Construction and application of a modified AAV-8 serotype targeting various retinal layers

Abstract

The present invention belongs to the field of genetic engineering and biotechnology, and relates to the screening, construction and application of a modified type of AAV-8 serotype for gene targeting and expression. The modified type of the AAV-8 serotype of the present invention inserts 10 amino acid sequences between the 590th amino acid and the 591st amino acid of the AAV-8 serotype capsid protein sequence as shown in SEQ ID NO.2, wherein the 3rd to 9th amino acids are amino acid sequences obtained by screening. The modified serotype of the present invention targets each layer of the retina, and the fluorescence effect after infection is stronger, and it can be clearly and intuitively observed that the exogenous gene can be efficiently expressed in each layer of the retina of the targeted tissue, including the retinal ganglion cell layer, the inner nuclear layer, the outer nuclear layer, the photoreceptor cell layer and the retinal pigment epithelial cell layer, and has better and more targeted tissue expression than the wild-type serotype when the same amount of virus is used.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and biotechnology, and specifically relates to the screening, construction and application of a modified AAV-8 serotype for gene targeting and expression. Background Art

[0002] AAV is a non-pathogenic parvovirus with DNA defects. The recombinant adeno-associated virus vector (rAAV) is derived from the non-pathogenic wild-type adeno-associated virus. It has the characteristics of weak immunogenicity, good safety, wide host range, high infection efficiency and strong tissue specificity. It is an ideal gene expression vector and has been approved by the FDA for clinical trials.

[0003] AAV virus capsid proteins are currently used in AAV virus packaging systems, and the most commonly used ones are traditional wild-type AAV1, 2, 5, 8, and 9 capsid proteins, which have different tissue or cell specificity. This type of natural wild-type AAV virus capsid can effectively target AAV to target tissues for the expression of exogenous genes, such as AAV1 can efficiently infect skeletal muscle cells, AAV2 can infect retinal cells, AAV8 can efficiently deliver exogenous genes to liver cells, and AAV9 has the ability to cross the blood-brain barrier and express exogenous genes in the central nervous system and brain, etc. The natural tropism of the virus determines the basis of targeted delivery therapy, and on the other hand, it also provides a platform for modifying these viral capsids to have higher penetration, longer-lasting expression time, and lower immunogenicity.

[0004] Given that AAV has been widely used in preclinical studies of various diseases, the top priority is to target the modification and optimization of natural wild-type AAV capsid proteins. Currently, there are many methods for modifying and optimizing AAV capsid proteins, including DNA shuffling technology, capsid protein amino acid site-directed mutagenesis, and artificial insertion and deletion of amino acid sequence modification of capsid proteins. Among them, the phage display system technology developed in recent years is an efficient and feasible screening method for screening specific targeted short peptides from random peptide libraries and inserting them into specific sites of wild-type AAV capsid proteins. Summary of the invention

[0005] One of the technical problems to be solved by the present invention is to screen a modified AAV-8 serotype for targeting and expressing various layers of the retina, including the retinal ganglion cell layer, inner nuclear layer, outer nuclear layer, photoreceptor cell layer and retinal pigment epithelial cell layer.

[0006] The second technical problem to be solved by the present invention is to provide a method for constructing the modified serotype. The present invention screened a new AAV-8 modified capsid AAV8-590MJ06 by vitreous injection. That is, by using the in vitro evolution screening method, 10 amino acids are inserted between the 590th amino acid and the 591st amino acid of the AAV-8 wild-type capsid, including 7 random amino acids, 2 protected amino acids LA at the 5' end, and 1 protected amino acid A at the 3' end. After a one-step virus packaging, each AAV virus capsid only has a unique 10 amino acid sequence insertion, corresponding to the genome sequence of its AAV. This virus is injected into the vitreous of mice. About 1 week later, the retina and choroid of the mice are taken, and their genomes are extracted and analyzed to observe whether there is AAV virus infiltrating from the vitreous to the various layers of the retina.

[0007] The third technical problem to be solved by the present invention is to provide the application of the modified serotype AAV8-590MJ06.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] In a first aspect of the present invention, a modified serotype of an AAV-8 type serotype for gene targeting and expression is provided.

[0010] The present invention provides a polypeptide, wherein a polypeptide consisting of 10 amino acids is inserted between the 590th amino acid and the 591st amino acid of the AAV-8 serotype capsid protein; the amino acid sequence of the AAV-8 serotype capsid protein is shown as SEQ ID NO.2; the polypeptide consisting of 10 amino acids contains a polypeptide with an amino acid sequence as shown in SEQ ID NO.5.

[0011] Preferably, the amino acid sequence at positions 1-590 is as shown in the amino acid sequence at positions 1-590 of the AAV-8 serotype capsid protein sequence shown in SEQ ID NO.2, the amino acid sequence at positions 593-599 among the amino acids at positions 591-600 is the amino acid sequence of the polypeptide sequence according to claim 1 as shown in SEQ ID NO.5, and the amino acid sequence at positions 601 and thereafter is the amino acid sequence at positions 591 and thereafter of the AAV-8 serotype capsid protein sequence shown in SEQ ID NO.2.

[0012] Preferably, the amino acid sequence of the polypeptide consisting of 10 amino acids is shown in SEQ ID NO.3.

[0013] The 10 amino acid sequence shown in SEQ ID NO.3 is inserted between the 590th amino acid and the 591st amino acid of the AAV-8 serotype capsid protein sequence shown in SEQ ID NO.2: LARGGQREGA, wherein the 1st, 2nd and 10th amino acids are protected amino acids, and the 3rd to 9th amino acids are amino acid sequences obtained by screening.

[0014] Preferably, the 10 amino acid sequences shown in SEQ ID NO.3 correspond to the base sequence shown in SEQ ID NO.4.

[0015] Preferably, the modified capsid protein sequence of the AAV-8 serotype for gene targeting and expression is shown as SEQ ID NO.1.

[0016] Preferably, the amino acid sequence LARGGQREGA inserted into the modified serotype is used for the capsid for AAV virus packaging, or for the connection and targeting of biological macromolecules, antibody drugs, peptides and chemical small molecules.

[0017] The present invention constructs an AAV serotype containing a 30-base sequence corresponding to the 10 amino acid sequences (7 random amino acid sequences and 3 protective amino acids) shown in SEQ ID NO.3, including a CAP gene, a REP gene, and a selective marker Ampicillin resistance gene; the corresponding 30 bases shown in SEQ ID NO.4 are inserted between the bases corresponding to the 590th amino acid and the 591st amino acid of the CAP protein. The Ampicillin resistance gene is an antibiotic resistance gene, and its purpose is to make the bacteria successfully introduced with the vector resistant to antibiotics, so that the AAV vector can be screened and amplified. The 10 amino acid sequences shown in SEQ ID NO.3 of the present invention are obtained by screening from thousands of polypeptides. During the research process, it was found that although some amino acid sequences differ from the present invention by only one amino acid, they cannot achieve the effect of the present invention, and even some polypeptides screened using a simulation system may not necessarily achieve the purpose of the present invention.

[0018] Accordingly, the present invention provides a nucleic acid, which contains the coding sequence of the above polypeptide, or the corresponding capsid protein sequence is shown in SEQ ID NO.1.

[0019] The present invention also provides an AAV capsid, which contains the above nucleic acid.

[0020] Preferably, the method for constructing the AAV capsid comprises the following steps:

[0021] Synthesize the nucleic acid whose sequence is shown in SEQ ID NO.6;

[0022] Adding protective bases cttgct and gcc to the 5' and 3' ends of the nucleic acid shown in SEQ ID NO.6, respectively;

[0023] Inserted between the nucleotides corresponding to the 590th and 591st amino acids of the AAV-8Cap gene to form the AAV capsid.

[0024] In a second aspect of the present invention, a method for constructing the above serotype is also provided, the method comprising the following steps:

[0025] In the first step, 21 random bases are synthesized and the protective bases cttgct and gcc are added to the 5' and 3' ends, and inserted into the base sequence corresponding to the 590th and 591st amino acids of the AAV-8Cap gene to form the AAV capsid;

[0026] In the second step, the above-mentioned AAV capsid vector was transformed by electroporation into several competent cells. Each competent cell was cultured overnight in Luria-Bertani (LB) medium. The next day, the bacterial liquid in each culture medium was mixed and inoculated into LB medium for shaking overnight. The remaining bacterial liquid was stored in glycerol (random fragment library); plasmids were extracted to obtain a mixed vector library, which was named pAAV8-590-7aa.

[0027] In the third step, the AAV virus was packaged and purified using the helper plasmid, REP2-AAV8 plasmid, pEGFP-N1 and pAAV8-590-7aa (10 ng);

[0028] The fourth step is to inject the AAV virus into the vitreous cavity of C57BL / 6J mice, take the retinal layer, extract the genomic DNA of the retinal layer, detect and sequence the 21 amino acids following the base sequence corresponding to the 590th amino acid of the AAV-8Cap gene;

[0029] In the fifth step, the sequencing results were analyzed and random sequences were amplified by PCR to determine the 10 amino acid sequences, which were inserted into positions 590 and 591 of the AAV-8Cap gene, packaged by AAV virus, and injected into the vitreous cavity of the eye to infect the retina, and the infection differences between AAV-8 and AAV8-590MJ06 were compared.

[0030] In the third aspect of the present invention, the use of the above serotype is also provided.

[0031] The polypeptide can be used to prepare drugs for treating retinal diseases, especially drugs that can improve retinal infection efficiency, increase retinal penetration efficiency or improve retinal recognition efficiency through vitreous injection.

[0032] The experimental results show that compared with the wild-type AAV-8 serotype, the application and comparative experiment of the modified serotype of AAV8-590MJ06 of the present invention in infecting various layers of the retina through intravitreal injection shows that:

[0033] The modified AAV-8 serotype can be injected into the vitreous cavity of the eyeball to better and more extensively infect all layers of the retina, including the retinal ganglion cell layer, inner nuclear layer, outer nuclear layer, photoreceptor cell layer, and retinal pigment epithelial cell layer ( Figure 3 ).

[0034] Preferably, the cells in the cell layer are selected from one or more of retinal ganglion isolated cells, Neuro2A cells, U251 cells, ARPE-19 cells, SH-SY-5Y cells, BV2 cells, HBMEC primary isolated cells, Jurkat cells, K562 cells, 293 cells or THP1 cells.

[0035] The detection of mRNA levels showed that compared with the wild-type AAV-8 serotype, the AAV-8 modified serotype had higher expression in the eye area and less organ leakage expression after intravitreal injection ( Figure 4 ).

[0036] The results of intravitreal injection of a virus with a single point mutation in the RGGQREG insertion sequence to infect retinal cells showed that the ability of the mutant serotype to infect the retina was reduced, indicating that the RGGQREG sequence is irreplaceable ( Figure 5 ).

[0037] The above results show that the AAV8-590MJ06 modified serotype of the present invention has good penetration in all layers of the retina, strong infectivity, wide application, low immunogenicity, high specificity, safety and high efficiency. At the same time, the fluorescence generated by the modified serotype is easy to observe and record, and can be used as a better tool for gene expression or gene delivery.

[0038] The above terms are explained as follows:

[0039] AAV capsid: AAV capsid is a protein capsid that can express adeno-associated virus. It is also called a capsid. The capsid is an oligomer formed by the subunits of the viral capsid protein. The function of the capsid is to encapsulate the genetic material of the virus.

[0040] AAV-8: A type of AAV originally isolated from rhesus monkeys. It is serologically unique, has minimal cross-reactivity with other serotypes, and is less immunogenic than AAV2.

[0041] AAV-8 serotype capsid protein: The protein capsid of the AAV-8 virus, which encapsulates the genetic material of AAV-8.

[0042] Wild-type AAV-8 serotype: antigens possessed by the natural AAV-8 virus without modification or alteration.

[0043] Gene targeting and expression: Directed delivery of target genes to target cells or tissues for gene expression.

[0044] Protective amino acids: connecting amino acids that connect the inserted amino acids to the original capsid amino acids, used to stabilize protein conformation and function.

[0045] Random amino acid sequence: A random base sequence synthesized from a random base sequence, which corresponds to a corresponding random amino acid sequence.

[0046] Compared with the prior art, the present invention has the following beneficial effects: the present invention includes a serotype capsid amino acid sequence, an insertion site and an insertion amino acid sequence. On the basis of the AAV-8 wild-type serotype, 7 random amino acid fragments and 3 protective amino acids, a total of 10 amino acids, are inserted together between the 590-591 amino acids of the AAV-8 wild-type capsid, and inserted into a random short peptide display library, and the random short peptide library is screened out in the target tissue by an in vitro expression method to quickly and effectively screen out a novel AAV modified capsid that can be injected into the vitreous body to infect various layers of the retina, including the retinal ganglion cell layer, the inner nuclear layer, the outer nuclear layer, the photoreceptor cell layer and the retinal pigment epithelial cell layer, with good penetration, strong infectivity, wide infection and low immunogenicity. After infection with the modified serotype of the present invention, the fluorescence effect is stronger, and it can be observed clearly and intuitively. The exogenous gene can be efficiently and widely expressed in various layers of the target tissue retina in vivo. When the same amount of virus is used, it has better and more extensive targeted tissue expression than the wild-type serotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1 This is a schematic diagram of the AAV8-590MJ06 serotype structure in Example 1 of the present invention.

[0049] Figure 2 This is a schematic diagram of the base sequencing results of the AAV8-590MJ06 serotype screened in Example 3 of the present invention.

[0050] Figure 3This is a schematic diagram of the virus packaged with the AAV8-590MJ06 serotype screened in Example 4 of the present invention and the virus packaged with the wild AAV8 serotype, which were injected into the vitreous cavity to infect the frozen sections of the mouse retina. The scale in the figure is 100 μm. Among them, Figure 3 (A) Representative of the group injected with wild-type AAV8 serotype-packaged viruses; Figure 3 (B) represents the injection of the virus group packaged with the AAV8-590MJ06 serotype screened by the present invention.

[0051] Figure 4 The virus packaged with the AAV8-590MJ06 serotype screened in Example 4 of the present invention and the virus packaged with the wild AAV8 serotype were injected into the vitreous cavity to detect the level of luciferase mRNA expressed by the virus in the mouse retina, brain, heart, liver, spleen, lung, kidney, muscle, small intestine, testicle and other organs.

[0052] Figure 5 The retinal infection status of frozen sections of mouse retina after intravitreal injection of the virus packaged with the AAV8-590MJ06 insertion sequence single point mutation serotype screened in Example 4 of the present invention. The scale in the figure is 100 μm. DETAILED DESCRIPTION

[0053] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0054] Example 1 Vector Construction

[0055] The specific methods and steps for sequence design and synthesis are as follows:

[0056] (1) Based on the gene information of Cap of AAV8 packaging plasmid in GeneBank, the BsmBI-AAV8 Cap (482-655aa)-BsmBI gene DNA fragment was designed and a double-stranded DNA molecule was synthesized;

[0057] (2) The double-stranded DNA molecules synthesized in step (1) were PCR-polymerized using the synthesized primers pAAV8-590-7aa-F and pAAV8-590-7aa-R to obtain PCR products.

[0058] In step (2), the PCR system is as follows: 32.5 μL H2O, 10 μL 5×Buffer (containing Mg2+), 4 μL dNTP (2.5 mM each), 1 μL forward primer Primer1 (+), 1 μL reverse primer Primer2 (-) (10 μM), 1 μL target gene template DNA, and 0.5 μL PrimeSTAR enzyme, constituting a reaction system;

[0059] The PCR program was as follows: denaturation at 98°C for 3 minutes; annealing at 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 1 minute, repeated for 30 cycles; and extension at 72°C for 10 minutes.

[0060] The specific method and steps for inserting the sequence into the site are as follows:

[0061] 1) Cut the viral vector Rep-AAV8-Cap with the restriction endonuclease BsmBI to recover the vector backbone;

[0062] 2) The PCR product of step (2) and the vector backbone of step 1) are recombined, transformed into Escherichia coli, positive bacteria are screened and their plasmids are extracted to obtain a recombinant vector.

[0063] In step 1), the enzyme digestion system is: BsmBI: 1 μL, buffer: 3 μL, Rep-AAV8-Cap plasmid: 1 μg, water is added to 30 μL; enzyme digestion is carried out at 37° C. for 4 hours.

[0064] In step 2), the recombination system is: recombinase: 15 μL, recovered PCR product DNA: 40 ng, recovered plasmid: 20 ng; after 30 min in a 42° C. water bath, transformed into E. coli.

[0065] pAAV8-590-7aa-F: AGGACCCTGTTACCGCCAAC (SEQ ID NO.7);

[0066] pAAV8-590-7aa-R:GATGTTTCAGGCCAAAGCCG (SEQ ID NO. 8);

[0067] like Figure 1 As shown, the constructed pAAV8-590MJ06 vector structure includes an ampicillin resistance gene, an AAV replication gene, and an AAV8 capsid protein gene, which includes a random base sequence corresponding to a 10-amino acid sequence: LARGGQREGA, as shown in SEQ ID NO.3.

[0068] Example 2 AAV virus packaging

[0069] (I) Cryopreservation of AAV-293 cells

[0070] As the number of passages increases, AAV-293 cells will experience a decline in growth status and mutations. In order to prevent such phenomena, we need to freeze a large number of cells at the beginning to ensure the stability and sustainability of the experiment. Cryopreservation during the logarithmic growth phase of cells increases the survival rate of cell recovery.

[0071] 1. Remove the cell culture supernatant and add PBS to wash away the residual culture medium;

[0072] 2. Add 0.25% trypsin and digest for 1-2 minutes. When the cells become round and the intercellular gap increases under the microscope, remove the trypsin, add fresh culture medium, blow and mix well, and transfer to a centrifuge tube.

[0073] 3. Cell counting: shake all the cells off, add 3mL 10% DMEM preheated at 37℃, and blow with a 10mL pipette for 6-8 times without leaving any dead corners. After that, aspirate all the cells and place them in a 15mL centrifuge tube. Take 50μL of the mixed cells in a 1.5mL eppendorf tube, add 450μL 10% DMEM, which is a 10-fold dilution, mix well, and take 10μL of cells to count in a counting plate. There are 4 large grids on the counting plate, each with 16 small grids. When counting, count all 4 large grids, divide the total by 4 (to get the number of cells per large grid), and then multiply by 10 (10-fold dilution), which is the actual n ten thousand / mL cell concentration.

[0074] 4. Centrifuge the cells at 1000 rpm / min for 5 min. Discard the supernatant.

[0075] 5. According to the cell counting results, add cell freezing solution (70% complete culture medium + 20% FBS + 10% DMSO) to resuspend the cells at a density of 3 x 106 cells / mL.

[0076] 6. Divide into cell cryopreservation tubes, place in cryopreservation boxes, and place in a -80℃ ultra-low temperature freezer.

[0077] 7. The next day, place the cells in a liquid nitrogen tank for long-term storage and keep records. During the storage process, resuscitate the cells from time to time to detect cell survival rate and observe cell status.

[0078] (II) Passaging of AAV-293 cells

[0079] When the cells grow to a confluence rate of 80%-90%, the cells need to be subcultured to expand the cell number and maintain a good growth state of the cells.

[0080] 1. Digest cells using the same method as for cryopreservation.

[0081] 2. After the cells are centrifuged, add complete culture medium to resuspend them.

[0082] 3. Depending on the specific situation, divide the cells into cell culture dishes and fill each dish with 10 mL of culture medium.

[0083] (III) Recovery of AAV-293 cells

[0084] When cells are passaged too many times, the cell condition deteriorates, or there is a cell contamination accident, it is necessary to discard and thaw the originally frozen cells.

[0085] 1. Set the water bath to 37-42°C.

[0086] 2. Check the cell bank records, take out the frozen cells from the liquid nitrogen tank according to the records (wear cotton gloves to prevent frostbite), quickly put them into the water bath and shake them quickly, try to dissolve the cell solution completely within 1-2 minutes.

[0087] 3. Transfer the cell solution to a 15 mL centrifuge tube and add 1 mL of fresh complete culture medium. Mix well and centrifuge at 1000 rpm / min for 5 min.

[0088] 4. Remove the supernatant, add 5 mL of fresh complete culture medium, mix the precipitate, and transfer to a 6 cm culture dish.

[0089] 5. Place the culture dish steadily in an incubator at 37°C, 5% CO2 and 95% relative humidity.

[0090] 6. Observe the cell survival rate on the second day. Change the culture medium for the cells. Observe the cell growth every day thereafter.

[0091] (IV) AAV packaging and concentration

[0092] 1. Plasmid Amplification

[0093] The constructed AAV vector, packaging plasmid and auxiliary plasmid need to be extracted in large quantities, with a concentration greater than 1μg / μL and an A260 / 280 ratio between 1.7 and 1.8 before they can be used for packaging. It is recommended to use the Qiagen large-scale extraction kit for large-scale endotoxin-free extraction of plasmids.

[0094] 2. Transfecting Cells with AAV-293

[0095] Aspirate the medium in the T75 bottle of AAV-293 cells, add 2mL of 0.25% trypsin taken out of the 4-degree refrigerator, and evenly cover the bottom of the bottle. Place it in a 37-degree incubator for 3-5 minutes, take it out, shake it to find that the cells are detached from the bottom, shake it all off, add 3mL of 10% DMEM preheated in a 37-degree water bath, and use a 10mL pipette to blow with a pipette gun. Blow 6-8 times with great force, leaving no dead corners. The bottle mouth is difficult to blow, so the pipette can be aimed at the bottle mouth, and the medium can be blown out with a small force to cover the cells close to the bottle mouth. After that, aspirate all the cells and place them in a 15mL centrifuge tube, take 50μL of the mixed cells in a 1.5mL eppendorf tube, add 450μL of 10% DMEM, that is, 10 times dilution, mix well, and take 10μL of cells to count in a counting plate. There are 4 large grids on the counting plate, and each large grid has 16 small grids. When counting, count all four large grids, divide the total by 4 (to get the number of cells per large grid), and then multiply by 10 (10-fold dilution), which is the actual n million / mL cell concentration. The day of subculture is recorded as the first day. If transfection is performed on the second day, 9-10 million / T75 should be plated; if transfection is performed on the third day, 3.5-4 million / T75 should be plated. Add 10mL of 10% DMEM culture medium to each bottle of T75. Observe the cell density on the day of transfection, and transfection can be performed when it is 80% to 90% full. There is no need to change the culture medium before transfection.

[0096] 3. Make fat transfer complex

[0097] Reagent Name Reagent Quantity

[0098] Vector plasmid 5μL (1.0μg / μL)

[0099] Packaging plasmid 5μL (1.0μg / μL)

[0100] Helper plasmid 5μL (1.0μg / μL)

[0101] Note: LipofiterTM transfection reagent is a product of Hanbio Biotech. For instructions, please refer to the LipofiterTM manual.

[0102] 4.AAV virus collection:

[0103] Virus particles are present in both packaging cells and culture supernatant. Both cells and culture supernatant can be collected to obtain the best yield.

[0104] 1 Prepare a dry ice ethanol bath (pour ethanol into a foam box filled with dry ice, or use liquid nitrogen instead of a dry ice ethanol bath) and a 37°C water bath;

[0105] 2. Collect the toxin-producing cells and the culture medium into a 15 mL centrifuge tube. When collecting cells, tilt the culture plate at a certain angle to scrape the cells into the culture medium;

[0106] 3 Centrifuge at 1000 rpm / min for 3 minutes to separate the cells and supernatant. Store the supernatant separately and resuspend the cells in 1 mL PBS.

[0107] 4 Transfer the cell suspension repeatedly between a dry ice ethanol bath and a 37°C water bath, and freeze and thaw four times. Shake slightly after each thawing. Note: Each solidification and thawing takes about ten minutes.

[0108] 5. AAV virus concentration:

[0109] 1 Centrifuge at 10,000 g to remove cell debris and transfer the supernatant to a new centrifuge tube.

[0110] 2. Mix the supernatants collected twice and filter them with a 0.45 μm filter to remove impurities.

[0111] 3. Add 1 / 2 volume of 1M NaCl, 10% PEG8000 solution, mix well, and incubate at 4°C overnight.

[0112] 4 Centrifuge at 12,000rpm for 2h, discard the supernatant, dissolve the virus precipitate with an appropriate amount of PBS solution, and filter and sterilize with a 0.22μm filter after complete dissolution.

[0113] 5. Add Benzonase nuclease to digest and remove residual plasmid DNA (final concentration is 50U / mL). Close the tube cap and invert several times to mix thoroughly. Incubate at 37℃ for 30 minutes;

[0114] 6 Filter with a 0.45 μm filter head and take the filtrate, which is the concentrated AAV virus.

[0115] 6. Purification of AAV

[0116] 1. Add solid CsCl to the virus concentrate until the density is 1.41 g / mL (refractive index 1.372);

[0117] 2. Add the sample to the ultracentrifuge tube and fill the remaining space of the tube with the pre-prepared 1.41 g / mL CsCl solution;

[0118] 3 Centrifuge at 175,000g for 24 hours to form a density gradient. Collect samples of different densities step by step in sequence and take samples for titer determination. Collect the fractions enriched with AAV particles;

[0119] 4Repeat the above process once.

[0120] 5) Place the virus into a 100 kDa dialysis bag and dialyze and desalt overnight at 4 degrees. This is the purified AAV virus.

[0121] AAV virus packaging titer determination (using Q-PCR method)

[0122] 1) Take 20 μL of concentrated virus solution, add 1 μL of RNAse-free DNAse, mix well, and react in a 37°C water bath for 30 min.

[0123] Centrifuge at 24°C, 12 000 rpm / min for 10 min, and take 10 μL of supernatant into another sterile 1.5 mL EP tube.

[0124] 3) Add 90 μL Dilution Buffer (1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 150 mM NaCl), mix well, and react in a metal bath at 37°C for 30 min.

[0125] 4) Cool naturally to room temperature, add 1 μL of proteinase K, and react in a 65°C water bath for 1 h.

[0126] React in a 5100℃ metal bath for 10 min and cool naturally to room temperature.

[0127] 6) Perform Q-PCR to detect the titer.

[0128] AAV virus storage and dilution

[0129] 1. Storage of viruses:

[0130] If you want to use the adeno-associated virus for experiments within a short period of time after receiving the virus solution, you can temporarily store the virus at 4°C; if you need to store it for a long time, please store it at -80°C (put the virus in a cryopreservation tube and seal it with sealing film).

[0131] 1) The virus can be stored at -80℃ for more than 6 months; however, if the virus is stored for more than 6 months, it is recommended to re-determine the virus titer before use.

[0132] 2) Repeated freezing and thawing will reduce the virus titer: Each freezing and thawing will reduce the virus titer by 10%; therefore, repeated freezing and thawing should be avoided as much as possible during the use of the virus. To avoid repeated freezing and thawing, it is recommended to package the virus according to the amount used each time after receiving it.

[0133] 2. Virus dilution:

[0134] If you need to dilute the virus, please take out the virus and place it in an ice bath to thaw, then use PBS buffer or serum-free medium to culture the target cells (containing serum or double antibodies will not affect virus infection). Mix well and store at 4°C after aliquoting (please try to use it up within three days) and use after aliquoting.

[0135] Safety precautions for AAV use

[0136] 1. It is best to use a biosafety cabinet when operating viruses. If you use an ordinary clean bench to operate viruses, please do not turn on the exhaust fan.

[0137] 2. Please wear lab coat, mask and gloves when handling viruses.

[0138] 3. Be especially careful not to generate aerosol or splash when handling viruses. If the clean bench is contaminated with viruses during operation, please wipe it clean immediately with 70% ethanol plus 1% SDS solution. Pipette tips, centrifuge tubes, culture plates, culture fluids, etc. that have come into contact with viruses should be soaked in 84 disinfectant or 1% SDS overnight and then discarded.

[0139] 4. When observing the infection of cells under a microscope, follow the following steps: Tighten the culture bottle or cover the culture plate. Clean the outer wall of the culture bottle with 70% ethanol and then observe and take pictures under the microscope. Before leaving the microscope bench, clean the microscope bench with 70% ethanol.

[0140] 5. If centrifugation is required, use centrifuge tubes with good sealing properties, or seal them with sealing film before centrifugation, and try to use a centrifuge in the tissue culture room.

[0141] 6. After removing gloves, wash your hands with soap and water.

[0142] Example 3 Viruses packaged with AAV capsids containing random sequences, intravitreal injection into mice and retinal extraction

[0143] 1. Anesthesia: 4.3% chloral hydrate 0.01mL / g;

[0144] 2. Mydriatic solution dilates the pupil, and methylcellulose keeps the ocular surface moist;

[0145] 3. Adjust the mouse head position and injection position: about 1mm behind the corneal limbus;

[0146] 4. Use a 33G syringe to make an incision, insert the needle tip vertically, then tilt it, and slowly push the AAV capsid protein library virus, each of which contains a specific amino acid sequence, into the mouse vitreous cavity. After injection, leave the needle in for 0.5-1 minute and quickly remove the needle;

[0147] 5. After about one week, the mice were anesthetized and killed, and the retina of the target tissue was taken to extract its genome, and the AAV capsid sequence that penetrated into the target tissue was sequenced and analyzed;

[0148] The AAV sequences contained in the genome were analyzed by sequencing results (see Figure 2), the box represents a total of 30 bases containing 21 random base sequences inserted by sequencing, among which the main peak of the sequencing results of the 21 random base sequences is relatively obvious, and the base sequence of cttgctcgcggaggccagagagaaggcgcc (SEQ ID NO.4) is obtained through reading analysis.

[0149] Example 4 Comparison of infection areas of eyeballs and organs after intravitreal injection of AAV-8 and AAV8-590MJ06 viruses with single-point mutations in the insertion sequence and capsid packaging

[0150] 1. Intravitreal injection (see Example 3)

[0151] 2. Retinal frozen sections: After 3-4 weeks of feeding, the virus-injected mice were killed by cervical dislocation, and their eyes were removed and fixed with 4% paraformaldehyde for about 1 day. They were dehydrated with 20% and 30% sucrose solutions; frozen sections were made with a thickness of 10 μm and fluorescence was observed under a fluorescence microscope.

[0152] 3. Figure 3 The same amount of virus was expressed in AAV-8 (A) and AAV8-590MJ06 (B) capsids respectively. The virus was injected into the vitreous and the expression effect was observed in retinal frozen sections. It was found that the virus packaged with AAV8-590MJ06 capsid had better infection efficiency in various retinal layers, including the retinal ganglion cell layer, inner nuclear layer, outer nuclear layer, photoreceptor cell layer and retinal pigment epithelial cell layer.

[0153] Further research of the present invention found that not all sequences similar to RGGQREG (SEQ IN NO 5) can achieve the expected results of the present invention. Some of the candidate polypeptides obtained by in vitro evolution screening methods cannot achieve good retinal infection efficiency as expected by the screening method after vitreous injection. For example, by detecting the mRNA level of luciferase in non-targeted tissues, it was found that the virus packaged by AAV8-590MJ06 capsid has more expression in the eyeball area and less leakage expression in non-targeted organs ( Figure 4 ). The virus packaged with the modified capsid of RGGQKEG (SEQ IN NO 9) was screened under the same model and conditions. The same amount of virus was injected into the vitreous body and the expression effect was observed by taking frozen sections of the retina. It was found that the retinal infection efficiency and penetration efficiency of the virus packaged with the capsid of RGGQKEG (SEQ IN NO 9) sequence were low, which did not meet expectations ( Figure 5 ).

[0154] The sequence involved in the present invention is as follows:

[0155] SEQ ID NO.1: (748aa)

[0156] MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEH

[0157] DKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSP

[0158] DSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDS

[0159] TWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPK

[0160] RLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVG

[0161] RSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGP

[0162] NTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQN

[0163] AARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNLARGGQREGATAPQIGTVNSQGALPGMVWQNRDVYLQG

[0164] PIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRW

[0165] NPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL

[0166] SEQ ID NO.2:(738aa)

[0167] MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEH

[0168] DKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSP

[0169] DSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDS

[0170] TWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPK

[0171] RLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVG

[0172] RSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGP

[0173] NTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQN

[0174] AARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTD

[0175] GNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNY

[0176] YKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL

[0177] SEQ ID NO.3:

[0178] LARGGQREGA

[0179] SEQ ID NO.4:

[0180] cttgctcgcggaggccagagagaaggcgcc

[0181] SEQ ID NO.5:

[0182] RGGQREG

[0183] SEQ ID NO.6:

[0184] cgcggaggccagagagaaggc

[0185] SEQ ID NO.7:

[0186] AGGACCCTGTTACCGCCAAC

[0187] SEQ ID NO.8:

[0188] GATGTTTCAGGCCAAAGCCG

[0189] SEQ ID NO.9:

[0190] RGGQKEG

Claims

1. A polypeptide, characterized in that A polypeptide consisting of 10 amino acids is inserted between amino acids 590 and 591 of the capsid protein of the AAV-8 serotype; The amino acid sequence of the AAV-8 serotype capsid protein is shown in SEQ ID NO. 2; The polypeptide composed of 10 amino acids includes a polypeptide having an amino acid sequence as shown in SEQ ID NO.

5.

2. The polypeptide according to claim 1, characterized in that The amino acid sequence at positions 1-590 is as shown in the amino acid sequence at positions 1-590 of the AAV-8 serotype capsid protein sequence shown in SEQ ID NO.2, the amino acid sequence at positions 593-599 among the amino acids at positions 591-600 is the amino acid sequence of the polypeptide sequence described in claim 1 as shown in SEQ ID NO.5, and the amino acid sequence at positions 601 and thereafter is the amino acid sequence at positions 591 and thereafter of the AAV-8 serotype capsid protein sequence shown in SEQ ID NO.

2.

3. The polypeptide according to claim 1, characterized in that The amino acid sequence of the polypeptide consisting of 10 amino acids is shown in SEQ ID NO.

3.

4. A nucleic acid, characterized in that The sequence of the nucleic acid is any one of the following: (1) a base sequence encoding the polypeptide according to claim 1; (2) a base sequence encoding the polypeptide of claim 2; (3) a base sequence encoding the polypeptide according to claim 3; or The corresponding capsid protein sequence is shown in SEQ ID NO.

1.

5. An AAV capsid, characterized in that The protein of the AAV capsid contains the polypeptide of any one of claims 1-3.

6. The AAV capsid according to claim 5, characterized in that The construction method includes the following steps: Synthesize the nucleic acid whose sequence is shown in SEQ ID NO.6; Adding protective bases cttgct and gcc to the 5' and 3' ends of the nucleic acid shown in SEQ ID NO.6, respectively; Inserted between the nucleotides corresponding to the 590th and 591st amino acids of the AAV-8Cap gene to form the AAV capsid.

7. The use of the nucleic acid according to claim 4, characterized in that: The nucleic acid is used in the preparation of a drug for treating retinal diseases; the drug for treating retinal diseases is a drug targeting various layers of the retina.

8. Use of the polypeptide according to any one of claims 1 to 3, characterized in that: The polypeptide is used in preparing medicine for treating retinal related diseases.

9. The use according to claim 8, characterized in that: The drug for treating retinal-related diseases is a drug that is injected into the vitreous body to improve retinal infection efficiency, increase retinal penetration efficiency, or improve retinal recognition efficiency.

10. The use according to claim 8, characterized in that: The cells in the cell layer are selected from one or more of retinal ganglion isolated cells, Neuro2A cells, U251 cells, ARPE-19 cells, SH-SY-5Y cells, BV2 cells, HBMEC primary isolated cells, Jurkat cells, K562 cells, 293 cells or THP1 cells.

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