CN118754952B

CN118754952B - Novel adeno-associated virus and application thereof

Info

Publication number: CN118754952B
Application number: CN202411245111.9A
Authority: CN
Inventors: 钟桂生; 储岑凤; 黄先玉
Original assignee: Shanghai Weimei Gene Technology Co ltd
Current assignee: Shanghai Weimei Gene Technology Co ltd
Priority date: 2024-09-06
Filing date: 2024-09-06
Publication date: 2025-01-10
Anticipated expiration: 2044-09-06
Also published as: CN118754952A

Abstract

The application relates to the field of biological medicine, in particular to a novel adeno-associated virus and application thereof. The application discloses adeno-associated virus AAV-WM34 capsid protein, which is obtained by inserting an amino acid sequence shown as SEQ ID No.4 between 588 th site and 589 th site of amino acid sequence of wild adeno-associated virus AAV2 capsid protein. Adeno-associated virus AAV-WM34 can have high-efficiency infection capability on retina tissues of eyes, and can be widely applied to gene therapy of related diseases.

Description

Novel adeno-associated virus and application thereof

Technical Field

The specification relates to the field of biological medicine, in particular to a novel adeno-associated virus and application thereof.

Background

Hereditary eye diseases are caused by gene defects, often appear as familial diseases, and cause blindness eye diseases to seriously affect the life quality of people. Among them, hereditary retinal diseases (IRDs) are rare blinding eye diseases caused by genetic variation, also known as retinal dystrophy, hereditary retinal degeneration, which are the first cause of legal blindness in china and the world, are various genetic diseases caused by pathogenic variation of key genes of retinal function, often manifested as retinal pigment epithelial cells (RPE) and photoreceptor cell damage or death, and there has been no effective therapeutic means. In recent years, along with the gradual maturation of gene therapy, clinical researches related to IRDs gene therapy are gradually accelerated, and particularly, gene supplementation therapy using AAV as a vector is expected to bring good prospects for blinding eye patients.

Retinal pigment epithelial cells (RPEs) are essential components of the retina and play a vital role in visual function. The structural and functional impairment of the retinal pigment epithelium can lead to a variety of retinal diseases such as Retinitis Pigmentosa (RP), stargardt disease (STGD), crystalline-like retinal degeneration (BCD), age-related macular degeneration (AMD), macular Dystrophies (MD), and the like. Retinitis pigmentosa is a common hereditary eye disease, closely related to photoreceptor degeneration of rods and cones and RPE cell degeneration. Traditional drug therapies are not effective against these diseases, whereas gene therapy can be used to correct or compensate for diseases caused by gene defects and abnormalities by delivering exogenous genes, small RNAs, or gene editing tools.

One key to gene therapy is delivery, where the effectiveness and innocuity of the vector is critical. Among them, adeno-associated virus (AAV) has been successful in the field of gene therapy as a safe and effective gene delivery vehicle. In recent years, researchers have developed experiments in the field of gene therapy using AAV as a vector in an attempt to treat retinopathy-related diseases by delivering normal RPE cell genes. AAV vectors are capable of stably expressing exogenous genes in cells for long periods of time, thereby correcting gene defects and abnormalities. However, how to optimize surgical administration is also a hot spot in recent years. Modes of administration include intravitreal injection, subretinal injection, and suprachoroidal injection. Subretinal cavity injection is still the most common route of administration for indications targeted at RPE or photoreceptor cells, although suprachoroidal cavity injection is also under active exploration, the current technical requirements are high. In addition, the location, bleb, time and patient factors associated with surgical injections all have an impact on the effectiveness and safety of the gene therapy drug. In order to overcome these problems, further optimization and development of AAV capsids is needed to obtain better AAV vectors which can be administered by intravitreal injection and are suitable for gene therapy of ocular diseases, simplify the surgical procedures, and promote the specificity and expression effect of tissue delivery to more effectively treat related ocular diseases such as hereditary retinopathy.

For ophthalmic, to date, eye disease gene therapy based on AAV viral vectors requires direct injection of the viral vector under the retina, which technique can only be performed in specialty hospitals with high levels of expertise and equipment, and this injection involves the risk of damaging delicate retinal tissue. Another disadvantage of this approach is that each injection can only target a small fraction of cells near the injection site due to the weak lateral diffusion capacity of AAV viruses. Therefore, development of a carrier capable of efficiently reaching retina through vitreous injection is needed, the operation difficulty is reduced, and the treatment effect is improved.

Disclosure of Invention

In order to solve the technical problems, the application provides a novel adeno-associated vector AAV-WM34 which can mediate the specific expression of a target gene in the whole retina of an adult mouse under the condition of vitreous injection.

The application provides adeno-associated virus AAV-WM34 capsid protein, which is obtained by inserting an amino acid sequence shown as SEQ ID No.4 between 588 th site and 589 th site of amino acid sequence of wild adeno-associated virus AAV2 capsid protein, wherein the amino acid sequence of wild adeno-associated virus AAV2 capsid protein is shown as SEQ ID No. 1.

The application also provides a polynucleotide which codes for the adeno-associated virus AAV-WM34 capsid protein.

The present application also provides a nucleic acid construct comprising the polynucleotide described above.

The application also provides a host cell comprising a construct as described above or a polynucleotide as described above that is heterologous integrated into the genome, or comprising an adeno-associated virus AAV-WM34 capsid protein as described above.

The application also provides an adeno-associated virus vector system, which comprises a packaging plasmid, an expression plasmid and an auxiliary plasmid, wherein the packaging plasmid is the nucleic acid construct.

The application also provides adeno-associated virus AAV-WM34, wherein the adeno-associated virus contains the adeno-associated virus AAV-WM34 capsid protein, or is obtained by virus packaging through the adeno-associated virus vector system.

The application also provides a cell which is an engineered cell obtained by transformation with the adeno-associated virus AAV-WM 34.

The application also provides a pharmaceutical composition, which comprises the adeno-associated virus AAV-WM34 and a pharmaceutically acceptable carrier or auxiliary material.

The application also provides application of the adeno-associated virus AAV-WM34 capsid protein, or the construct, or the host cell, or the adeno-associated virus vector system, or the adeno-associated virus AAV-WM34, or the cell, or the pharmaceutical composition in preparing medicines for preventing and/or treating diseases.

The application has the beneficial effects of (1) providing a novel adeno-associated vector AAV-WM34, which can mediate the specific expression of a target gene in the RPE cells of adult mice under the condition of vitreous injection, and (2) the novel adeno-associated vector AAV-WM34 mediates the expression of the target gene more flexibly and efficiently than the traditional transgenic method, and has the advantages of more convenient application and lower cost.

Drawings

The application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:

FIG. 1 is a plasmid map of RC-WM34 according to some embodiments of the present application.

FIG. 2 is a block diagram of a computer-simulated AAV-WM34 according to some embodiments of the application, where a is a schematic diagram of AAV-WM34 sequences in FIG. 2 and b is a simulated diagram of AAV-WM34 structures in FIG. 2.

FIG. 3 is a diagram of a retinal section of a mouse injected with rAAV-WM34-CMV-EGFP virus, EGFP green for fluorescent protein expressed by the virus infection and DAPI blue for nuclear staining, according to some embodiments of the application. The result shows that the rAAV-WM34-CMV-EGFP has higher infection characteristic on RPE cells, and can be used for ophthalmic gene therapy.

FIG. 4 shows infection of rAAV-WM34-CMV-EGFP and AAV2-CMV-EGFP in eye tissue according to some embodiments of the application, FIG. 4 shows a graph of retinal sections of mice injected with rAAV-WM34-CMV-EGFP and AAV2-CMV-EGFP viruses, EGFP is green showing fluorescent protein expressed by viral infection, DAPI is blue staining of nuclei, FIG. 4 b is a statistical graph of distribution of EGFP signals in retinal tissue, and results show that EGFP signals of WM34 are concentrated in the RPE layer, EGFP signals of AAV2 are concentrated in the INL and RGC layers, indicating that WM34 is capable of specifically infecting RPE cells.

Detailed Description

In order to more clearly illustrate the technical solutions of the embodiments of the present specification, the drawings that are required to be used in the description of the embodiments will be briefly described below. It is apparent that the drawings in the following description are only some examples or embodiments of the present specification, and it is possible for those of ordinary skill in the art to apply the present specification to other similar situations according to the drawings without inventive effort. Unless otherwise apparent from the context of the language or otherwise specified, like reference numerals in the figures refer to like structures or operations.

As used in this specification and the claims, the terms "a," "an," "the," and/or "the" are not specific to a singular, but may include a plurality, unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that the steps and elements are explicitly identified, and they do not constitute an exclusive list, as other steps or elements may be included in a method or apparatus.

A flowchart is used in this specification to describe the operations performed by the system according to embodiments of the present specification. It should be appreciated that the preceding or following operations are not necessarily performed in order precisely. Rather, the steps may be processed in reverse order or simultaneously. Also, other operations may be added to or removed from these processes.

The term "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof.

In some embodiments, the amino acid sequence of the adeno-associated virus AAV-WM34 capsid protein may be as shown in SEQ ID No. 3. In some embodiments, the amino acid sequence of the adeno-associated virus AAV-WM34 capsid protein may have 85% and greater sequence identity to SEQ ID No.3, as well as functions of the amino acid sequence defined by SEQ ID No. 3.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single or double stranded form, and polymers thereof. Unless specifically limited otherwise, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implies conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J.biol.chem.260:2605-2608 (1985); and Rossolini et al, mol.cell.probes 8:91-98 (1994)).

In some embodiments, the nucleotide sequence of the polynucleotide may be as shown in SEQ ID No. 2.

"Construct" as used herein refers to a polynucleotide capable of carrying at least one polynucleotide fragment. The construct may deliver a fragment of the nucleic acid, each polynucleotide, into a host cell. It may comprise at least one expression cassette comprising regulatory sequences for the correct expression of the polynucleotide incorporated therein. Polynucleotides to be introduced into a cell (e.g., polynucleotides encoding a product of interest or a selectable marker) may be inserted into an expression cassette of a vector for expression therefrom. Constructs according to the application may exist in circular or linear (linearized) form and also include expression construct fragments. The term "construct" also encompasses artificial chromosomes or similar individual polynucleotides that allow transfer of exogenous nucleic acid fragments.

In some embodiments, the host cell does not include fertilized eggs of plant cells and animal cells.

In some embodiments, the host cell may be selected from any of mammalian cells (e.g., CHO, COS, and N2A), human cells (human cervical cancer cells such as HELA, and human embryonic kidney cells such as HEK 293T), bacterial cells (e.g., escherichia coli, streptomyces, salmonella typhimurium), fungal cells (e.g., yeast), insect cells (e.g., sf 9). In some embodiments, preferably, the host cell may be an animal cell, and more preferably, a human cell. In some embodiments, the host cell may be a passaged cell or a primary cell, i.e., a cell isolated directly from an organism (e.g., a human). In some embodiments, the host cell may be an adherent cell or a cell grown in suspension, i.e., a suspension.

In some embodiments, the packaging plasmid may further comprise a rep gene fragment of an adeno-associated virus. Wherein the rep gene fragment comprises an intron comprising a transcription termination sequence.

In some embodiments, the expression plasmid may comprise a heterologous nucleotide sequence encoding a product of interest.

In some embodiments, the product of interest may be a nucleic acid or a protein. The desired product may be selected as desired, for example, by selecting different desired products according to different targets, as is conventional in the art. The above-mentioned heterologous nucleotide sequence encoding a product of interest is a construct comprising a nucleic acid encoding a product of interest. The construct is constructed by inserting a nucleic acid encoding the product of interest into a suitable expression vector, which can be selected by a person skilled in the art, for example, from any of the above-mentioned expression vectors selected from the group consisting of pAAV-CAG, pAAV-TRE, pAAV-EF1a, pAAV-GFAP, pAAV-Lgr5, pAAV-Sox2, pAAV-Syn or pAAV-CMV expression vectors.

In some embodiments, the packaging plasmid, expression plasmid, helper virus plasmid are transformed into a cell, and the nucleic acid sequences therein are all integrated into the cell to produce the adeno-associated virus.

The term "packaging" refers to a series of intracellular processes that result in the assembly and encapsulation of AAV particles.

In some embodiments, the adeno-associated virus AAV-WM34 further comprises a heterologous nucleotide sequence encoding a product of interest.

As representative examples of suitable cells, any one selected from mammalian cells (such as CHO, COS and N2A), plant cells, human cells (human cervical cancer cells such as HELA and human embryonic kidney cells such as HEK293 FT), bacterial cells (such as escherichia coli, streptomyces, salmonella typhimurium), fungal cells (such as yeast), insect cells (such as Sf 9). Preferably, the host cell is an animal cell, and more preferably a human cell. The cells are either passaged cells or primary cells, i.e., cells isolated directly from an organism (e.g., a human). The cells are adherent cells or cells grown in suspension, i.e., in suspension.

The adjuvants include various excipients and diluents, which are not essential active ingredients and which are not excessively toxic after administration. The adjuvant comprises sterile water or physiological saline, stabilizer, excipient, antioxidant (ascorbic acid, etc.), buffer (phosphoric acid, citric acid, other organic acids, etc.), antiseptic, surfactant (PEG, tween, etc.), chelating agent (EDTA, etc.), or binder. The adjuvant also comprises other low molecular weight polypeptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharide, monosaccharide, mannitol or sorbitol. The adjuvant is selected from physiological saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose or sugar alcohol isotonic solution when used in injectable aqueous solution. The injectable aqueous solution contains a solubilizing agent. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG) and/or nonionic surfactants (Tween 80 or HCO-50).

In the pharmaceutical composition provided by the application, the adeno-associated virus AAV-WM34 is a single active ingredient, and can also be combined with one or more other active ingredients useful for treating eye diseases to form a combined preparation.

The amount of active ingredient in the pharmaceutical composition is a safe and effective amount which should be adjustable to one skilled in the art, for example, the adeno-associated virus AAV-WM34 and the amount of active ingredient to be administered in the pharmaceutical composition will depend on the weight of the patient, the type of application, the condition and severity of the disease.

In some embodiments, the pharmaceutical composition is one or more of an injection, a tablet, a capsule, an aerosol, an eye drop, or a nose drop.

The term "preventing and/or treating" (and grammatical variations thereof) refers to attempting to alter the natural course of a disease in a treated individual, and may be for the purpose of preventing or clinical intervention performed during the course of a clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating a disease state, and eliminating or improving prognosis.

In some embodiments, the disease may be an ophthalmic disease. In some embodiments, preferably, the ophthalmic disease may be a retinal disease. In some embodiments, more preferably, the ophthalmic disease may be selected from one or more of retinitis pigmentosa, stargardt disease, crystalline-like retinal degeneration, age-related macular degeneration, or macular dystrophy.

In some embodiments, the drug may be a vitrectomy drug. In some embodiments, the drug may be targeted to the retinal pigment epithelial cells.

The application also provides a method of treating an ocular disease comprising administering to a subject in need thereof an effective amount of an adeno-associated virus, host cell, vector system or pharmaceutical composition of the application.

In the present application, the adeno-associated virus or host cell or vector system or pharmaceutical composition of the application may be administered to a patient. One skilled in the art will be able to determine the appropriate mode of administration and dosage.

The delivery of one or more therapeutic genes by the adeno-associated viruses provided herein may be used alone or in combination with other therapeutic methods or therapeutic components.

Adeno-associated viruses of the application are useful for infecting cells, thereby delivering genes and/or linked (e.g., without limitation, covalently linked) biologically active polypeptides to the cells. Accordingly, the present application provides a method of delivering a heterologous gene to a cell by allowing one or more adeno-associated viruses of the application to infect a cell, wherein the adeno-associated viruses comprise one or more heterologous genes.

The application also provides a method of producing a stable adeno-associated viral vector producer cell line comprising:

(a) Introducing an adeno-associated viral vector as defined herein into a culture of mammalian host cells, and/or,

(B) Selecting within the culture a mammalian host cell having a nucleic acid sequence encoded on a vector integrated into the endogenous chromosome of the mammalian host cell.

AAV vector producing cells are mammalian cells. In some embodiments, the mammalian cell is selected from HEK293 cells, CHO cells, jurkat cells, K562 cells, perC6 cells, heLa cells, or derivatives thereof. In some embodiments, the mammalian host cell is a HEK293 cell, or is derived from a HEK293 cell. In some embodiments, the HEK293 cell is a HEK293T cell.

The genomic sequences of the various serotypes of AAV and the sequences of the native ITR, rep proteins and capsid proteins are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. The disclosure of which is incorporated herein by reference for AAV nucleic acid and amino acid sequences.

In the compounds of the application and their uses, when adeno-associated virus is used in combination with other therapeutic agents, the active compound is co-administered with the other therapeutic agents. By "co-administration" is meant simultaneous administration via the same or different routes, or sequential administration via the same or different routes, in the same formulation or in two different formulations.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent companies. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Example 1 acquisition of novel adeno-associated Virus AAV-WM34

A. Construction of Rep-Cap plasmid of AAV-WM34

The serotype of AAV is determined by the sequence of the Cap protein in the Rep-Cap plasmid required for AAV packaging, and therefore AAV produced by different Cap sequence packages has different infectivity. The existing natural serotype is modified, a random peptide Seq ID No.4: SGRDLTP (fig. 2 a) with a length of 7 amino acids is inserted between the 588R and 589Q sites of the amino acid sequence of AAV2, and the nucleotide sequence of the random peptide is TCTGGTCGTGATCTGACGCCG (Seq ID No. 5), so that a novel AAV serotype plasmid is constructed, which is named RC-WM34 (fig. 1), the nucleotide sequence of which is Seq ID No.2, and the amino acid sequence of which is Seq ID No.3. The outer surface of the capsid shows the presence of the capsid fragment from the inserted polypeptide and indicates that the triple-axis protrusion of AAV-WM34 is affected by the insertion sequence (b in fig. 2).

Seq ID No.1:

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL*

Seq ID No.2:

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGATCTGGTCGTGATCTGACGCCGCAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA

Seq ID No.3:

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRSGRDLTPQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL*

B. preparation and purification of rAAV-WM34 virus

The sequence encoded by the Cap gene of WM34 was synthesized by the biotechnology limited company of su Jin Weizhi and was constructed by plasmid to obtain the Rep-Cap plasmid of AAV-WM34, RC-WM34, which was verified to be correct. And co-transferring the obtained RC-WM34 and a genome plasmid pAAV-CMV-EGFP expressing a green fluorescent protein EGFP into HEK-293T cells in proper quantity, and purifying the AAV virus by adopting iodixanol density gradient centrifugation, wherein the final product is rAAV-WM34-CMV-EGFP. Virus titers were measured at 2e+13 GC/mL to the appropriate concentration and placed at-80 ℃ for use.

Example 2-live experience of novel adeno-associated Virus rAAV-WM34 in the visual System

Anesthetized adult mice were placed under a split microscope and 1-2 drops of topiramate eye drops were placed on the eyeball to dilate the pupil. The eye is coated with levofloxacin eye ointment, left hand elbow forceps are used for supporting and fixing the eye from the optic nerve disk, a right hand 1mL syringe or a glass needle is used for puncturing the cornea to reduce intraocular pressure, and paper towels are used for gently wiping periocular fluid. 1.1 mu L of virus is sucked by using a microinjector, eyeballs are fixed by using left hand-held forceps, the needle is inserted at an angle of 50 DEG between the posterior edge of the corneosclera and the plane of the iris, and rAAV-WM34-CMV-EGFP virus is slowly injected with the titer of 1.1E13GC/mL. After the injection is completed, the glass needle stays for 30 seconds and is slowly pulled out. Erythromycin ointment is smeared at the wound, the mice are put on a mouse cage in a water bath kettle at 41 ℃ for heat preservation, and after the mice wake up, the mice are moved to a mouse house for feeding. After 21 days, mice were sacrificed by spinal dislocation, eye tissue was removed, and after fixation in 4% PFA for 1h, the eyes were punctured into the cornea with a 1mL syringe, and aqueous humor was discharged. The eyeballs were placed in a small petri dish containing a PBS solution and the eye tissue dissected under a microscope. The cornea was grasped with a pointed forceps, the cornea was cut off along the circumference of the corneosclera with an ophthalmic scissors from the cornea opening, the lens was removed with forceps, and the optic nerve was retained by about 2mm. The dissected eye cup was placed in 4% PFA and fixed at 4 ℃ for 12h. The fixed eye cup was placed in 30% sucrose and dehydrated at 4 ℃ for 12h. Then embedding eyeball tissues, freezing and slicing, and picking out the slice with good effect and complete tissues under a fluorescence microscope for staining. Immunofluorescence results indicated that rAAV-WM34-CMV-EGFP could infect retinal pigment epithelial cells (RPE), and the results are shown in FIG. 3. Meanwhile, the application also carries out analysis on the condition of the rAAV-WM34 and AAV2 virus infected retina cells, the immunofluorescence result is shown as a in figure 4, and by carrying out statistical analysis on the distribution condition of EGFP signals of the tissue sections of the eyeballs of the WM34 and AAV2 verified animals in retina tissues, the EGFP signals of the WM34 are concentrated in an RPE layer, the EGFP signals of the AAV2 are mainly concentrated in INL and RGC layers, and the result is shown as b in figure 4, so that the WM34 can specifically infect the RPE cells.

While the basic concepts have been described above, it will be apparent to those skilled in the art that the foregoing detailed disclosure is by way of example only and is not intended to be limiting. Although not explicitly described herein, various modifications, improvements, and adaptations to the present disclosure may occur to one skilled in the art. Such modifications, improvements, and modifications are intended to be suggested within this specification, and therefore, such modifications, improvements, and modifications are intended to be included within the spirit and scope of the exemplary embodiments of the present invention.

Meanwhile, the specification uses specific words to describe the embodiments of the specification. Reference to "one embodiment," "an embodiment," and/or "some embodiments" means that a particular feature, structure, or characteristic is associated with at least one embodiment of the present description. Thus, it should be emphasized and should be appreciated that two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various positions in this specification are not necessarily referring to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of the present description may be combined as suitable.

In some embodiments, numbers describing the components, number of attributes are used, it being understood that such numbers being used in the description of embodiments are modified in some examples by the modifier "about," approximately, "or" substantially. Unless otherwise indicated, "about," "approximately," or "substantially" indicate that the number allows for a 20% variation. Accordingly, in some embodiments, numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and employ a method for preserving the general number of digits. Although the numerical ranges and parameters set forth herein are approximations that may be employed in some embodiments to confirm the breadth of the range, in particular embodiments, the setting of such numerical values is as precise as possible.

Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments of this specification. Other variations are possible within the scope of this description. Thus, by way of example, and not limitation, alternative configurations of embodiments of the present specification may be considered as consistent with the teachings of the present specification. Accordingly, the embodiments of the present specification are not limited to only the embodiments explicitly described and depicted in the present specification.

Claims

1. An adeno-associated virus AAV-WM34 capsid protein, characterized in that the adeno-associated virus AAV-WM34 capsid protein is a capsid protein obtained by inserting an amino acid sequence shown as SEQ ID No.4 between the 588 th site and the 589 th site of the amino acid sequence of wild adeno-associated virus AAV2 capsid protein;

The amino acid sequence of the wild adeno-associated virus AAV2 capsid protein is shown in SEQ ID No. 1.

2. The adeno-associated virus AAV-WM34 capsid protein according to claim 1, wherein the amino acid sequence of the adeno-associated virus AAV-WM34 capsid protein is shown in SEQ ID No. 3.

3. A polynucleotide encoding the adeno-associated virus AAV-WM34 capsid protein of claim 1 or 2.

4. A polynucleotide according to claim 3, wherein the nucleotide sequence of the polynucleotide is shown in SEQ ID No. 2.

5. A nucleic acid construct comprising the polynucleotide of claim 3 or 4.

6. A host cell comprising the construct or genome of claim 5 integrated with the heterologous polynucleotide of claim 3 or 4, or comprising the adeno-associated virus AAV-WM34 capsid protein of claim 1 or 2, excluding fertilized eggs of plant and animal cells.

7. An adeno-associated viral vector system comprising a packaging plasmid, an expression plasmid, and a helper plasmid, wherein the packaging plasmid is the nucleic acid construct of claim 5.

8. An adeno-associated virus AAV-WM34 comprising an adeno-associated virus AAV-WM34 capsid protein according to claim 1 or 2, or obtainable by viral packaging of an adeno-associated virus vector system according to claim 7.

9. A cell, wherein the cell is an engineered cell obtained by transformation using adeno-associated virus AAV-WM34 of claim 8, and wherein the cell does not include fertilized eggs of plant cells and animal cells.

10. A pharmaceutical composition comprising adeno-associated virus AAV-WM34 according to claim 8 and a pharmaceutically acceptable carrier or adjuvant.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is one or more of an injection, a tablet, a capsule, an aerosol, an eye drop, or a nose drop.

12. Use of an adeno-associated virus AAV-WM34 capsid protein according to claim 1 or 2, or a construct according to claim 5, or a host cell according to claim 6, or an adeno-associated virus vector system according to claim 7, or an adeno-associated virus AAV-WM34 according to claim 8, or a cell according to claim 9, or a pharmaceutical composition according to claim 10, for the preparation of a medicament for the treatment of a disease, which is retinitis pigmentosa.

13. The use according to claim 12, wherein the medicament is a vitrectomy drug.