-type adeno-associated virus control group.

[0028] Figure 4 The graph shows the detection results of fluorescence intensity and location of the adeno-associated virus variant in this test case and the wild-type adeno-associated virus in the control group;

[0029] Figure 5 The graph shows the fluorescence intensity results of the adeno-associated virus variant in this test case and the wild-type adeno-associated virus in the control group. Detailed Implementation

[0030] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as 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, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0031] Example 1

[0032] This embodiment provides an adeno-associated virus variant (AAV2-MutB), the preparation of which includes the following steps:

[0033] 1. Plasmid construction

[0034] The MutB sequence was synthesized by gene synthesis (Sangon Biotech Co., Ltd.). The synthesized sequence was cloned into the pAAV2-Rep2 / Cap2 plasmid by double digestion with BsiWI and XcmI. The amino acids at positions 561-588 of Cap2, as shown in SEQ ID NO.1, were replaced with the mutant sequence shown in SEQ ID NO.3 to construct the pAAV2-mutant plasmid vector. The ligation product was transformed into competent E. coli DH5α cells, and single colonies were picked for enzyme digestion verification and sequencing verification.

[0035] The sequence alignment results of the MutB sequence of the adeno-associated virus variant capsid protein and the wild-type AAV2 capsid protein are shown in the figure below. Figure 1 As shown in SEQ ID NO. 1, the amino acid sequence of the adeno-associated virus variant capsid protein differs from that of the wild-type AAV2 capsid protein in that the wild-type AAV2 capsid protein sequence is replaced by two amino acids, D578 and E581. The amino acid sequence of the wild-type adeno-associated virus capsid protein Cap2 is shown in SEQ ID NO. 2.

[0036] MutB sequence: DEEEIRTTNPVATEQYGDVSENLQRGNR (as shown in SEQ ID NO.3).

[0037] 2. Packaging of mutant viruses such as AAV2-WT and AAV2-MutB

[0038] The correctly sequenced AAV2-WT plasmid and AAV2-mutant plasmid were extracted and transfected into HEK293 cells using a viral packaging three-plasmid system. The plasmid map is shown below. Figure 2 As shown, it includes the following steps:

[0039] A cell transfection

[0040] (1) One day before transfection, HEK293 cells were digested with trypsin and counted. The cells were then seeded into T175 cell culture flasks so that their aggregation reached 80%-90% on the day of transfection.

[0041] (2) For each flask of cells, use 3 mL of CaCl2 to dilute the packaging plasmid, pHelper, and ITR-CMV-Luciferase-BGHpA-ITR to a total of 42 μg;

[0042] (3) Add 3 mL of 2x HBSS to the plasmid dilution buffer and mix. Let stand at room temperature for 10 minutes.

[0043] (4) Add the above mixture directly to the cell culture flask, shake the culture flask, and mix gently;

[0044] (5) Incubate at 37°C and 5% CO2 for 72 hours.

[0045] B virus purification

[0046] (1) Viral lysis

[0047] 1) Repeatedly pipet to detach the cells, then centrifuge at 3000g for 10 minutes to separate the supernatant and cells;

[0048] 2) Add 0.245 mL of PEG 8000 to each milliliter of supernatant, mix well, and incubate overnight at 4°C. Resuspend the cells in 2.5 mL of Trislysis buffer, repeat freeze-thaw cycles 5 times, and then incubate overnight at 37°C.

[0049] 3) After 12 hours, centrifuge the supernatant after PEG 8000 concentration at 10°C and 3000g for 15 minutes. After removing the supernatant, resuspend the precipitate in 1.5 mL of Tris lysis buffer. Add DNase to the cell suspension that has been incubated overnight at 37°C, mix well by pipetting, incubate at 37°C for 30 minutes, mix the suspension again by pipetting, and centrifuge at 10°C and 2000g for 15 minutes. Combine the two supernatants into one centrifuge tube.

[0050] (2) Ultracentrifugation

[0051] 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 in sequence. Collect the virus suspension and balance it with Trislysis buffer.

[0052] 2) Centrifuge at 10℃, 230000g, for 18 hours at an ultra-high speed of 8°C and 9°C.

[0053] (3) Virus purification

[0054] 1) Take an ultrafiltration tube and wet the filter membrane with 1 mL of Tris lysis buffer;

[0055] 2) Carefully aspirate the 40% Iodixanol liquid layer from the ultracentrifuge tube using a pipette, avoiding aspirating protein, and transfer it to the ultrafiltration tube;

[0056] 3) Add an appropriate amount of Tris lysis buffer and mix well by pipetting. Centrifuge at 4500g for 20min. Repeat this step until Iodixanol is removed.

[0057] 4) Add 1 mL of Tris lysis buffer and pipette for 40-50 times to form a virus suspension, then transfer to an EP tube;

[0058] 5) Use a 5mL syringe to draw the virus suspension from the EP tube, filter it through a 0.22μm filter, collect 20ul of virus solution as a test sample, and then dispense it into 102μL tubes to obtain the adeno-associated virus variant.

[0059] Comparative Example 1

[0060] This comparative example provides a wild-type adeno-associated virus (AAV2-WT), which differs from the preparation method in Example 1 only in that the adeno-associated virus capsid protein sequence uses the wild-type AAV2 capsid protein sequence.

[0061] Comparative Example 2

[0062] This comparative example provides an adeno-associated virus variant (AAV2-Mut21), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.4.

[0063] Comparative Example 3

[0064] This comparative example provides an adeno-associated virus variant (AAV2-Mut22), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.5.

[0065] Comparative Example 4

[0066] This comparative example provides an adeno-associated virus variant (AAV2-Mut23), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.6.

[0067] Comparative Example 5

[0068] This comparative example provides an adeno-associated virus variant (AAV2-Mut24), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.7.

[0069] Comparative Example 6

[0070] This comparative example provides an adeno-associated virus variant (AAV2-Mut25), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.8.

[0071] Comparative Example 7

[0072] This comparative example provides an adeno-associated virus variant (AAV2-Mut26), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.9.

[0073] Comparative Example 8

[0074] This comparative example provides an adeno-associated virus variant (AAV2-Mut27), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.10.

[0075] Comparative Example 9

[0076] This comparative example provides an adeno-associated virus variant (AAV2-Mut28), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.11.

[0077] Comparative Example 10

[0078] This comparative example provides an adeno-associated virus variant (AAV2-Mut29), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.12.

[0079] Comparative Example 11

[0080] This comparative example provides an adeno-associated virus variant (AAV2-Mut30), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.13.

[0081] Comparative Example 12

[0082] This comparative example provides an adeno-associated virus variant (AAV2-Mut31), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.14.

[0083] Comparative Example 13

[0084] This comparative example provides an adeno-associated virus variant (AAV2-Mut32), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.15.

[0085] Comparative Example 14

[0086] This comparative example provides an adeno-associated virus variant (AAV2-Mut33), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.16.

[0087] Comparative Example 15

[0088] This comparative example provides an adeno-associated virus variant (AAV2-Mut34), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.17.

[0089] Comparative Example 16

[0090] This comparative example provides an adeno-associated virus variant (AAV2-Mut35), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.18.

[0091] Comparative Example 17

[0092] This comparative example provides an adeno-associated virus variant (AAV2-Mut36), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.19.

[0093] Comparative Example 18

[0094] This comparative example provides an adeno-associated virus variant (AAV2-Mut37), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.20.

[0095] Comparative Example 19

[0096] This comparative example provides an adeno-associated virus variant (AAV2-Mut38), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.21.

[0097] Comparative Example 20

[0098] This comparative example provides an adeno-associated virus variant (AAV2-Mut39), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.22.

[0099] Comparative Example 21

[0100] This comparative example provides an adeno-associated virus variant (AAV2-Mut40), which differs from the preparation method of Example 1 only in that amino acids 561-588 of Cap2 shown in SEQ ID NO.1 are replaced with the mutant sequence shown in SEQ ID NO.23.

[0101] The amino acid substitution sequences at positions 561-588 of the adeno-associated virus variant Cap2 are shown in Table 1.

[0102] Table 1

[0103]

[0104]

[0105] Test case

[0106] 1. Virus titer detection

[0107] (1) Viral lysis

[0108] 1) Take 20 μL of the virus samples from Example 1 and Comparative Examples 1-21 respectively;

[0109] 2) Add 1 μL each of 10% SDS, 0.5 mol / L EDTA, and proteinase K, and mix well;

[0110] 3) Incubate at 56°C for 1 hour in a constant temperature mixer, then incubate at 90°C for 10 minutes;

[0111] 4) Take 2 μL of lysis buffer, dilute it 500 times, and set aside.

[0112] (2) Preparation of standards for standard curves

[0113] Take 2×10 of the original solution 12 Plasmids of copies / mL were diluted with ultrapure water (ddH2O) to create six gradients as templates for standards. (2×10⁻⁶) 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.

[0114] (3) Absolute quantitative qPCR

[0115] 1) Take 0.2 mL PCR tubes and prepare the following reaction system, making 3 tubes for each viral lysis dilution product. 2×qPCRMix 10 μL; forward and reverse primers 0.4 μL each; viral lysis dilution product 5 μL; ddH2O 4.2 μL.

[0116] Amplification primers:

[0117] Forward primer 5'-TCATATGCCAAGTACGCCCC-3' (SEQ ID NO.24)

[0118] Reverse primer 5'-CCCGTGAGTCAAACCGCTAT-3' (SEQ ID NO.25)

[0119] 2) PCR amplification

[0120] Pre-denaturation: 95℃, 2 min;

[0121] 40 cycles: 95℃, 15s; 60℃, 60s.

[0122] 3) qPCR data processing: Virus titer = dilution factor * viral gene array copy number.

[0123] Experimental results are as follows Figure 3 As shown in the figure, the present invention transfects the AAV2-WT virus packaging three-plasmid system (containing three plasmids: pAAV2-Rep2 / Cap2, pAAV2-ITR-CMV-Luciferase-BGH pA-ITR, and pHelper) and the AAV2-mutant virus packaging three-plasmid system (containing three plasmids: pAAV2-mutant plasmid, pAAV2-ITR-CMV-Luciferase-BGH pA-ITR, and pHelper) into HEK293 cells in equal amounts. Among them, the total amount of AAV2-MutB virus particles is increased by 63.6% compared with AAV2-WT, and the total amount of AAV2-MutA virus particles is significantly higher than that of the adeno-associated virus variant (AAV2-Mut21-40) prepared in Comparative Examples 2-21, proving that the adeno-associated virus variant prepared by the present application can significantly improve the AAV2 virus packaging capacity.

[0124] 2. Detection of infectivity in mouse tissues

[0125] The adeno-associated virus variant prepared in Example 1 and the adeno-associated virus prepared in Comparative Example 1 were used to detect their infectivity in mouse tissues using a mouse model. The detection method included the following steps:

[0126] (1) Mouse virus injection

[0127] A. Preparation before the experiment

[0128] Preparation materials: 8 healthy 4-6 week old C57BL / 6J mice (from Cyagen Guangzhou Biotechnology Co., Ltd.), mouse restraints, 1mL centrifuge tubes, alcohol swabs, virus (thawed in an ice bath), and physiological saline.

[0129] B mice were fixed

[0130] 1) First, lift the mouse by its tail and place it on the lid of the cage;

[0131] 2) Then the mouse is placed in the restraint, the lid is closed, and the tail is left sticking out.

[0132] 3) Wipe the mouse's tail with an alcohol swab to dilate its blood vessels;

[0133] C virus injection

[0134] 1) Dilute AAV2-WT and AAV2-MutB viruses to 5×10⁻⁶ mcg using physiological saline. 9 vg / ul;

[0135] 2) Draw 200 μL of virus into a 1 mL syringe and inject 1 × 10⁻⁶ virus into each mouse. 11 vg virus, of which AAV2-WT virus was injected into 3 C57BL / 6J mice and AAV2-MutB virus was injected into 4 C57BL / 6J mice;

[0136] 3) Straighten the mouse's tail to make its red veins clearly visible, insert the needle at 1 / 3 of the distance from the tail, slowly inject the virus, and then pull it out. Press the injection point with an alcohol swab for about 1 minute to stop the bleeding.

[0137] D Animal Recovery

[0138] Remove the mouse from the restraints, observe its condition, and then return it to its original cage.

[0139] (2) Mouse in vivo imaging

[0140] In vivo imaging of mice was performed 8 days after viral injection, and included the following steps:

[0141] Intraperitoneal injection of mouse fluorescent substrate D-Luciferin

[0142] 1) Prepare D-Luciferin working solution (15 mg / mL) using DPBS, and filter aseptically through a 0.2 μm filter membrane;

[0143] 2) Inject the working solution at a rate of 10 μL / g body weight;

[0144] 3) Perform downstream anesthesia procedures 10-15 minutes after intraperitoneal injection;

[0145] Mouse B was anesthetized

[0146] 1) Turn on the anesthesia machine;

[0147] 2) Adjust the induction concentration to about 3%, and after about 1 minute, the anesthetic will fill the induction box;

[0148] 3) Place the animal in the induction box and close it. Wait approximately 2-3 minutes for the animal to be fully anesthetized.

[0149] 4) Adjust the concentration range to 1-1.5%, remove the animal from the induction box, place its head / nose in the anesthesia mask to fix it, and check whether the animal is completely anesthetized (you can pinch the animal's paws or tail with two fingers; if the animal does not react, it indicates that the animal is completely anesthetized, and you can start the surgery and other experiments at this time).

[0150] 5) After the animal experiment is completed, turn off the evaporator;

[0151] 6) Keep the animal breathing in pure oxygen for about 5-10 minutes to help it recover quickly.

[0152] In vivo imaging of mouse C

[0153] 1) Open the software and select the imaging field of view;

[0154] 2) Focus the sample: Focus the sample, selecting 10-20mm for mice;

[0155] 3) Confirm exposure time: Shoot a black and white outline of the sample; the exposure time is the default.

[0156] 4) Take a picture of the Luciferase-labeled sample (or the self-luminous sample labeled with luminescent bacteria), select "Luminous Photography": Exposure time Auto, Binning 8*8;

[0157] 5) Preview the sample: Check the sample placement;

[0158] 6) Start taking pictures: Obtain the image results according to the preset parameters.

[0159] (3) Mouse recovery

[0160] After taking photos, observe the animal's condition and then return it to its original cage.

[0161] Experimental results are as follows Figure 4 and Figure 5As shown in the figure, C57BL / 6J mice injected with AAV2-MutB virus exhibited significantly stronger Luciferase protein expression than C57BL / 6J mice injected with AAV2-WT virus. The results showed that the average Luciferase fluorescence value of AAV2-WT mice was 4.57E+03, while that of AAV2-MutB mice was 5.4E+04. The average Luciferase fluorescence value of AAV2-MutB mice was 11.8 times that of AAV2-WT mice. Furthermore, the Luciferase fluorescent protein expression site was mainly in the liver region, indicating that AAV2-MutB can rapidly infect mouse liver tissue and rapidly express the protein. In conclusion, this demonstrates that the MutB amino acid sequence can significantly enhance the AAV2 virus's ability to infect mouse tissues.

[0162] In summary, the adeno-associated virus variant prepared by the present invention significantly enhances the AAV2 virus packaging capacity and mouse tissue infection capacity compared to the wild type.

[0163] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified. SEQUENCE LISTING <110> Cyagen (Guangzhou) Biotechnology Co., Ltd. <120> A mutant of adeno-associated virus capsid protein and its application <130> <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 735 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 1 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190 Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445 Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 450 455 460 Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly 465 470 475 480 Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn 485 490 495 Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505 510 Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp 515 520 525 Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys 530 535 540 Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr 545 550 555 560 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575 Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr 580 585 590 Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp 595 600 605 Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr 610 615 620 Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys 625 630 635 640 His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn 645 650 655 Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln 660 665 670 Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys 675 680 685 Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr 690 695 700 Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr 705 710 715 720 Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 2 <211> 735 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190 Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445 Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 450 455 460 Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly 465 470 475 480 Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn 485 490 495 Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505 510 Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp 515 520 525 Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys 530 535 540 Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr 545 550 555 560 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575 Gly Asp Val Ser Glu Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr 580 585 590 Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp 595 600 605 Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr 610 615 620 Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys 625 630 635 640 His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn 645 650 655 Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln 660 665 670 Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys 675 680 685 Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr 690 695 700 Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr 705 710 715 720 Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 3 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 3 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 1 5 10 15 Gly Asp Val Ser Glu Asn Leu Gln Arg Gly Asn Arg 20 25 <210> 4 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 4 Asp Cys Asn Glu Ile Ala Ala Thr Asn Pro Val Ala Thr Glu Gln Tyr 1 5 10 15 Gly Glu Ala Ala Thr Asn Leu Gln Arg Gly Asn Arg 20 25 <210> 5 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 5 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gly Tyr 1 5 10 15 Gly Glu Ala Ala Thr Asn Leu Gln Arg Gly Gly His 20 25 <210> 6 <211> 29 <212> PRT <213> Artificial Sequence[[ID=�0]] <220> <223> Artificial sequence <400> 6 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ser Thr Glu Gln Tyr 1 5 10 15 Gly Glu Val Ser Thr Asn Leu Gln Arg Gly Thr Arg Ile 20 25 <210> 7 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 7 His Cys Glu Asn Glu Ile Ala Ala Thr Asn Pro Val Ser Thr Glu Gln ^1 5 10 15 Tyr Gly Glu Ala Ala Thr Asn Leu Gln Arg Gly Asn Thr 20 20 25 <210> 8 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 8 Asp Glu Gln Glu Ile Ala Thr Thr Asn Pro Val Ala Thr Glu Gly Tyr 1 5 10 15 Gly Glu Val Ser Thr Asn Leu Gln Arg Ala Asn Gly 20 25 <210> 9 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 9 Asp Glu Glu Glu Ile Arg Ala Thr Asn Pro Val Ser Thr Glu Gln Tyr 1 5 10 15 Gly Glu Ala Ala Thr Asn Leu Gln Arg Gly Asn Met 20 25 <210> 10 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 10 Asp Glu Gln Glu Ile Ala Thr Thr Asn Pro Val Ala Thr Glu Val Tyr 1 5 10 15 Gly Glu Ala Ala Thr Asn Leu Gln Arg Ala Asn Gln Arg Gly 20 25 30 <210> 11 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 11 Thr Glu Glu Glu Ile Ala Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 1 5 10 15 Gly Asp Val Ser Glu Asn Leu Gln Arg Gly Asn His Ser 20 25 <210> 12 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 12 Asp Ser Gly Glu Ile Ala Ala Thr Asn Pro Val Ser Thr Glu Gln Tyr 1 5 10 15 Gly Glu Val Ser Thr Asn Leu Gln Arg Gly Thr Arg Asp 20 25 <210>= 13 <211> 28 <212> PRT <213> Artificial Sequence<000067​​​​​​Asp Glu Ser Glu Ile Ala Ala Thr Asn Pro Val Ala Thr Glu Gln Tyr 1 5 10 15 Gly Glu Val Ser Thr Asn Leu Gln Arg Gly Asn Ala 20 25 <210> 14 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 14 Ala Asp Cys Asn Glu Ile Ala Ala Thr Asn Pro Val Ala Thr Glu Gln 1 5 10 15 Tyr Gly Glu Ala Ser Thr Asn Leu Gln Arg Gly Asn Ala Lys 20 25 30 <210> 15 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 15 Ala Glu Asp Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 1 5 10 15 Gly Asp Val Ser Thr Asn Leu Gln Arg Gly Asn Ile 20 25 <210> 16 <211> 29 <212> PRT [[ID=५८]]<213> Artificial Sequence <220> <223> Artificial sequence <400> 16 天冬氨酸-谷氨酸-谷氨酸-谷氨酸-异亮氨酸-精氨酸-丙氨酸-苏氨酸-天冬酰胺-脯氨酸-缬氨酸-丝氨酸-苏氨酸-谷氨酸-谷氨酰胺-酪氨酸 1 5 10 15 甘氨酸-谷氨酸-丙氨酸-丙氨酸-苏氨酸-天冬酰胺-亮氨酸-谷氨酰胺-精氨酸-甘氨酸-天冬酰胺-丙氨酸-组氨酸 20 25 <210> 17 <211> 29 <212> PRT <213> 人工序列 <220> <223> Artificial sequence <400> ;17 天冬氨酸-丝氨酸-甘氨酸-谷氨酸-异亮氨酸-丙氨酸-苏氨酸-苏氨酸-天冬酰胺-脯氨酸-缬氨酸-丝氨酸-苏氨酸-谷氨酸-谷氨酰胺-酪氨酸 1 5 10 15 甘氨酸-谷氨酸-丙氨酸-丙氨酸-苏氨酸-天冬酰胺-亮氨酸-谷氨酰胺-精氨酸-甘氨酸-甲硫氨酸-脯氨酸-组氨酸 20 25 <210> 18 <211> 29 <212> PRT <213> 人工序列 <220> <223> Artificial sequence <400> 18 丙氨酸-丝氨酸-甘氨酸-谷氨酸-异亮氨酸-丙氨酸-丙氨酸-苏氨酸-天冬酰胺-脯氨酸-缬氨酸-丙氨酸-苏氨酸-谷氨酸-缬氨酸-酪氨酸 1 5 10 15<000073\0>甘氨酸-谷氨酸-丙氨酸-丙氨酸-苏氨酸-天冬酰胺-亮氨酸-谷氨酰胺-精氨酸-甘氨酸-天冬酰胺-丙氨酸-组氨酸 20 25 <210> 19 <211> 28 <212> PRT <213> 人工序列 <220> <223> Artificial Sequence <400> 19 Asp Glu Glu Glu Ile Ala Thr Thr Asn Pro Val Ser Thr Glu Gln Tyr 1 5 10 15 Gly Glu Ala Ala Thr Asn Leu Gln Arg Gly Gly Arg 20 25 <210> 20 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 20 Thr Glu Glu Glu Ile Ala Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 1 5 10 15 Gly Asp Val Ser Glu Asn Leu Gln Arg Gly Asn Ile 20 25 <210> 21 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 21 Ala Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Ser Tyr 1 5 10 15 Gly Glu Val Ser Thr Asn Leu Gln Arg Gly Gly His 20 25 <210> 22 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 22 Ser Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 1 5 10 15 Gly Glu Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln 20 25 <210> 23 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 23 Ser Val Ser Gly Glu Ile Ala Thr Thr Asn Pro Val Ala Thr Glu Val 1 5 10 15 Tyr Gly Glu Ala Ser Thr Asn Leu Gln Arg Gly Asn Ala His 20 25 30

Claims

1. A mutant of an adeno-associated virus capsid protein, characterized in that, Compared to the amino acid sequence of the wild-type AAV capsid protein, substitutions occurred at amino acid positions 578 and 581. The amino acid sequence of this mutant is shown in SEOID NO.

2.

2. A nucleic acid molecule encoding the mutant of claim 1.

3. An expression cassette, recombinant vector, or transgenic cell comprising the nucleic acid molecule as described in claim 2.

4. A recombinant adeno-associated virus variant, characterized in that, The recombinant adeno-associated virus variant comprises a mutant of the adeno-associated virus capsid protein of claim 1 or a nucleic acid molecule of claim 2.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of the expression cassette, recombinant vector, or transgenic cell as described in claim 3, or the recombinant adeno-associated virus variant as described in claim 4.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition further includes any one or a combination of at least two of pharmaceutically acceptable carriers, excipients, or diluents.

7. The use of the mutant of the adeno-associated virus capsid protein of claim 1, the nucleic acid molecule of claim 2, the expression cassette, recombinant vector or transgenic cell of claim 3, the recombinant adeno-associated virus variant of claim 4, or the pharmaceutical composition of claim 5 or 6 in the preparation of products that deliver nucleic acid molecules encoding functional gene products to cells and / or tissues.

Images