Adeno-associated virus mutant and its application

Abstract

The present invention belongs to the field of biomedicine technology, and discloses an adeno-associated virus mutant and its application. The amino acid sequence of the adeno-associated virus capsid protein mutant of the present invention includes a sequence as shown in any one of SEQ ID No.1‑6. The present invention uses the adeno-associated virus capsid protein mutant and its expression vector, host cell, and recombinant adeno-associated virus in the preparation of a drug delivery tool for preventing and / or treating retinal diseases. The adeno-associated virus capsid protein mutant of the present invention has retinal targeting, and the retinal targeting is increased by about 48.27 times at most, with good specificity. The present invention can promote the AAV-based gene therapy method towards large-scale and socialized application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an adeno-associated virus mutant and application thereof. Background Art

[0002] Adeno-associated virus (AAV) is a small non-enveloped virus that encapsulates a linear single-stranded DNA genome. It belongs to the genus Dependovirus of the family Parvoviridae and requires a helper virus (usually adenovirus) to participate in replication. The AAV genome is a single-stranded DNA fragment contained in a non-enveloped viral capsid and can be divided into three functional regions: two open reading frames (Rep gene, Cap gene) and inverted terminal repeats (ITR). Recombinant adeno-associated virus vectors (rAAV) are derived from non-pathogenic wild-type adeno-associated viruses. Due to its advantages such as wide host range, non-pathogenicity, low immunogenicity, long-term stable expression of foreign genes, good diffusion performance and stable physical properties, it is widely used as a gene transfer vector in gene therapy and vaccine research. In medical research, rAAV has been used in the study of gene therapy for a variety of diseases (including in vivo and in vitro experiments), such as gene function research, construction of disease models, and preparation of gene knockout mice.

[0003] As a cutting-edge biotechnology, AAV gene therapy has shown great potential in the treatment of retinal diseases. For example, Luxturna, the first FDA-approved AAV gene therapy drug, was successfully used to treat Leber congenital amaurosis type 2 (LCA2) caused by a specific gene mutation. It significantly improved patients' vision by delivering the healthy RPE65 gene to retinal cells. However, despite these breakthroughs, the application of AAV to treat retinal diseases still faces many challenges.

[0004] The current limitations of AAV vectors in the treatment of retinal diseases mainly include insufficient transduction efficiency, weak targeting, and possible immune response problems. These limitations may lead to poor treatment effects or adverse reactions, such as subretinal injections that may cause bleeding, inflammation, and even retinal detachment. In addition, the lateral diffusion capacity of AAV vectors is limited, and each injection can only target cells near the injection point, which limits the breadth and depth of treatment. The use of vitreous injection requires low surgical skills, but the infection effect is not as good as retinal injection. Improving the retinal targeting of AAV is particularly necessary and urgent. By engineering the AAV capsid, its affinity for specific retinal cells can be enhanced, thereby improving the transduction efficiency and the accuracy of treatment. In addition, improving the retinal targeting of AAV can also help reduce unnecessary side effects, such as reducing the risk of immune response by reducing transduction of non-target tissues. This is of great significance for achieving long-term and stable treatment effects and improving the quality of life of patients. Therefore, continuously optimizing the design of AAV vectors and improving their specificity and safety are the key to promoting the application of AAV gene therapy in the field of retinal diseases.

[0005] In summary, although AAV is one of the most widely used and safest gene therapy vectors, further improvements are still needed in terms of lower doses and more efficient retinal targeting through vitreous injection. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an adeno-associated virus mutant with retinal targeting and its application.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides an adeno-associated virus capsid protein mutant, the amino acid sequence of which includes a sequence as shown in any one of SEQ ID No.7-12.

[0009] The adeno-associated virus capsid protein mutant of the present invention has retinal targeting, and the mutant has a stronger infection effect on cells of the GCL and INL layers (such as Ganglion cells, Muller cells, etc.). Compared with the control group AAV8, the retinal targeting is increased by up to about 48.27 times, with good specificity, and also shows good effects in NHP (Nonhuman primate).

[0010] As a preferred embodiment of the adeno-associated virus capsid protein mutant of the present invention, a targeting peptide is inserted into the amino acid sequence; the amino acid sequence of the targeting peptide is any one of the sequences shown in SEQ ID No. 1-6.

[0011] In a second aspect, the present invention provides a nucleic acid encoding the adeno-associated virus capsid protein mutant.

[0012] As a preferred embodiment of the nucleic acid of the present invention, its nucleotide sequence comprises a nucleotide sequence as shown in any one of SEQ ID No.13-18.

[0013] In a third aspect, the present invention provides an expression vector comprising the nucleic acid.

[0014] In a fourth aspect, the present invention provides a host cell comprising the expression vector.

[0015] In a fifth aspect, the present invention provides a host cell that expresses the adeno-associated virus capsid protein mutant.

[0016] In a sixth aspect, the present invention provides a recombinant adeno-associated virus, including the adeno-associated virus capsid protein mutant.

[0017] As a preferred embodiment of the recombinant adeno-associated virus of the present invention, it also includes a heterologous target gene.

[0018] As a further preferred embodiment of the recombinant adeno-associated virus of the present invention, the heterologous target gene encodes any gene product of interfering RNA, aptamer, endonuclease, and guide RNA.

[0019] In the seventh aspect, the present invention provides a method for preparing a recombinant adeno-associated virus, comprising introducing at least the following components into a host cell: 1) the nucleic acid or the expression vector, 2) an adeno-associated virus helper plasmid, and 3) a plasmid containing two terminal inverted repeat sequences.

[0020] In an eighth aspect, the present invention provides rAAV prepared by the above method.

[0021] In a ninth aspect, the present invention provides a pharmaceutical composition comprising the recombinant adeno-associated virus or the rAAV, and a pharmaceutically acceptable carrier.

[0022] In the tenth aspect, the present invention uses the adeno-associated virus capsid protein mutant, the expression vector, the host cell, the recombinant adeno-associated virus, and the rAAV in the preparation of a drug or preparation for delivering a gene product to cells or tissues of a subject.

[0023] As a preferred embodiment of the application of the present invention, the cells are retinal cells; preferably, the retinal cells include at least one of Ganglion cells, Muller cells, Bipolar cells, Horizontal cells, rod cells, cone cells, and RPE cells; and the tissue is an eye.

[0024] In the eleventh aspect, the present invention uses the adeno-associated virus capsid protein mutant, the expression vector, the host cell, the recombinant adeno-associated virus, and the rAAV in the preparation of a drug delivery tool for preventing and / or treating retinal diseases.

[0025] As a preferred embodiment of the use of the present invention, the retinal diseases include retinitis pigmentosa, Leber hereditary optic neuropathy (LHON), Leber congenital amaurosis and color blindness.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The adeno-associated virus capsid protein mutant of the present invention has good targeting to different retinal cell types, and the retinal targeting is increased by up to about 48.27 times. The recombinant adeno-associated virus vector constructed using the AAV capsid protein mutant of the present invention has higher specificity, better safety, and a wide range of applications. The present invention has developed a new AAV gene therapy product with a simpler and more effective injection method to meet the needs of more different patients and promote AAV-based gene therapy methods towards large-scale and socialized applications. This will be of great significance in the future for improving the benefits of gene therapy and serving a large number of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 To analyze the targeting of different serotypes to the cynomolgus monkey retina for NGS testing (28 days);

[0029] Figure 2 Immunofluorescence sections were used to detect the ability of different serotypes to infect different types of cells in the retina of crab-eating monkeys (28 days). DETAILED DESCRIPTION

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0031] Unless the context clearly indicates otherwise, the term "or" refers to a single element of the listed alternative elements and the term "and / or" refers to any one, any two, any three, any more or all of the listed alternative elements.

[0032] The term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In the text, when the term "comprising" or "including" is used, unless otherwise indicated, it also covers the situation consisting of the stated elements, integers or steps. For example, when referring to a polypeptide "comprising" a specific sequence, it is also intended to cover a polypeptide consisting of the specific sequence.

[0033] "Adeno-associated virus (AAV)" is a non-enveloped icosahedral capsid virus of the Parvoviridae family, including a single-stranded DNA viral genome. The Parvoviridae family includes the genus Dependovirus, which includes AAV, which relies on the presence of a helper virus such as adenovirus for its replication. Due to its relatively simple structure, ability to infect a variety of cells (including quiescent and dividing cells) without integrating into the host genome, and its relatively mild immunogenic characteristics, AAV has been shown to be useful as a biological tool for expressing a gene of interest in vitro or in vivo. AAV-based expression vectors are also contemplated herein, including recombinant AAV (rAAV) with a gene of interest for therapeutic purposes.

[0034] The wild-type AAV viral genome is a linear, single-stranded DNA (ssDNA) molecule with a length of about 5000 nucleotides (nt). The AAV viral genome typically includes two inverted terminal repeats (ITRs), which cap the viral genome at the 5' and 3' ends, respectively, and provide a replication origin for the viral genome. These ITRs have a characteristic T-shaped hairpin structure and have multiple functions, including but not limited to acting as a starting point for DNA replication by acting as a primer for the endogenous DNA polymerase complex of the host viral replication cell.

[0035] The wild-type AAV viral genome also includes the Rep gene and the Cap gene, which encode four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40) and three capsid proteins or structural proteins (VP1, VP2, VP3), respectively. The Rep protein is associated with viral replication and packaging, while the capsid protein assembles to form the protein shell of AAV or the AAV capsid. Alternative splicing and alternate start codons and promoters result in the production of four different Rep proteins from a single open reading frame in the Rep gene and three capsid proteins from a single open reading frame in the Cap gene.

[0036] In the context of AAV, the terms "viral capsid protein" or "capsid protein" used refer to the protein of AAV that is capable of self-assembly to produce AAV particles, also known as coat protein or VP protein. The VP protein includes three subunits VP1, VP2 and VP3, and thus the changes in the VP protein mutant relative to the wild-type VP protein can be reflected in the changes in the amino acid sequences of the VP1, VP2 and VP3 subunits. Accordingly, in this article, "capsid protein mutants" include VP protein mutants, and also VP1 VP2 and / or VP3 subunit mutants. Due to the consistency of the amino acid sequences between the VP1, VP2 and VP3 subunits expressed from the same Cap gene, when the coding sequence in the Cap gene is altered, for example, when the coding sequence of the VP1 subunit is altered, the amino acid sequences of the expressed VP2 and VP3 subunits are also changed.

[0037] The term "serotype" used in the context of AAV is used to refer to the difference in the capsid protein of AAV that is serologically different from other AAV serotypes. The determination of serological uniqueness is based on the reactivity between an antibody and one AAV, but lacks cross-reactivity with other or another AAV. This cross-reactivity difference is usually due to differences in the capsid protein sequence (or its subunit sequence) / antigenic determinants (for example, due to differences in the VP1, VP2 and / or VP3 sequences of serotype AAV8). A variety of AAV serotypes have been discovered, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12, and their mutants.

[0038] "Recombinant AAV vector" refers to an AAV genome derived by removing some wild-type genes (e.g., Rep genes and Cap genes) from the AAV genome using molecular biological methods and replacing them with heterologous nucleic acid sequences (e.g., coding sequences of proteins or RNA for therapeutic purposes). Typically, for recombinant AAV vectors, one or two inverted terminal repeat (ITR) sequences of the AAV genome are retained. In most cases, recombinant AAV vectors are replication-defective and lack sequences encoding functional Rep and Cap proteins in their viral genomes. These replication-defective AAV particles may lack most of the parental coding sequences and essentially carry only one or two AAV ITR sequences and target nucleic acids for delivery to cells, tissues, organs or organisms. AAVs including recombinant AAV vectors are referred to herein as recombinant AAV (rAAV).

[0039] "Amino acid changes" herein include amino acid substitutions, deletions or insertions. The number of amino acid changes in the mutant sequence relative to the parent sequence can be calculated as the sum of the number of amino acid substitutions, the number of deleted amino acids and the number of inserted amino acids.

[0040] As used herein, the terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably to refer to nucleotide polymers. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include (but are not limited to) DNA, RNA and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide. "Isolated nucleic acid molecule" refers to a nucleic acid molecule that is separated from its natural environment (such as the intracellular environment) and is substantially free of one or more substances that are normally associated with it in nature, such as proteins, nucleic acids, lipids, carbohydrates, cell membranes, etc., or is an artificially prepared (such as artificially synthesized) nucleic acid molecule.

[0041] The term "expression vector" refers to a nucleic acid molecule comprising various expression elements for expressing a target protein or target RNA in a host cell. For expression vectors for expressing a target protein in eukaryotic cells, these expression elements generally include promoters, enhancers, polyadenylation signal sequences, etc. For convenience of amplification in Escherichia coli, the expression vector generally also includes an Escherichia coli replicon sequence. In addition, the expression vector may also include an antibiotic resistance gene or a selective marker gene (e.g., ampicillin resistance gene (AmpR), thymidine kinase gene (TK), kanamycin resistance gene (KanR), neomycin resistance gene (NeoR) etc.) for screening and a multiple cloning site (MCS) for insertion of the target gene.

[0042] The term "host cell" refers to cells in which an expression vector can be maintained and / or replicated, including prokaryotic cells and eukaryotic cells, such as bacteria (such as E. coli), fungi (yeast), insect cells (such as SF9) and mammalian cells (such as HEK-293T).

[0043] When referring to a pharmaceutical composition, the term "pharmaceutically acceptable carrier" is used to refer to a solid or liquid diluent, filler, antioxidant, stabilizer, or other substance that can be safely administered, is suitable for administration to humans and / or animals without excessive adverse side effects, and is suitable for maintaining the activity of the drug or active agent located therein. Depending on the route of administration, various different carriers well known in the art can be used, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer, emulsifiers, isotonic saline, and / or pyrogen-free water, etc.

[0044] The "targeting" of AAV or rAAV refers to the phenomenon that when it is introduced into the body, it is relatively concentrated in a specific tissue or organ. For example, the targeting can be manifested as a higher concentration in tissue A than in tissue B. This targeting can be reflected by detecting the content or concentration of its genome in different tissues or organs.

[0045] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0046] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials and reagents used, unless otherwise specified, can be obtained from commercial sources. The nucleotide sequence of the AAV8 serotype capsid protein is NC_006261.1 (GenBank).

[0047] Example 1: Screening of novel mutants

[0048] (1) Construction of AAV8 serotype mutant library backbone plasmid:

[0049] The backbone vector of the AAV8 serotype mutant library contains a CAG promoter, Intron, a mutant AAV8 serotype capsid protein sequence [the VP1 sequence after D584 is removed, and the D584 nucleic acid sequence GAT and the polyA front sequence constitute the restriction site BsrG I (TGTACA), which is used for subsequent backbone restriction linearization] and poly A. The above sequence is synthesized by gene synthesis and inserted between the ITRs of the AAV vector plasmid to form the backbone vector of the AAV8 serotype mutant library.

[0050] (2) Construction of mutant Rep-CAP vector

[0051] By introducing a stop codon at the N-terminus of the VP1, VP2, and VP3 proteins of the CAP sequence in the AAV8 serotype, the Rep-CAP vector can normally express the Rep protein and the AAP protein, but cannot express the VP1, VP2, and VP3 proteins of the CAP, thereby avoiding contamination of the CAP sequence in the parent. The above sequence is synthesized by gene synthesis and inserted to replace the CAP sequence of the AAV8 serotype Rep-CAP vector.

[0052] (3) Construction of random heptapeptide plasmid library

[0053] F:TACGGTATCGTGGCAGATAACTTGCAGCAGCAAAACNNKNNKNNKN NKNNKNNKNNKACGGCTCCTCAAATTGGAACTG;

[0054] R: CGGTTTATTGATTAACAATCGATTACAGATTACGGGTGAGGTAACG.

[0055] Using the vector of AAV8 capsid protein as a template, PCR amplification was performed to obtain a fragment containing a random sequence (using the above-mentioned primers F and R). The fragments were subjected to gel electrophoresis and gel recovery to obtain the size fragments of the purified random 7-peptide library nucleic acid fragments. The fragments were connected to the AAV8 capsid protein mutant library backbone vector (BsrG I enzyme digestion and gel recovery purification) by Gibson homologous recombination connection. The connected vector was purified by a PCR product purification kit and digested with Plasmid-SafeDNase enzyme to remove the unconnected fragments. Finally, after purification by a PCR product purification kit, the constructed AAV8 mutant plasmid library was obtained.

[0056] (4) Production of random heptapeptide virus library

[0057] The mutant Rep-Cap plasmid constructed in step (2), the AAV8 serotype mutant plasmid library constructed in step (3), and the pHelper plasmid were co-transfected into HEK-293T cells, and the adeno-associated virus was purified by iodixanol gradient ultracentrifugation. The virus titer was measured at 10 12 GC / Ml-10 13 GC / mL is the appropriate titer, and the AAV8 serotype mutant virus library is obtained and placed at -80°C for future use.

[0058] (5) Screening of mutants

[0059] (5.1) Animal injection and dissection

[0060] Animal experiments were conducted using cynomolgus monkeys. Each eye was injected intravitreally with 1E12GC of the AAV8 serotype mutant virus library prepared in step (4). The animals were dissected and organs were collected 28 days after the injection. The samples were immediately frozen in liquid nitrogen and used for subsequent RNA extraction experiments.

[0061] (5.2) Total RNA extraction and RT-PCR

[0062] Grinding of samples: Precool the grinder 10 minutes in advance and set the grinding parameters. Take out the animal tissue samples stored in the -80℃ refrigerator, add an appropriate amount of TransZol Up according to the ratio of 1mL TransZol Up for every 50mg-100mg tissue, then add two clean and sterile 3mm grinding ceramic beads and wrap with sealing film. Place the sample in the 24-well grinding adapter and balance it, tighten the screw, and press the cover closing button. Start the grinding program, take out the sample after the instrument runs, observe the sample grinding particle size, if there are no large pieces of tissue remaining, you can proceed with the subsequent extraction operation. The ground sample is centrifuged at 4℃, 12,000×g for 2 minutes, and the supernatant is transferred to a new 1.5mL RNase-free EP tube with corresponding labels.

[0063] Extraction of total RNA from samples: Refer to the instructions of TransZol Up Plus RNA Kit (Beijing Quanshijin, catalog number: ER501) for details. For every 1 mL of TranZol up, add 0.2 ml of RNA Extraction Agent, shake vigorously for 5 minutes, and centrifuge at 12,000×g, 4°C for 10 minutes. At this time, the sample is divided into three layers. Transfer the colorless aqueous phase to a new 1.5mL RNase-free EP tube, add an equal volume of anhydrous ethanol (precipitate may appear at this time), and gently invert to mix; add the obtained solution and precipitate to the centrifuge column, centrifuge at 12,000×g at room temperature for 30s, and discard the filtrate; add 500μL CB9, centrifuge at 12,000×g at room temperature for 30s, and discard the filtrate; repeat the above steps once; add 500μL WB9, centrifuge at 12,000×g at room temperature for 30s, and discard the filtrate; repeat the above steps once; centrifuge at 12,000×g at room temperature for 2min to completely remove residual ethanol; place the centrifuge column in a 1.5mL RNase-free EP tube, add 30μL-50μL (depending on the size of the tissue) RNase-free Water in the center of the centrifuge column, and let it stand at room temperature for 1min; centrifuge at 12,000×g at room temperature for 1min to elute RNA;

[0064] Determination of sample nucleic acid concentration: Use a micro-nucleic acid quantifier detector to detect RNA concentration, record the concentration, OD260 / 280, OD260 / 230, and store the RNA at -80°C.

[0065] RT-PCR: RNA samples were extracted and the first-strand cDNA was synthesized using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, R312-01). Then two rounds of PCR amplification were performed using NEB Q5 (the first round used outer primers for amplification; the second round used the first round product recovered from the gel as a template and amplified using NGS primers). The PCR products of the corresponding band size recovered from the gel were sent to the company for NGS sequencing;

[0066] NGS sequencing, data analysis, and selection of candidate vectors: After sequencing, sequencing data analysis was performed, and sequences with high frequency rankings and that appeared multiple times in multiple samples were selected as candidates. 30,000 candidate sequences were selected from the first round for oligo pool synthesis. The oligo pool was used instead of the random 7-peptide primers mentioned above to re-construct the second round of plasmid library and virus library. The screening and enrichment process was repeated, and finally candidate sequences were selected for vector construction and related verification work.

[0067] AAV capsid protein mutants 1-6 were screened out, and the amino acid sequences of their VP1 were shown as SEQ ID No.7-SEQ ID No.12, and the nucleotide sequences were shown as SEQ ID No.13-SEQ ID No.18, respectively; the amino acid sequences of the targeting peptides in VP1 were shown as SEQ ID No.1 to SEQ ID No.6, respectively.

[0068] Example 2: Construction of AAV capsid protein mutants and production of viruses

[0069] (1) Plasmid construction

[0070] (1.1) Construction of mutant serotype GOI plasmid:

[0071] The CAP sequence of the mutant serotype GOI plasmid is located between the ITRs. The plasmid is mainly used for NGS sequencing detection of mixed viruses. The construction method is as follows;

[0072] The AAV8 serotype mutant library backbone plasmid was digested with BsrG I, gel electrophoresis was performed, and a band with a fragment size of 6815 bp was cut out for gel recovery to obtain the digested backbone fragment.

[0073] According to the Cap sequence of mutant 1, the following primers were designed. The specific steps were as follows: AAV8 capsid protein plasmid was used as a template and YJ478-F+V8-Stop-R primers were used for PCR amplification to obtain the target product, which was named YJ478-1. The backbone fragment and YJ478-1 were mixed by the following steps and proportions to recombinantly construct the GOI plasmid YJ478 containing the capsid protein of mutant 1;

[0074] According to the Cap sequence of mutant 2, the following primers were designed. The specific steps were as follows: AAV8 capsid protein plasmid was used as a template and YJ487-F and V8-Stop-R primers were used for PCR amplification to obtain the target product, named YJ487-1. The backbone fragment and YJ487-1 were mixed by the following steps and proportions to recombinantly construct the GOI plasmid YJ487 containing the capsid protein of mutant 2;

[0075] According to the Cap sequence of mutant 3, the following primers were designed. The specific steps were as follows: AAV8 capsid protein plasmid was used as a template and YJ489-F and V8-Stop-R primers were used for PCR amplification to obtain the target product, which was named YJ489-1. The backbone fragment and YJ489-1 were mixed by the following steps and proportions to recombinantly construct the GOI plasmid YJ489 containing the capsid protein of mutant 3;

[0076] According to the Cap sequence of mutant 4, the following primers were designed. The specific steps were as follows: AAV8 capsid protein plasmid was used as a template and YJ491-F+V8-Stop-R primers were used for PCR amplification to obtain the target product, which was named YJ491-1. The backbone fragment and YJ491-1 were mixed by the following steps and proportions to recombinantly construct the GOI plasmid YJ491 containing the capsid protein of mutant 4;

[0077] According to the Cap sequence of mutant 5, the following primers were designed. The specific steps are as follows: AAV8 capsid protein plasmid was used as a template and YJ498-F and V8-Stop-R primers were used for PCR amplification to obtain the target product, which was named YJ498-1. The backbone fragment and YJ498-1 were mixed by the following steps and proportions to recombinantly construct the GOI plasmid YJ498 containing the capsid protein of mutant 5;

[0078] According to the Cap sequence of mutant 6, the following primers were designed. The specific steps are as follows: AAV8 capsid protein plasmid was used as a template and YJ499-F+V8-Stop-R primers were used for PCR amplification to obtain the target product, which was named YJ499-1. The backbone fragment and YJ499-1 were mixed by the following steps and proportions to recombinantly construct the GOI plasmid YJ499 containing the capsid protein of mutant 6;

[0079] The primers involved in the construction of the GOI vector containing the AAV capsid protein mutant are shown in Table 1:

[0080] Table 1 Primer information

[0081]

[0082] (1.2) Construction of mutant serotype RC plasmid:

[0083] The mutant serotype RC plasmid is mainly used to produce virus together with the GOI plasmid carrying the reporter gene for the verification of a single virus. The construction method is as follows:

[0084] The AAV9 Rep-CAP plasmid was double-digested with Smi I and BshT I, and the fragment band of about 5000 bp was cut out for gel electrophoresis and gel recovery to obtain the digested backbone fragment.

[0085] According to the Cap sequence of mutant 1, the following primers were designed. The specific steps were as follows: plasmid YJ478 was used as a template and primers YJ929-F1+YJ930-R were used for amplification and gel recovery to obtain the target product YJ930-1. Plasmid YJ478 was used as a template and primers YJ930-F+YJ929-R2 were used for amplification and gel recovery to obtain the target product YJ930-2. The backbone fragment, YJ930-1, and YJ930-2 were mixed in the following steps and proportions to recombinantly construct the Rep-CAP plasmid of mutant 1;

[0086] According to the Cap sequence of mutant 2, the following primers were designed. The specific steps were as follows: plasmid YJ487 was used as a template and primers YJ929-F1+YJ931-R were used for amplification and gel recovery to obtain the target product YJ931-1. Plasmid YJ487 was used as a template and primers YJ931-F+YJ929-R2 were used for amplification and gel recovery to obtain the target product YJ931-2. The backbone fragment, YJ931-1, and YJ931-2 were mixed in the following steps and proportions to recombinantly construct the Rep-CAP plasmid of mutant 2;

[0087] According to the Cap sequence of mutant 3, the following primers were designed. The specific steps were as follows: using plasmid YJ489 as a template, using primers YJ929-F1+YJ934-R to amplify and gel-recover to obtain the target product YJ934-1; using plasmid YJ489 as a template, using primers YJ934-F+YJ929-R2 to amplify and gel-recover to obtain the target product YJ934-2; by mixing the backbone fragment, YJ934-1, and YJ934-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 3 can be recombined and constructed;

[0088] According to the Cap sequence of mutant 4, the following primers were designed, and the specific steps were as follows: plasmid YJ491 was used as a template, and primers YJ929-F1+YJ929-R were used for amplification and gel recovery to obtain the target product YJ929-1; plasmid YJ491 was used as a template, and primers YJ929-F+YJ929-R2 were used for amplification and gel recovery to obtain the target product YJ929-2; the backbone fragment, YJ929-1, and YJ929-2 were mixed by the following steps and proportions to recombinantly construct the Rep-CAP plasmid of mutant 4;

[0089] According to the Cap sequence of mutant 5, the following primers were designed. The specific steps were as follows: using plasmid YJ498 as a template, using primers YJ929-F1+YJ933-R to amplify and gel-recover to obtain the target product YJ933-1; using plasmid YJ498 as a template, using primers YJ933-F+YJ929-R2 to amplify and gel-recover to obtain the target product YJ933-2; by mixing the backbone fragment, YJ933-1, and YJ933-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 5 can be recombined and constructed;

[0090] According to the Cap sequence of mutant 6, the following primers were designed, and the specific steps were as follows: plasmid YJ499 was used as a template, and primers YJ929-F1+YJ932-R were used for amplification and gel recovery to obtain the target product YJ932-1; plasmid YJ499 was used as a template, and primers YJ932-F+YJ929-R2 were used for amplification and gel recovery to obtain the target product YJ932-2; the backbone fragment, YJ932-1, and YJ932-2 were mixed by the following steps and proportions to recombinantly construct the Rep-CAP plasmid of mutant 6;

[0091] The primers involved in the construction of the Rep-CAP vector of the above-mentioned AAV capsid protein mutant are shown in Table 2:

[0092] Table 2 Primer information

[0093]

[0094]

[0095] (1.3) Plasmid construction steps

[0096] Take a clean 200μL PCR tube, mark it and place it on an ice box. Prepare the reaction solution with the above enzyme-cut backbone and each target fragment at a backbone: fragment molar ratio of 1:3, and react in a PCR instrument at 50℃ for 30min for recombination connection. Take 50μL competent cells and thaw them on ice. Mix 10μL of the connection product with DH5α competent cells and place them on ice for 20-30 minutes; heat shock at 42℃ for 45 seconds; quickly place them on ice for 2 minutes, add 400μL of recovery SOC medium (without antibiotics), and culture at 37℃ and 200rpm for 1h; evenly apply it to Amp resistance plate (50μg / ml) and culture at 37℃ for 14 hours. Select monoclonal bacteria, expand the culture in 4mL liquid LB medium (Amp+ resistance), and culture at 37℃ for 14 hours.

[0097] The bacterial solution was centrifuged at 12000rpm for 1 minute, and the supernatant medium was discarded; 250μL of buffer P1 / RNaseA mixture was added, and the bacteria were resuspended by high-speed vortexing; 250μL of buffer P2 was added, and the solution was inverted 8-10 times; 350μL of buffer P3 was added, and the solution was immediately inverted and mixed 8-10 times to completely neutralize the solution; centrifuged at 13000rpm for 10 minutes, and the supernatant was passed through the column; centrifuged at 12000 for 1 minute, and the waste liquid was discarded, 500μL of PW1 was added, and the waste liquid was discarded; 600μL of PW2 was added, and the supernatant was discarded; 600μL of PW2 was added, and the supernatant was discarded; the supernatant was idling at 12000rpm for 2 minutes; 30-50μL of 55℃ preheated eluent was added, and the solution was allowed to stand for 2 minutes, and the solution was centrifuged at 12000rpm for 1 minute. Use a micro-volume nucleic acid quantifier to detect the concentration.

[0098] The obtained plasmid was tested for concentration, and 10 μL of the positive plasmid identified by enzyme digestion was sent for sequencing, and the positive plasmid was stored at -20°C. The sequencing results showed that the obtained plasmid could encode the variant capsid protein VP1.

[0099] (2) Packaging and purification of mutant serotype viruses

[0100] (2.1) Mutant serotype GOI plasmid virus packaging

[0101] The mutant serotype GOI plasmids of each group (control group serotype and AAV mutants 1-6 of the present invention), the mutant Rep-CAP vector used in library construction, and the pHelper plasmid were co-transfected into HEK-293T cells, and the AAV virus was purified by iodixanol gradient ultracentrifugation. The virus titer was measured to be around 1E+13GC / mL, which was an appropriate titer, and the cells were placed at -80°C for use.

[0102] (2.2) Mutant serotype RC plasmid virus packaging

[0103] The Rep-Cap plasmid (RC plasmid) of each group (control group serotype and AAV mutants 1-6 of the present invention), the GOI plasmid expressing green fluorescent protein (EGFP), and the pHelper plasmid were co-transfected into HEK-293T cells, and the AAV virus was purified by iodixanol gradient ultracentrifugation. The virus titer was measured to be around 1E+13GC / mL, which was an appropriate titer and was placed at -80°C for use.

[0104] Example 3: Comparative test of various indicators of mutant serotypes

[0105] (1) NGS analysis of the retinal infection ability of each serotype mutant

[0106] Male cynomolgus monkeys were used in animal experiments and were used after passing the AAV8 pre-stored neutralizing antibody test before the experiment. Different serotype mutants and control serotypes (mutant, control serotype GOI plasmids) were injected into the vitreous with equal viral doses (the total mixed virus dose was controlled to be 1E12GC / eye), and different tissue parts of the eyes were peeled and sampled at 4 weeks. Finally, tissue RNA was extracted, RT-PCR and NGS sequencing were performed. By analyzing the NGS data, the expression multiples of each serotype mutant relative to the control AAV8 were determined.

[0107] (2) Immunofluorescence section observation of the retinal infection ability of each serotype mutant

[0108] Each serotype was injected into the vitreous of 4 monkeys at a viral dose of 1E12GC (GOI plasmid: scAAV.CAG.EGFP.WPREs.SV40pA) per eye. The samples were dissected 4 weeks later and placed in 50mL and 15mL centrifuge tubes containing 4% PFA, respectively, and post-fixed for more than 24 hours. The samples were soaked in 30% sucrose solution for dehydration for more than three days. After OCT embedding and cryosectioning, immunofluorescence staining and filming were performed as follows.

[0109] A. Blocking: Add 4% NGST (200 μL per three retinal samples per slide) to a 24-well plate wrapped in tin foil (protected from light) and incubate at room temperature for 30 minutes. Remove the blocking solution with a pipette and start all steps from the blocking step to avoid drying of the samples.

[0110] B. Primary antibody incubation: Refer to the instructions of the primary antibody and dilute the primary antibody with immunostaining primary antibody diluent (4% NGST) according to the appropriate ratio. Add the diluted primary antibody immediately after blocking and incubate at 4°C overnight. Take out and recover the primary antibody the next day, wash 3 times with PBS x1, PBSx1, and PBSx1, each time for 10 minutes. (The primary antibodies used are: Tuj1-Mouse antibody (Biolegend, Cat#801202, 1:1000), GFP-Chicken antibody (Invitrogen, Cat#A10262, 1:1000), DAPI (sigma D8417, 1:5000)).

[0111] C. Secondary antibody incubation: Use the secondary antibody corresponding to the primary antibody, dilute the secondary antibody with 4% NGST according to the appropriate ratio. Add the diluted secondary antibody to the sample and incubate at room temperature for 1.5 hours. Wash 3 times with PBS x1, PBS x1, PBSx1, 10 minutes each time. If the background of the result is high, the washing time can be appropriately extended and the number of washes can be increased. (The primary antibodies used are: Goat anti-Chicken, Alexa FluorTM 488(Invitrogen,Cat#A11039,1:500);Goat anti-Mouse,AlexaFluor TM 555(Invitrogen,Cat#A-21424,1:500).

[0112] D. Sealing: After removing the excess PBS solution, drop 150 μL of sealing solution on each slide, and be careful not to dry the sample too much. Tilt the cover glass to contact the surface of the sealing solution, and it is not easy to produce bubbles. Put it in a drying box to dry overnight.

[0113] E. Imaging: Fluorescence images were taken by Zeiss LSM980 confocal microscope imaging system.

[0114] The targeting effects of different serotypes in the retina of the same cynomolgus monkey were analyzed by NGS. Figure 1 It can be seen that the 6 candidate targeting serotypes obtained from the retinal screening have better targeting effects at the mRNA level than wild-type AAV8. The best results are mutants 1 and 2, which are 44.39 times and 48.27 times that of AAV8, respectively. Other serotype mutants also have an improvement of 16-40 times, indicating that the overall screening effect is good, and further proves the enrichment effect of mRNA candidate sequences.

[0115] To further confirm the retinal targeting effect of candidate serotype mutants and determine the infection effects of different serotype mutants on different types of retinal cells, each serotype was packaged with the same reporter gene and injected into the vitreous of cynomolgus monkeys. The results are as follows Figure 2 As shown. From the results, it can be seen that the infection effect of the six candidate serotype mutants is indeed better than that of the control AAV8 under the same dose conditions, and the serotype mutants generally have a stronger infection effect on cells in the GCL and INL layers (such as Ganglion cells, Muller cells, etc.). Among them, the stronger infection effects are mutants 1, mutant 2 and mutant 6, which are consistent with the detection results of mRNA levels. Compared with the positive control AAV2 7m8, although the overall infection intensity of the mutant is not as strong, AAV2 7m8 infection is mainly concentrated in the ONL layer (photoreceptor cells, such as cones and rods), which is different from the main cell population infected by the mutant, suggesting the characteristics of different serotypes, and forming their own advantages and characteristics in the application of different levels of retinal treatment.

[0116] In addition, it was found from the candidate sequences that the mutant serotype has certain sequence characteristics or motifs, such as the high frequency of amino acid "RR" and "KK" sequences, which may be related to the infection effect and targeting characteristics. Further analysis or expansion of research in this area will be carried out in the future. In short, the current serotype mutant has a better retinal targeting effect than the wild-type AAV8, and has the ability to infect different types of retinal cells compared with the control AAV2 7m8, showing relatively good application value and prospects.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An adeno-associated virus capsid protein mutant, characterized in that: The amino acid sequence thereof includes a sequence as shown in any one of SEQ ID No. 7-12.

2. A nucleic acid encoding the adeno-associated virus capsid protein mutant according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleotide sequence thereof comprises the nucleotide sequence shown in any one of SEQ ID Nos. 13-18.

4. An expression vector, characterized in that: It comprises the nucleic acid according to claim 2 or 3.

5. A host cell, characterized in that It comprises the expression vector according to claim 4.

6. A host cell, characterized in that It expresses the adeno-associated virus capsid protein mutant described in claim 1.

7. A recombinant adeno-associated virus, characterized in that: It includes the adeno-associated virus capsid protein mutant described in claim 1.

8. The recombinant adeno-associated virus according to claim 7, characterized in that Heterologous genes of interest are also included.

9. The recombinant adeno-associated virus according to claim 8, characterized in that The heterologous target gene encodes any gene product of interfering RNA, aptamer, endonuclease, and guide RNA.

10. A method for preparing a recombinant adeno-associated virus, characterized in that: The method comprises introducing at least the following components into a host cell: 1) the nucleic acid according to claim 2 or 3 or the expression vector according to claim 4, and 2) an adeno-associated virus helper plasmid.

11. rAAV prepared by the method according to claim 10.

12. A pharmaceutical composition comprising the recombinant adeno-associated virus according to any one of claims 7 to 9 or the rAAV according to claim 11, and a pharmaceutically acceptable carrier.

13. Use of the adeno-associated virus capsid protein mutant of claim 1, the expression vector of claim 4, the host cell of claim 5 or 6, the recombinant adeno-associated virus of any one of claims 7 to 9, and the rAAV of claim 11 in the preparation of a drug or preparation for delivering a gene product to the retinal cells or eyes of a subject.

14. The use according to claim 13, characterized in that The retinal cells include at least one of Ganglion cells, Muller cells, Bipolar cells, Horizontal cells, rod cells, cone cells, and RPE cells.

15. Use of the adeno-associated virus capsid protein mutant of claim 1, the expression vector of claim 4, the host cell of claim 5 or 6, the recombinant adeno-associated virus of any one of claims 7 to 9, and the rAAV of claim 11 in the preparation of a drug delivery tool for preventing and / or treating retinal diseases.

16. The use according to claim 15, characterized in that The retinal diseases include retinitis pigmentosa, Leber hereditary optic neuropathy, Leber congenital amaurosis and achromatopsia.

Images