An AAV viral vector for nervous system drug delivery

Abstract

The present invention relates to a novel AAV vector comprising an insertion of the polypeptide sequence WPTSYDA corresponding to amino acid position 588 of AAV9, with H527 mutated to Y and R533 mutated to S. Furthermore, the promoter used to express the target gene is hSyn. Compared with wild-type AAV9 vectors, this AAV9.7YS mutant has improved blood-brain barrier penetration and, after intravenous injection, is more potent in infecting the nervous system, while exhibiting lower infection efficiency in peripheral tissues (heart, liver, and skeletal muscle). The present invention also relates to methods for packaging and preparing the disclosed viral vector and its use in gene therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a novel AAV virus for delivering drugs to the nervous system. Background Art

[0002] Adeno-associated virus (AAV) is a small, non-pathogenic virus found in humans and other mammals. AAV consists of an icosahedral capsid approximately 26 nm in diameter and a 4.7 kb single-stranded DNA genome. Due to its efficient infectivity of both dividing and non-dividing cells and its specific tissue tropism in vivo, recombinant AAV (rAAV) has been developed as a gene delivery vector. The discovery of numerous AAV serotypes and variants from humans and primates has greatly facilitated the development of AAV vectors, providing a wide range of options for targeting different tissues and cells.

[0003] Of all the AAV serotypes discovered to date, AAV9 is highly sought after in gene therapy applications due to its high tissue tropism for the liver, heart, and skeletal muscle. Furthermore, it has the ability to cross the blood-brain barrier (BBB) ​​and infect nerve cells, making it widely used in gene therapy for neurological diseases. Currently, Novartis' ZOLGENSMA gene therapy drug, based on AAV9, has been approved by the FDA for marketing. This gene therapy uses AAV9 to carry the therapeutic gene SMN1 for the treatment of spinal muscular atrophy patients under the age of two. Clinical trial evidence shows that Zolgensma can not only save patients' lives, but also enable some patients to thrive like healthy children. After a single treatment, Zolgensma's efficacy can be maintained for more than five years, providing a potential "once-and-for-all" cure for SMA patients (spinal muscular atrophy).

[0004] AAV capsid protein determines the tissue cell specificity of AAV. Therefore, since AAV was proven to have clinical application prospects, designing new AAV capsids to obtain new characteristics has been a goal that researchers have been pursuing. With the advancement of technology, the strategy for developing new capsids has gradually evolved. At present, the capsid protein development strategies are mainly divided into rational design, directed evolution and computer-aided design. Improving the targeting and efficient transduction of target tissue cells is an important strategy to reduce toxic side effects in AAV gene therapy. Although AAV9 can cross the blood-brain barrier and infect nerve cells, its infection efficiency is still very low. After intravenous injection of AAV9 virus, a large amount of AAV9 virus will infect myocardial, skeletal muscle and liver cells. The proportion of AAV9 virus that crosses the blood-brain barrier and infects nerve cells is still relatively small. This phenomenon has been verified in many laboratories at home and abroad.

[0005] Therefore, there is an urgent need in this field to develop a new AAV vector that can efficiently infect nerve cells, inefficiently infect muscle, myocardial and liver cells, and has a stronger ability to cross the blood-brain barrier. Summary of the Invention

[0006] In order to solve the problems described in the background art, the present invention provides a novel AAV viral vector that can efficiently infect nerve cells, inefficiently infect muscle, myocardial and liver cells, and has a stronger ability to cross the blood-brain barrier.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The PCR site-directed insertion technology was used to perform site-directed insertion of a specific amino acid sequence SEQ ID NO: 4 (wherein, the specific nucleic acid sequence was SEQ ID NO: 5) after the 558th position of the AAV9 capsid sequence, i.e., the AAV9 wild-type capsid protein VP1 amino acid sequence SEQ ID NO: 2 (wherein the DNA sequence of the AAV9 wild-type capsid protein VP1 was SEQ ID NO: 3); the inserted AAV9 viral vector was then transformed and the plasmid was extracted to obtain an AAV9 capsid protein sequence of which the amino acid sequence was SEQ ID NO: 6 and the DNA sequence was SEQ ID NO: 7.

[0009] Based on the above sequence, site-directed mutagenesis primers spanning the mutation site were designed, and then PCR technology was used to perform site-directed mutagenesis on sites 527 and 533 on the AAV9 capsid sequence. The amino acid H527 of the AAV9 capsid protein was mutated to Y, and the amino acid R533 was mutated to S. Finally, the mutated AAV9 viral vector was transformed and the plasmid was extracted to obtain the site-directed mutagenesis AAV9 capsid protein sequence, of which the amino acid sequence is SEQ ID NO: 8 and the DNA sequence is SEQ ID NO: 9.

[0010] Based on the above sequence, the promoter of the target gene of the AAV viral vector was changed to the hSyn promoter using PCR technology. The hSyn promoter sequence was SEQ ID NO: 10. The vector was then transformed and the plasmid was extracted to obtain an AAV viral vector with a sequence of SEQ ID NO: 1.

[0011] The optimized novel AAV viral vector SEQ ID NO: 1 was packaged by the following method: HEK293T cells were cultured at a cell density of 80-90%; pHelper, pAAV9 / pAAV9.7YS and pAAV-LUC were mixed with Neofect transfection reagent and added to HEK293T cells, mixed and cultured for 72 hours; the cell pellet and supernatant were separated by centrifugation, the cell pellet was lysed by repeated freeze-thaw method to release the AAV virus, and finally the AAV crude extract was purified by iodixanol density gradient ultracentrifugation to obtain the AAV virus.

[0012] The packaged new AAV virus was ultrafiltrated and concentrated, and the titer of the AAV virus was determined by real-time fluorescence quantitative PCR technology. The calculation formula was copy number (copies / μL) = concentration (ng / μL)*10^(-9)*6.02*10^23 / base pair number bp*650, where 6.02*10^23 is Avogadro's constant, that is, the number of molecules in 1 mol of substance. Subsequently, the lg value of the genome copy number was used as X and the Ct value was used as Y to establish a standard curve to calculate the titer of the sample.

[0013] Western blotting was used to detect the expression of three components of AAV virus capsid: VP1, VP2, and VP3, so as to evaluate the packaging and purification effect of AAV virus.

[0014] The tissue and organ distribution of the new AAV viral vector was evaluated in mice. The method was to inject AAV virus carrying luciferase through the mouse tail vein and detect the distribution of AAV virus in the mouse body by small animal live imaging; the brain, heart, liver, spleen, lung, kidney, and muscle tissues of the mice were obtained to detect the enzyme activity of luciferase and the copy number of the AAV viral genome in each tissue and organ to evaluate the specificity of the new AAV vector AAV9.7YS in infecting the nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Agarose gel electrophoresis diagram of AAV9 and AAV9.7YS capsid protein particles.

[0016] Figure 2 Standard curve for AAV virus titer detection and titers of AAV9 and AAV9.7YS samples.

[0017] Figure 3 Western blot was used to detect AAV virus capsid proteins VP1, VP2, and VP3.

[0018] Figure 4 Distribution of AAV9 and AAV9.7YS viruses in living mice.

[0019] Figure 5 These are the brain imaging and quantitative results after AAV9 and AAV9.7YS infection of mice.

[0020] Figure 6 This is a diagram of luciferase enzyme activity in various tissues and organs of mice.

[0021] Figure 7 This is a diagram of the AAV9 and AAV9.7YS viral genome copy numbers in various tissues and organs of mice. DETAILED DESCRIPTION

[0022] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0023] The purchase manufacturers and reagent formulas of the reagents and kits involved in the examples are as follows:

[0024] 5×Prime STAR @ Buffer (Mg 2+ Plus): Takara; Prime STAR @ HS DNA Polymerase: Takara; dNTP Mixture: Takara;

[0025] DpnI enzyme: Takara;

[0026] Plasmid extraction kit: Beijing Zhuangmeng Company;

[0027] DMEM medium: Thermo Fisher Scientific;

[0028] Neofect transfection reagent: Beijing Mayin Technology Co., Ltd.;

[0029] Fetal bovine serum: Biological Industries

[0030] 10×PBS-MK buffer 100mL: 10×100mL PBS solution + 0.2g MgCl2·6H2O + 0.19g KCl, dissolved;

[0031] PBS: Weigh 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 and dissolve them in 800 mL distilled water. Adjust the solution to 7.4 with HCl and finally add distilled water to 1 L. Autoclave and store at 4°C.

[0032] 0.5% phenol red solution: weigh 0.1 g of phenol red powder in 20 mL of 50% ethanol and heat to dissolve;

[0033] 60% iodixanol stock solution: Stem cell;

[0034] 0.001% PF68: Sigma-Aldrich;

[0035] 2X SYBR Green Mix Buffer: TransGen Biotech;

[0036] 5× loading buffer: 200 mM DTT, 4% SDS, 100 mM Tris-HCl pH 6.8, 0.2% bromophenol blue, 20% glycerol;

[0037] 30% Acrylamide solution (Acr:Bis = 29:1): Dissolve 29g acrylamide and 1g methylenebisacrylamide in 60ml of water. Heat to 37°C to dissolve, then add water to a final volume of 100ml. Sterilize by filtering through a 0.45µm filter and store in a brown bottle at room temperature.

[0038] 1M Tris-HCl pH 6.8: Weigh 121.1 g Tris into a 1000 ml beaker, add approximately 800 ml of deionized water, and stir thoroughly to dissolve. Adjust the pH to 6.8 with concentrated HCl, and bring the solution to 1000 ml. Sterilize by autoclaving and store at room temperature.

[0039] 10% SDS: Dissolve 100 g of SDS (Sodium dodecyl sulfate crystals) in 900 mL of purified water. Heat to 68°C to dissolve the SDS crystals. Add purified water to 1 L, aliquot, and store at room temperature.

[0040] 10% APS: Dissolve 0.5 g APS in 5 ml deionized water and store at 4°C in the dark.

[0041] TEMED: Thermo Fisher Scientific;

[0042] Transfer buffer: 24 mM Tris, 20% methanol, 190 mM glycine;

[0043] Nitrocellulose membrane: GE Healthcare;

[0044] 1× TBST: Tris-HCl (1 M, pH 7.5) 50 mL, NaCl 8 g, KCl 0.2 g, Tween 0.5 ml, distilled water to 1 L;

[0045] CAP primary antibody: American Research Products;

[0046] ECL reaction solution: Thermo Fisher Scientific;

[0047] 3% chloral hydrate anesthesia solution: add 3 g of chloral hydrate powder to 100 mL of PBS buffer, mix well and filter sterilize through a 0.22 μM filter;

[0048] 30mg / ml Luciferase substrate working solution: Add 90mg Luciferin powder to 3mL PSB buffer, mix well, filter through a 0.22μM filter to sterilize, and store in the dark.

[0049] Genomic DNA Kit (containing RNase A) (EE101-01): Transgene;

[0050] RIPA lysis buffer: Beijing Solebow Technology Co., Ltd.;

[0051] 4% paraformaldehyde: Beijing Solebow Technology Co., Ltd.;

[0052] 0.05% Triton X-100 solution: Sigma-Aldrich; BSA: Beijing Biolab Technology Co., Ltd.;

[0053] Anti-fluorescence quenching mounting fluid: Invitrogen.

[0054] Example 1

[0055] A specific sequence (amino acid sequence SEQ ID NO: 4, DNA sequence SEQ ID NO: 5) was inserted into the AAV9 capsid protein sequence (AAV9 wild-type capsid protein VP1 amino acid sequence SEQ ID NO: 2, AAV9 wild-type capsid protein VP1 DNA sequence SEQ ID NO: 3) after position 558. The specific steps are as follows:

[0056] (1) Design a pair of primers containing a specific insertion sequence, the front primer sequence is SEQ ID NO: 11 (sequence is CACCAGAGTGCCCAATGGCCCACCAGCTACGACGCCGCACAGGCGCAGACC), the rear primer sequence is SEQ ID NO: 12 (sequence is

[0057] GGTCTGCGCCTGTGCGGCGTCGTAGCTGGTGGGCCATTGGGCACTCTGGTG);

[0058] (2) Using PCR technology to insert a specific fragment at a specific position, the reaction system is shown in Table 1, and the reaction procedure is shown in Table 2:

[0059] Table 1 PCR reaction system

[0060]

[0061]

[0062] Table 2 PCR reaction procedure

[0063]

[0064] (4) Add 1 μL of DpnI enzyme to the PCR product to digest the original PCR template containing methylation sites and incubate at 37°C for 3 h;

[0065] (5) Take 5 μL of the enzyme digestion product for transformation, pick the colonies and shake them, and obtain the AAV9 viral vector with the specific sequence inserted. The amino acid sequence is SEQ ID NO: 6 and the DNA sequence is SEQ ID NO: 7.

[0066] Example 2

[0067] The H527 on the AAV9 viral vector inserted with a specific sequence was mutated to Y and the R533 was mutated to S. The specific steps are as follows:

[0068] (1) Design a pair of primers containing the target mutation site, the forward primer sequence is SEQ ID NO: 13 (sequence: CCTGCTATGGCCAGCCGCAAAGAAGGAGAGGACCGATTCTTTCCTTTGTCT), and the rear primer sequence is SEQ ID NO: 14 (sequence: AGACAAGGAAAGAATCGGTCCTCTCCTTCTTTGCGGCTGGCCATAGCAGG);

[0069] (2) Amplifying the mutant fragment using PCR technology, the reaction system and procedure are the same as in Example 1;

[0070] (3) Add 1 μL of DpnI enzyme to the PCR product, mix well, and digest at 37°C for 3 h;

[0071] (4) Take 5 μL of the enzyme-digested product for transformation, pick the colonies and shake them, and obtain the mutant AAV9 viral vector with the specific sequence inserted (amino acid sequence SEQ ID NO: 8, nucleic acid sequence SEQ ID NO: 9).

[0072] Example 3

[0073] The promoter of the target gene on the mutant AAV9 viral vector with a specific sequence inserted is replaced with the hSyn promoter, and the steps are as follows:

[0074] (1) Primers were designed based on the hSyn promoter sequence. The forward primer sequence was SEQ ID NO: 15 (CAGAAGCTTAGTGCAAGTGGGTTTTAG), and the rear primer sequence was SEQ ID NO: 16 (CAGCTCGAGCTGCGCTCTCAGGCACGA).

[0075] (2) Amplify the hSyn promoter (hSyn promoter sequence is SEQ ID NO: 10) using PCR technology. The reaction system is shown in Table 1 and the reaction procedure is shown in Table 3 (check whether the table represents the operation steps):

[0076] Table 3 PCR reaction procedure

[0077]

[0078] (3) The PCR amplification products were subjected to 1% agarose gel electrophoresis, and the single and relatively bright PCR band was excised and recovered;

[0079] (4) Select restriction endonucleases Hind III and Xba I to digest the recovered DNA fragments and AAV vectors in a 37°C water bath for 2-3 h. After digestion, observe the digestion effect by agarose gel electrophoresis, and recover the target fragments for subsequent experiments.

[0080] (5) Take the PCR product after enzyme digestion and connect it with the AAV viral vector. The ratio of vector to DNA fragment will affect the connection efficiency. Generally speaking, a molar ratio of vector to DNA fragment of 1:3 to 1:8 will have a better connection efficiency. The connection reaction is as follows: take an appropriate amount of PCR product and vector, add 2 μL of 10× connection buffer, add 1 μL of T4 DNA ligase, and make up to 20 μL with ddH2O. Set the metal bath temperature to 16°C and place the connection reaction solution in the metal bath for 16 hours;

[0081] (6) All ligation products are used for transformation, and the colonies are picked and shaken to obtain AAV viral vectors containing the hSyn promoter.

[0082] Example 4

[0083] Transformation of various plasmids and extraction of plasmid DNA

[0084] (1) Take 2 μL of AAV plasmid containing hSyn promoter, pAAV9 plasmid, and pAAV9.7YS plasmid and add them to 100 μL of competent Escherichia coli respectively;

[0085] (2) Place on ice for 30 min, heat shock at 42°C for 40 s, and then quickly place on ice for 2 min after the heat shock.

[0086] (3) Add 500 μL of pre-cooled LB medium without resistance and culture at 37°C on a shaker at 220 rpm for 1 h;

[0087] (4) After recovery, centrifuge at 3000 rpm for 3 min to precipitate the bacteria, discard part of the supernatant, retain about 80 μL of culture medium, resuspend the E. coli pellet, and use a coating rod to evenly spread it on an LB solid culture plate containing the corresponding antibiotics, and culture it upside down at 37°C overnight;

[0088] (5) Plasmid DNA was extracted using the endotoxin-free plasmid extraction kit provided by Tiangen Biochemical Technology Co., Ltd. The extracted plasmid was analyzed by agarose gel electrophoresis. The results were as follows: Figure 1 As shown, the AAV9.7YS capsid protein plasmid is derived from the AAV9 capsid protein plasmid by inserting the sequence SEQ ID NO: 3 after the Q588 position and by mutating H527 to Y and R533 to S. Both plasmids are approximately 7.3 kb in size, as determined by agarose gel electrophoresis. The extracted plasmid is of good quality, approximately 7.3 kb in size, indicating that the plasmid construction was correct.

[0089] Example 5

[0090] The packaging method of AAV9-LUC (sequence is SEQ ID NO: 3) and AAV9.7YS-LUC virus, the specific steps are as follows:

[0091] (1) HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and incubated in a cell culture incubator at 37°C and 5% CO2 until the logarithmic growth phase. The cells were collected and counted. The cells were inoculated in a 10 cm diameter cell culture dish with an inoculum size of 4 × 10 6 cells, and continue culturing for 20 h until the cell density reaches 80-90%;

[0092] (2) 2 h before plasmid transfection, fresh DMEM medium was replaced for HEK293T cells;

[0093] (3) Mix 10 μg / dish of pHelper, 8 μg / dish of pAAV9 or pAAV9.7YS, and 6 μg / dish of core plasmid pAAV-LUC, add 500 μL of DMEM medium to dilute the plasmid, and dilute Neofect transfection reagent with 500 μL of DMEM medium. Let it stand at room temperature for 5 minutes.

[0094] (4) Mix the diluted plasmid and transfection reagent suspension evenly and let it stand at room temperature for 20 minutes;

[0095] (5) Remove HEK293T cells from the cell culture incubator, add the transfection working solution dropwise to the cells, shake in a cross shape, mix well, and place the cells in the cell culture incubator for further culture;

[0096] (6) After 16-24 hours, gently replace the culture medium and continue culturing;

[0097] (7) After 72 h, the cell culture medium was collected and the adherent cells were harvested using a cell spatula. The cells were centrifuged at 1000 rpm for 10 min. If the cell pellet was not used immediately for downstream experiments, it could be stored at -80 °C for later use.

[0098] (8) The supernatant after centrifugation was mixed with PEG8000 solution and allowed to stand at 4°C for 12-16 h to precipitate the virus in the culture supernatant. The supernatant was centrifuged at 3000 rpm for 90 min, and the precipitate was resuspended in PBS.

[0099] (9) Use repeated freezing and thawing to lyse the cell pellet: Place the cell pellet in a -80°C refrigerator, then quickly transfer it to a 37°C water bath to thaw it. If necessary, use a shaker to lyse the cells more fully. Each cycle takes about 15 minutes. After repeated freezing and thawing 3 times, the virus released by the cells can be collected.

[0100] (10) Combine the AAV viruses obtained in steps (9) and (10), add universal nuclease to a concentration of 50 U / mL, and digest at 37°C for 2 h;

[0101] (11) Centrifuge at 10,000 g for 10 min at room temperature, and transfer the supernatant to a new 4 ml centrifuge tube in a biosafety cabinet to obtain crude AAV9-LUC and AAV9.7YS-LUC virus extracts.

[0102] Example 6

[0103] The purification method of the crude AAV9-LUC and AAV9.7YS-LUC virus extracts in Example 5 is as follows:

[0104] (1) Prepare iodixanol solutions of different concentrations, as shown in Table 5:

[0105] Table 5 Iodixanol solution formulas of different concentrations

[0106]

[0107] (2) Add the crude AAV9-LUC and AAV9.7YS-LUC virus extracts to a 10 ml ultracentrifuge tube and add 0.001% PF68;

[0108] (3) Layer the tubes in the following order: 3 mL of 15% iodixanol solution; 2 mL of 25% iodixanol solution; 2 mL of 40% iodixanol solution; 1 mL of 60% iodixanol solution, and then add 1X PBS-MK to the top of the tube to level it.

[0109] (4) Ultracentrifugation at 60,000 rpm and 18°C ​​for 2 h;

[0110] (5) Carefully remove the ultracentrifuge tube, carefully aspirate the colorless 40% iodixanol layer with a syringe, and collect it into a clean EP tube or a 15 mL centrifuge tube to obtain the purified AAV9-LUC and AAV9.7YS-LUC viruses.

[0111] Example 7

[0112] Ultrafiltration concentration of AAV9-LUC and AAV9.7YS-LUC viruses, the specific steps are as follows:

[0113] (1) Add the iodixanol solution containing AAV9-LUC and AAV9.7YS-LUC viruses collected in Example 6 to a 100 kDa ultrafiltration centrifuge tube, add PBS to the bottom of the tube, and mix thoroughly;

[0114] (2) Centrifugation at 4000 rpm, 4°C, for approximately 20 min;

[0115] (3) Discard the filtrate in a biosafety cabinet and place it in a dedicated waste container. Continue to add 1X PBS to the bottom of the ultrafiltration centrifuge tube and mix thoroughly.

[0116] (4) Centrifugation at 4000 rpm, 4°C, for approximately 15 min;

[0117] (5) Repeat steps (3) and (4) 1-2 times;

[0118] (6) Use a 200 μL pipette to repeatedly pipette the remaining liquid in the ultrafiltration tube until evenly distributed. Transfer and dispense into virus storage tubes, labeling the tubes with the name and date. Store at -80°C for long-term storage. For short-term storage (no more than 1 week), store at 4°C.

[0119] Example 8

[0120] The specific steps for determining the titer of AAV9-LUC and AAV9.7YS-LUC viruses are as follows:

[0121] (1) The ITR sequence on the viral core plasmid vector was selected to design real-time fluorescence quantitative PCR primers. The forward primer sequence was SEQ ID NO: 17 (GGAACCCCTAGTGATGGAGTT), and the rear primer sequence was SEQ ID NO: 18 (sequence: CGGCCTCAGTGAGCGA);

[0122] (2) The AAV9-LUC and AAV9.7YS-LUC virus samples to be tested and the standard plasmid ssAAV-EGFP were used as template plasmids for real-time fluorescence quantitative PCR amplification. The real-time fluorescence quantitative PCR reaction system is shown in Table 6, and the reaction procedure is shown in Table 7:

[0123] Table 6 Real-time fluorescence quantitative PCR reaction system

[0124]

[0125] Table 7 Real-time fluorescence quantitative PCR reaction program

[0126]

[0127] (3) The experimental data of real-time fluorescence quantitative PCR is entered into the following formula for calculation:

[0128] Copy number (copies / μL) = concentration (ng / μL) × 10^(-9) × 6.02 × 10^23 / base pairs × 650;

[0129] Among them, 6.02×10^23 is Avogadro's constant, that is, the number of molecules in 1 mol of substance,

[0130] Then, the lg value of the genome copy number is the X axis and the Ct value is the Y axis to establish a standard curve. Figure 2 As shown, the titers of AAV9-LUC and AAV9.7YS-LUC virus samples were calculated. The AAV virus titer was detected by real-time fluorescence quantitative PCR, and the correlation coefficient R of the standard curve was drawn. 2 >0.99, the fitting is good. By calculating the virus titer, the titer of AAV9-LUC is 5.01×10 6 The titer of AAV9.7YS-LUC virus was 1.28×10 7, indicating that the titer of the modified AAV9.7YS virus is not affected, AAV9.7YS can still be packaged into AAV virus, and the titer is even higher than AAV9.

[0131] Example 9

[0132] The specific steps for identification of AAV9 and AAV9.7YS viral capsid protein expression are as follows:

[0133] (1) Each AAV virus sample was taken from the EP tube to a titer of 2 × 10^9 vg, and PBS buffer was added to keep the volume and concentration of each sample consistent;

[0134] (2) Add 5× loading buffer to each sample, mix well, heat at 100°C for 5 min, and then immediately place on ice or store at -20°C until use;

[0135] (3) Prepare a separation gel with an acrylamide concentration of 10% and a stacking gel with an acrylamide concentration of 5%. The formula of the separation gel is shown in Table 10, and the formula of the stacking gel is shown in Table 11:

[0136] Table 10 Separation gel formula

[0137]

[0138] Table 11 Concentrating gel formula

[0139]

[0140] (4) Load the sample using a 20 μL pipette and perform polyacrylamide gel electrophoresis;

[0141] (5) Adjust the voltage to 80V. After about 30 minutes, the sample will run to the junction of the stacking gel and the separation gel. At this time, the pre-stained protein marker will have 1-3 distinct color-separated bands. Adjust the voltage to 120V and continue electrophoresis. Stop electrophoresis when the 55kDa pre-stained protein marker band reaches the bottom edge of the gel. This step takes about 2 hours.

[0142] (6) Add transfer buffer to the enamel plate used for transfer, and then soak the filter paper and nitrocellulose membrane in the transfer buffer;

[0143] (7) Carefully remove the gel and fix the gel into a sandwich structure in the following order: positive electrode / white plywood / sponge pad / filter paper / nitrocellulose membrane / gel / filter paper / sponge pad / black plywood / negative electrode. During the whole process, make sure that there are no bubbles between the layers to avoid affecting the transfer;

[0144] (8) Connect the power supply correctly and use a constant current of 300 mA to transfer the membrane for 2-2.5 hours. Place the membrane transfer device in an ice-water mixture to ensure that the membrane transfer device is kept at a low temperature during the entire transfer process to avoid unnecessary damage to the sample and the device.

[0145] (9) After the transfer is completed, the nitrocellulose membrane is quickly removed and placed in a small box containing 5% skim milk powder prepared in 1× TBST to block nonspecific binding sites. The membrane is incubated on a shaker at room temperature for 1 h.

[0146] (10) Wash the membrane with 1× TBST until no milk remains in the 1× TBST buffer, approximately 3 min;

[0147] (11) Add CAP primary antibody at a dilution ratio of 1:3000 and incubate overnight at 4°C. The next day, wash the membrane three times with 1×TBST at room temperature on a shaker for 10 min each time.

[0148] (12) Add secondary antibody at a dilution ratio of 1:3000 and incubate on a shaker at room temperature for 1 h;

[0149] (13) Wash the membrane three times with 1× TBST on a shaker at room temperature for 10 min each time;

[0150] (14) The chemiluminescence instrument was used for development. The experimental results are shown in Figure 3 After iodixanol density gradient centrifugation, AAV9 and AAV9.7YS viruses were analyzed by Western blot for AAV capsid proteins. The results showed that the VP antibody could recognize the AAV9 and AAV9.7YS capsid proteins. Three bands were clearly visible in the AAV9 and AAV9.7YS viruses, corresponding to the AAV capsid proteins VP1, VP2, and VP3, respectively, meeting the typical characteristics of AAV viruses. The modified AAV9.7YS capsid protein bands were higher than those of the AAV9 capsid protein, indicating that the AAV capsid protein quality was increased after modification. This indicates that the AAV9-LUC and AAV9.7YS-LUC viruses were successfully packaged and purified effectively.

[0151] Example 10

[0152] AAV9-LUC and AAV9.7YS-LUC viruses were injected into mice via tail vein injection. The specific steps are as follows:

[0153] (1) Fix a mouse weighing approximately 20 g on a mouse fixing table;

[0154] (2) Use a 1 mL insulin syringe to draw 1 × 10 11 vg of AAV9-LUC and AAV9.7YS-LUC viruses, making sure to exclude air, were injected into the tail vessels of mice;

[0155] (3) After the injection, wait for 5 seconds, slowly withdraw the needle, and quickly press the injection site with a dry cotton ball. Then quickly put the mouse back into the cage and continue feeding.

[0156] Example 11

[0157] One week after the tail vein injection of AAV9-LUC and AAV9.7YS-LUC viruses, the distribution of AAV9-LUC and AAV9.7YS-LUC viruses in mice can be observed using the IVIS Spectrum Small Animal In vivo Imager. The specific steps are as follows:

[0158] (1) Weigh the mice and determine the specific dosage of luciferin and 3% chloral hydrate based on the weight;

[0159] (2) Generally speaking, the injection dose of Luciferin is 1.5 mg / 10 g, that is, a 20 g mouse is intraperitoneally injected with 100 μL of 30 mg / mL Luciferase enzyme substrate working solution;

[0160] (3) After 10 minutes, inject the anesthetic. Generally, the injection volume is 0.1 mL / 10 g. For a 20 g mouse, 200 μL of 3% chloral hydrate anesthetic solvent is injected intraperitoneally.

[0161] (4) In vivo imaging of small animals can be performed 10 minutes after the substrate Luciferin is injected. Figure 4 In vivo mouse imaging revealed that fluorescence signals in the AAV9-LUC group were primarily located in the back muscles and liver, while those in the AAV9.7YS-LUC group were primarily located in the brain, with no fluorescence signals observed in other tissues. This suggests that the AAV9.7YS vector can more effectively penetrate the mouse blood-brain barrier and achieve higher gene transduction efficiency in the nervous system.

[0162] (5) After the mouse in vivo imaging was completed, the mouse brain tissue was taken for tissue imaging. The imaging results are shown in Figure 5 Figure A shows that after the in vivo imaging of small animals, brain tissues of two groups of mice were taken for tissue imaging. The imaging results showed that the fluorescence signal of the AAV9.7YS group was significantly higher than that of the AAV9 group, that is, the fluorescence signal of the AAV9.7YS-LUC group was significantly higher than that of the AAV9-LUC group in the mouse brain tissue; Figure B performed quantitative analysis of the fluorescence signal, and the fluorescence signal of the brain of the AAV9.7YS group was significantly higher than that of the AAV9 group (n=3, ***P<0.001).

[0163] Example 12

[0164] After AAV9-LUC and AAV9.7YS-LUC virus injection, the heart, liver, spleen, lung, kidney, muscle, and brain tissues of the mice were collected to extract proteins from each tissue and detect the activity of luciferase in each tissue. This can then be used to evaluate the distribution of AAV9-LUC and AAV9.7YS-LUC viruses in the mice and determine the tissue tropism of the AAV virus. The specific steps are as follows:

[0165] (1) Take approximately 100 mg of tissue and place it in an EP tube containing 1 mL of PBS for washing;

[0166] (2) Centrifugation at 3000 rpm for 3 min;

[0167] (3) The precipitate was added to a homogenization tube containing 400 μL of RIPA lysis buffer and 6 small ceramic beads;

[0168] (4) Place the tissue in an electric homogenizer and homogenize it: homogenize for 10 seconds, then rest for 10 seconds, for a total of 3 times. If large pieces of tissue still exist after 3 homogenizations, continue homogenizing for 1-2 more rounds.

[0169] (5) Place the homogenized suspension on ice and incubate for 10 min. Mix on an oscillator for three times.

[0170] (6) Centrifuge at 12000 rpm for 10 min;

[0171] (7) Transfer the supernatant to a new EP tube, then adjust the concentration of mouse tissue total protein to a consistent 25 μg / μL, a total of 20 μL, and add it to the assay tube;

[0172] (8) Prepare fresh substrate working solution: dilute Luciferin substrate 50-fold in assay buffer and store in the dark.

[0173] (9) In a dark room, turn on the fluorescence detector. Add 50 μL of substrate reaction solution to each test tube, mix quickly, and test on the instrument after 10 seconds to obtain the enzyme activity value of Luciferase in each tissue organ. The results are shown in Figure 6Proteins from major tissues and organs of mice in the AAV9 and AAV9.7YS groups were extracted, and luciferase activity in the tissues was detected using a reporter gene. The results showed that luciferase activity in the brain tissue of the AAV9.7YS group was approximately 55 times that of the AAV9 group, while the enzyme activity in the heart, liver, spleen, kidney, and skeletal muscle of the AAV9.7YS group was significantly lower than that of the AAV9 group (n=3, *P<0.05, **P<0.001). There was no significant difference in lung tissue between the two groups. This further indicates that the modified AAV9.7YS virus has a stronger ability to infect the nervous system, but has a lower infection efficiency in peripheral tissues (such as the heart, spleen, liver, and skeletal muscle).

[0174] Example 13

[0175] After AAV9-LUC or AAV9.7YS-LUC virus injection, the heart, liver, spleen, lung, kidney, muscle, brain, and spinal cord tissues of the mice were collected to extract genomic DNA from each tissue and organ. The tissue distribution of the AAV virus was determined by detecting the copy number of the AAV9 or AAV9.7YS viral genome in each tissue and organ. The specific steps are as follows:

[0176] (1) Take about 25 mg of mouse tissue and use Genomic DNA from mouse tissues was extracted using the Genomic DNA Kit (containing RNase A) (EE101-01);

[0177] (2) Designing primers for the conserved gene GAPDH sequence in the mouse genome: the forward primer sequence is SEQ ID NO: 19CATCACTGCCACCCAGAAGACTG, and the rear primer sequence is SEQ ID NO: 20ATGCCAGTGAGCTTCCCGTTCAG; the primer sequence for the ITR of the AAV virus is the same as that in Example 8;

[0178] (3) Using the genomic DNA extracted from mouse tissue as a template, qPCR amplification of the GAPDH sequence and ITR sequence was performed, respectively. The qPCR system and reaction procedure were the same as in Example 8;

[0179] (4) Based on the CT values ​​obtained by amplification, the copy number diagram of AAV9 and AAV9.7YS viral genomes in various tissues and organs of mice can be obtained. The results are shown in Figure 7 The mouse tissue genome was extracted, and the AAV viral genome copy number in mouse tissues was detected by qPCR. There was no significant difference in the AAV genome copy number in brain, heart, liver, spleen and kidney tissues, while in lung and muscle tissues, the AAV9.7YS group was significantly lower than the AAV9 group (n=3, *P<0.05, **P<0.001).

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[0192] Sequence Listing <110> China Three Gorges University <120> An AAV viral vector for nervous system drug delivery <130> <160> 20 <210> 1 <211> 5604 <212> DNA <213> AAV viral vectors <400> SEQ ID NO: 1 <210> 2 <211> 650 <212> PRT <213> Amino acid sequence of AAV9 wild-type capsid protein VP1 <400> SEQ ID NO: 2 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIK <210> 3 <211> 650 <212> DNA <213> DNA sequence of AAV9 wild-type capsid protein VP1 <400> SEQ ID NO: 3 <210> 4 <211> 7 <212> PRT <213> Specific amino acid sequence <400> SEQ ID NO: 4 WPTSYDA <210> 5 <211> twenty one <212> DNA <213> Specific nucleic acid sequence <400> SEQ ID NO: 5 TGGCCCACCAGCTACGACGCC <210> 6 <211> 657 <212> PRT <213> AAV9 capsid protein amino acid sequence <400> SEQ ID NO: 6 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQWPTSYDAAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIK <210> 7 <211> 1971 <212> DNA <213> DNA sequence of AAV9 capsid protein <400> SEQ ID NO: 7 <210> 8 <211> 657 <212> PRT <213> Amino acid sequence of the AAV9 capsid protein with site-directed mutagenesis <400> SEQ ID NO: 8 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASYKEGEDSFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQWPTSYDAAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIK <210> 9 <211> 1971 <212> DNA <213> Amino acid sequence of the AAV9 capsid protein with site-directed mutagenesis <400> SEQ ID NO: 9 <210> 10 <211> 448 <212> DNA <213> hSyn <400> SEQ ID NO:10 AGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG <210> 11 <211> 51 <212> DNA <213> Forward primer sequence containing a specific insertion sequence <400> SEQ ID NO:11 CACCAGAGTGCCCAATGGCCCACCAGCTACGACGCCGCACAGGCGCAGACC <210> 12 <211> 51 <212> DNA <213> Reverse primer sequence containing a specific insertion sequence <400> SEQ ID NO:12 GGTCTGCGCCTGTGCGGCGTCGTAGCTGGTGGGCCATTGGGCACTCTGGTG <210> 13 <211> 51 <212> DNA <213> The forward primer sequence containing the target mutation site <400> SEQ ID NO: 13 CCTGCTATGGCCAGCCGCAAAGAAGGAGAGGACCGATTCTTTCCTTTGTCT <210> 14 <211> 51 <212> DNA <213> The rear primer sequence containing the target mutation site <400> SEQ ID NO: 13 AGACAAAGGAAAGAATCGGTCCTCTCCTTCTTTGCGGCTGGCCATAGCAGG <210> 15 <211> 27 <212> DNA <213> Promoter preprimer sequence <400> SEQ ID NO: 15 CAGAAGCTTAGTGCAAGTGGGTTTTAG <210> 16 <211> 27 <212> DNA <213> Post-promoter primer sequence <400> SEQ ID NO: 16 CAGCTCGAGCTGCGCTCTCAGGCACGA <210> 17 <211> twenty one <212> DNA <213> Fluorescence quantitative PCR primer forward primer sequence <400> SEQ ID NO: 17 GGAACCCCTAGTGATGGAGTT <210> 18 <211> 16 <212> DNA <213> Fluorescence quantitative PCR primer sequence <400> SEQ ID NO: 18 CGGCCTCAGTGAGCGA <210> 19 <211> twenty three <212> DNA <213> Preprimer sequence of the conserved gene GAPDH sequence <400> SEQ ID NO: 19 CATCACTGCCACCCAGAAGACTG <210> 20 <211> twenty three <212> DNA <213> Primer sequence after primer of conserved gene GAPDH sequence <400> SEQ ID NO: 20 ATGCCAGTGAGCTTCCCGTTCAG

Claims

1. A site-directed mutagenesis of an AAV9 capsid protein, characterized in that: (1) Inserting a specific amino acid sequence of SEQ ID NO: 4 after the 558th position of the amino acid sequence of SEQ ID NO: 2 of the wild-type AAV9 capsid protein VP1, then transforming the inserted AAV9 viral vector and extracting the plasmid to obtain an AAV9 capsid protein sequence of SEQ ID NO: 6 and a DNA sequence of SEQ ID NO: 7; (2) PCR technology was used to perform site-directed mutagenesis on sites 527 and 533 of the AAV9 capsid sequence. The amino acid H527 of the AAV9 capsid protein was mutated to Y, and the amino acid R533 was mutated to S. Finally, the mutated AAV9 viral vector was transformed and the plasmid was extracted to obtain the site-directed mutated AAV9 capsid protein sequence, of which the amino acid sequence was SEQ ID NO: 8 and the DNA sequence was SEQ ID NO: 9.

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