CN118667036B

CN118667036B - Fusion adeno-associated virus capsid protein, adeno-associated virus and application thereof

Info

Publication number: CN118667036B
Application number: CN202411139738.6A
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: 2024-08-20
Filing date: 2024-08-20
Publication date: 2025-01-10
Anticipated expiration: 2044-08-20
Also published as: CN118667036A

Abstract

The application relates to the field of biological medicine, in particular to fusion type adeno-associated virus capsid protein, adeno-associated virus and application thereof. The application provides a fusion type adeno-associated virus AAV-WM14 capsid protein, which comprises amino acid fragments of serotypes AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV10 and AAV13, wherein the capsid protein comprises an amino acid sequence shown as SEQ ID NO. 1. The fusion type adeno-associated virus provided by the application can be injected and administrated in a vitreous body, can efficiently infect retina photoreceptor cells in eyes, can be delivered to cochlea, and efficiently infect inner hair cells and supporting cells, and can be widely applied to gene therapy of related diseases.

Description

Fusion adeno-associated virus capsid protein, adeno-associated virus and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to fusion adeno-associated virus capsid protein, 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, and are also the first cause of legal blindness in China and the world, and there is no effective therapeutic means yet. Along with the development of scientific technology, the gene therapy provides a new treatment thought for the blinding eye disease, and is expected to bring good prospect for the blinding eye disease patients.

The retina is the multi-layered sensory tissue that is arranged in the back of the eye. Its main function is to convert light energy into a change in potential through a process called light transduction. The photosensitive element of the retina is a photoreceptor cell. The retina contains two types of photoreceptors, rod cells and cone cells. Rod cells (about 1.2 hundred million in the human eye) are responsible for night vision, whereas cone cells (about 600 to 700 tens of thousands in the human eye) are responsible for vision and color vision. The photoreceptor cells are capable of converting optical stimuli into nerve impulse cells. Dysplasia, dysfunction or premature death of retinal photoreceptor cells can lead to vision loss. Such as hereditary retinal diseases (IRDs), mainly including retinal pigment degeneration (RP) (also known as rod cell dystrophy), cone/rod dystrophy (CD/CRD), leber Congenital Amaurosis (LCA), macular Dystrophy (MD), and achromatopsia (rod cell monochromaticity), etc. Currently, over 270 different genes are known to cause IRDs, and gene therapy is the only currently effective treatment and intervention from some point of view.

One key to gene therapy is delivery, where the effectiveness and safety of the vector is critical. Among them, adeno-associated virus (AAV) has been successful in the field of gene therapy as a safe gene delivery vehicle. Hereditary retinal diseases are a type of blinding eye diseases caused by heredity, and no effective treatment means exists at present. In recent years, researchers have developed several clinical trials in the field of gene therapy using AAV as a vector to treat such diseases by delivering normal genes. However, the current injection method mainly injects the virus vector directly under retina, the operation has high technical requirement, the risk of damaging retina tissues exists, the virus has weak lateral diffusion capability, and only a small part of cells near the injection point can be infected by the virus after each injection. Thus, there is a need to develop better novel AAV viral vectors suitable for gene therapy of ocular diseases to more effectively treat similar hereditary ocular diseases. In addition to IRDs, retinal degenerative diseases and congenital glaucoma, which are genetic eye diseases related to genes, some gene therapy tests are currently underway. Gene therapy is an emerging therapeutic means and has important application prospect in the field of ophthalmology.

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, due to the weak lateral spreading capacity of AAV viruses, each injection of virus can only target a small fraction of cells near the injection site. Therefore, it is necessary to develop AAV vectors which can efficiently reach the inside of retina by intravitreal injection, thereby reducing the difficulty of surgery and improving the therapeutic effect.

Gene therapy for ophthalmic diseases such as RP still relies on subretinal vector delivery, which can lead to retinal detachment with serious attendant risks, and often fails to achieve widespread photoreceptor transduction. Existing AAV vectors are difficult to efficiently infect photoreceptor cells by intravitreal injection.

Disclosure of Invention

In order to solve the defects in the prior art, the application provides a novel adenovirus with mutated capsid protein, which can be injected and administrated in a vitreous body to efficiently infect retina photoreceptor cells in eyes, and in addition, the novel adenovirus is delivered to cochlea to efficiently infect inner hair cells and supporting cells, thereby being widely applied to gene therapy of related diseases.

The first aspect of the application provides a fusion type adeno-associated virus AAV-WM14 capsid protein, wherein the fusion type adeno-associated virus AAV-WM14 capsid protein comprises amino acid fragments of serotypes AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV10 and AAV13, and the capsid protein comprises an amino acid sequence shown as SEQ ID NO. 1.

In a second aspect, the application provides a nucleic acid encoding the fusion adeno-associated virus AAV-WM14 capsid protein of the first aspect, said nucleic acid comprising the nucleotide sequence depicted in SEQ ID NO. 2.

In a third aspect the application provides a construct comprising a nucleic acid of the second aspect described above.

In a fourth aspect the application provides a host cell comprising the construct of the third aspect, or having the nucleic acid of the second aspect integrated into the genome of the host cell, or comprising the fusion adeno-associated virus AAV-WM14 capsid protein of the first aspect.

In a fifth aspect, the application provides a fusion adeno-associated virus AAV-WM14, wherein the capsid structure of the fusion adeno-associated virus AAV-WM14 comprises the fusion adeno-associated virus AAV-WM14 capsid protein of the first aspect.

In a sixth aspect, the application provides a cell transformed with the fusion adeno-associated virus AAV-WM14 of the fifth aspect.

In a seventh aspect, the present application provides a fusion adeno-associated viral vector system comprising a packaging plasmid comprising the nucleic acid of the second aspect.

In an eighth aspect the application provides a medicament or conjugate comprising the fusion adeno-associated virus AAV-WM14 of the fifth aspect described above.

The ninth aspect of the present application provides the use of the fusion adeno-associated virus AAV-WM14 capsid protein of the first aspect, or the nucleic acid of the second aspect, or the construct of the third aspect, or the host cell of the fourth aspect, or the fusion adeno-associated virus AAV-WM14 of the fifth aspect, or the cell of the sixth aspect, or the fusion adeno-associated virus vector system of the seventh aspect, or the medicament or conjugate of the eighth aspect, in the manufacture of a medicament for gene therapy.

The application has the beneficial effects that:

Compared with the traditional transgenic method, the novel adeno-associated vector AAV-WM14 mediated target gene expression is more flexible and efficient, is more convenient to apply and has lower cost;

The application provides a novel adeno-associated vector AAV-WM14 which can mediate the high-efficiency expression of a target gene in adult mouse retina photoreceptor cells under the condition of vitreous injection;

the AAV-WM14 has higher infection efficiency on inner ear supporting cells of adult mice, and the infection efficiency is higher than AAV2.

Drawings

FIG. 1 shows a flow chart of in vivo screening of DNA family shuffling libraries.

FIG. 2 shows a plasmid map of RC-WM 14.

FIG. 3 shows the Cap amino acid sequence of AAV-WM14 aligned with the amino acid sequences of DNA shuffling of the 13 parental serotypes.

FIG. 4 shows the infection of photoreceptor cells with AAV-WM 14-CAG-EGFP. The figure is a frozen section of eyeball tissue injected with rAAV-WM14-CAG-EGFP virus. The green color shows the fluorescent protein expressed by the virus infection, and the blue color shows the nuclear staining. The result shows that the rAAV-WM14-CAG-EGFP virus has higher infection to retina photoreceptor cells and can be used for ophthalmic gene therapy.

FIG. 5 shows the infection of AAV-WM14, AAV2, AAV2.7m8 in the eye tissue. The figure is a frozen section of eyeball tissue of rAAV-WM14-CAG-EGFP, AAV2-CAG-EGFP and AAV2.7m8-CAG-EGFP virus. The green color shows the fluorescent protein expressed by the virus infection, and the blue color shows the nuclear staining.

FIG. 6 shows statistics of infection of photoreceptor cells with AAV-WM 14-CAG-EGFP. The figure is a statistical graph of the efficiency of injecting rAAV-WM14-CAG-EGFP, AAV2-CAG-EGFP and AAV2.7m8-CAG-EGFP viruses to infect photoreceptor cells, and the result shows that the AAV-WM14-CAG-EGFP viruses have high infection efficiency to photoreceptor cells.

FIG. 7 shows AAV-WM14 and AAV2 infection of adult mouse cochlear hair cells. The figure shows the results of cochlear tissue plating staining of injected rAAV-WM14-CAG-EGFP and AAV2 virus. Green shows EGFP fluorescent protein expressed by virus infection, red is Myo7a staining, i.e. hair cell staining. The results show that the rAAV-WM14-CAG-EGFP efficiently infects inner hair cells after adult mice are injected.

FIG. 8 shows AAV-WM14 and AAV2 infection of adult mouse cochlear support cells. The figure shows the results of cochlear tissue plating staining of injected rAAV-WM14-CAG-EGFP and AAV2 virus. Green shows EGFP fluorescent protein expressed by virus infection, and magenta shows SOX2 staining, i.e. support cell staining. The results indicate that the rAAV-WM14-CAG-EGFP is capable of infecting supporting cells after injection into adult mice.

FIG. 9 shows statistics of infection of adult mice with AAV-WM14 and AAV2 on intra-cochlear hair cells and supporting cells. Wherein, figure A is intra-cochlear hair cell statistics of injected rAAV-WM14-CAG-EGFP and AAV2 virus infection. Panel B shows statistics of cochlear support cells infected with injected rAAV-WM14-CAG-EGFP and AAV2 virus. The result shows that the rAAV-WM14-CAG-EGFP can efficiently infect inner hair cells after being injected into adult mice, and has the capability of infecting supporting cells.

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure of the present invention, which is to be read in light of the specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before further describing embodiments of the present invention, it is to be understood that the scope of the invention is not limited to the specific embodiments described below, and that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the invention, as the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

The application reforms the existing natural serotypes, uses capsid sequence of 13 AAV serotypes (AAV 1-13) found on human and non-human primate as parent to carry on DNA family reorganization, get DNA family reorganization library plasmid, pack the library plasmid virus, screen in vivo, get the Cap sequence with optimal infectivity, thus provide a brand new adeno-associated virus with mutated capsid protein, can be injected and dosed in vivo, infect retina photoreceptor cell in eyes with high efficiency, in addition, the new adeno-associated virus of the application is delivered to cochlea, can infect inner hair cell and supporting cell with high efficiency, can apply to the gene therapy of the related disease extensively.

In the present application, AAV is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof.

Under electron microscopy, the nucleocapsid of adeno-associated virus is generally nearly circular. The near circular capsid is in fact a closed icosahedral symmetrical hollow capsid made up of a plurality of protein capsomeres arranged such that genomic nucleic acid is enclosed therein. An icosahedral symmetrical structure comprises three rotationally symmetrical modes, 3-heavy, 2-heavy, 5-heavy symmetry (3-, 2-,5-fold symmetry). The symmetrical three-dimensional structure is provided with a 3-weight symmetrical axis passing through the center points of two opposite surfaces of virus particles, wherein the shell particles rotate around the 3-weight symmetrical axis for three times for resetting to form a triangular surface, a 2-weight symmetrical axis (edge) is provided, the shell particles rotate around the 2-weight symmetrical axis for two times for resetting to form two intersecting triangular surfaces, and the shell particles rotate around the 5-weight symmetrical axis for 72 times for resetting to form a pentamer (pentamel) passing through the 5-weight symmetrical axis. Therefore, the icosahedral symmetrical shell consists of 20 equilateral triangular faces, wherein every 2 triangular faces are intersected to form edges, 30 edges are totally formed, and every 5 triangular faces are connected to form 12 vertexes.

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.

The application firstly provides a fusion type adeno-associated virus AAV-WM14 capsid protein, wherein the fusion type adeno-associated virus AAV-WM14 capsid protein comprises amino acid fragments of serotypes AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV10 and AAV 13.

In the present application, the amino acid fragment refers to a sequence composed of one or more amino acids.

In a specific embodiment of the application, the capsid protein comprises a first fragment comprising an amino acid fragment from AAV7, a second fragment comprising an amino acid fragment from AAV8, a third fragment comprising an amino acid fragment from AAV1 or AAV6, a fourth fragment comprising an amino acid fragment from AAV8 or AAV10, a fifth fragment comprising an amino acid fragment from AAV1 or AAV6, a sixth fragment comprising an amino acid fragment from AAV8, a seventh fragment comprising an amino acid fragment from AAV10, an eighth fragment comprising an amino acid fragment from AAV4, a ninth fragment comprising an amino acid fragment from AAV10, a tenth fragment comprising an amino acid fragment from AAV8, an eleventh fragment comprising an amino acid fragment from AAV10, a twelfth fragment comprising an amino acid fragment from AAV2, a thirteenth fragment comprising an amino acid fragment from AAV7, a fourteenth fragment comprising an amino acid fragment from AAV2 or AAV3 or AAV13, connected in that order.

The first fragment is amino acid 1-197 of capsid protein, including the amino acid sequence shown in SEQ ID NO. 3.

SEQ ID NO.3:

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPAKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPS

The second fragment is 198-223 amino acids of capsid protein, which comprises the amino acid sequence shown in SEQ ID NO. 4.

SEQ ID NO.4:

GVGPNTMAAGGGAPMADNNEGADGVG

The third fragment is 224 th-272 th amino acid of capsid protein, comprising the amino acid sequence shown in SEQ ID NO. 5.

SEQ ID NO.5:

NASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDN

The fourth fragment is amino acid 273-313 of capsid protein, the first comprises the amino acid sequence shown in SEQ ID NO. 6.

SEQ ID NO.6:

TYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRL

The fifth segment is 314 th-411 th amino acid of capsid protein, which comprises the amino acid sequence shown in SEQ ID NO. 7.

SEQ ID NO.7:

NFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNF

The sixth fragment is amino acid 412-451 of capsid protein, comprising the amino acid sequence shown in SEQ ID NO. 8.

SEQ ID NO.8:

QFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQ

The seventh segment is 452-456 amino acids of capsid protein, including the amino acid sequence shown in SEQ ID NO. 9.

SEQ ID NO.9:

STGGT

The eighth fragment is 457-459 amino acids of capsid protein, which comprises the amino acid sequence shown in SEQ ID NO. 10.

SEQ ID NO.10:

AGT

The ninth segment is 460-469 th amino acid of capsid protein, which comprises the amino acid sequence shown in SEQ ID NO. 11.

SEQ ID NO.11:

QQLLFSQAGP

The tenth fragment is 470 th amino acid of capsid protein, comprising the amino acid sequence shown in SEQ ID NO. 12.

SEQ ID NO.12:

N

The eleventh fragment is 471-491 amino acid of capsid protein, which comprises the amino acid sequence shown in SEQ ID NO. 13.

SEQ ID NO.13:

NMSAQAKNWLPGPCYRQQRVS

The twelfth segment is amino acid 492-628 of capsid protein, which comprises the amino acid sequence shown in SEQ ID NO. 14.

SEQ ID NO.14:

KTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDG

The 629-705 amino acids of the thirteenth segment capsid protein comprise the amino acid sequence shown in SEQ ID NO. 15.

SEQ ID NO.15:

NFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSN

The fourteenth fragment is 706-737 amino acids of capsid protein, comprising the amino acid sequence as shown in SEQ ID NO. 16.

SEQ ID NO.16:

YNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL*

The means of fusion between amino acid fragments includes, but is not limited to, direct fusion, use of a short peptide linker, use of a flexible linker, use of a rigid linker, fusion to a domain, fusion of a peptide tag, use of a chaperone, use of a fusion enzyme, and in particular embodiments of the application, direct fusion between fragments is possible.

In a specific embodiment of the application, the capsid protein comprises the amino acid sequence shown in SEQ ID No. 1.

SEQ ID NO.1:

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPAKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL*

The x represents termination of the amino acid sequence, corresponding to a stop codon.

Stop codons refer to three consecutive nucleotide sequences on messenger RNA (mRNA) that indicate that ribosomes stop translation during protein synthesis, in the standard genetic code there are three stop codons, UAA (amber stop codon), UGA (opioid stop codon), UAG (amber stop codon), which do not encode any amino acids but serve as signals during protein synthesis, allowing ribosomes to recognize and stop extension of the polypeptide chain. When ribosomes move along the mRNA and encounter these specific triplets, they trigger the binding of release factors (release factors) that promote the release of polypeptide chains and the disintegration of the ribosome.

The application also provides a nucleic acid which codes for the fusion adeno-associated virus AAV-WM14 capsid protein.

The nucleic acid refers to a molecule carrying genetic information in organisms and mainly comprises two types of deoxyribonucleic acid and ribonucleic acid.

As will be appreciated by those skilled in the art, codons have degeneracy such that the amino acid sequence of the fusion adeno-associated virus AAV-WM14 capsid protein described above can correspond to a variety of nucleotide sequences, each of which can be translated into the fusion adeno-associated virus AAV-WM14 capsid protein of the application.

In one embodiment of the application, the nucleic acid comprises the nucleotide sequence shown as SEQ ID NO. 2.

SEQ ID NO.2:

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACAACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGATGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGCAAAGAAGAGACCGGTAGAGCCGTCACCTCAGCGTTCCCCCGACTCCTCCACGGGCATCGGCAAGAAAGGCCAGCAGCCCGCCAGAAAGAGACTCAATTTCGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCTCAACCTCTCGGAGAACCTCCAGCAGCGCCCTCTGGTGTGGGACCTAATACAATGGCTGCAGGTGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATGCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGCACCTGGGCCTTGCCCACCTACAATAACCACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGACAACACCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAATTGGGGATTCCGGCCCAAGAGACTCAACTTCAAACTCTTCAACATCCAAGTCAAGGAGGTCACGACGAATGATGGCGTCACAACCATCGCTAATAACCTTACCAGCACGGTTCAAGTCTTCTCGGACTCGGAGTACCAGTTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTGTTCATGATTCCCCAGTACGGCTACCTAACACTCAACAACGGTAGTCAGGCCGTGGGACGCTCCTCCTTCTACTGCCTGGAATACTTTCCTTCGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAAAGCCTGGACCGGCTGATGAACCCCCTCATCGACCAGTACCTGTACTACCTGTCTCGGACTCAGTCCACGGGAGGTACCGCAGGAACTCAGCAGTTGCTATTTTCTCAGGCCGGGCCTAATAACATGTCGGCTCAGGCCAAAAACTGGCTACCCGGGCCCTGCTACCGGCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCCAAGATTCCTCACACGGATGGCAACTTTCACCCGTCTCCTTTGATGGGCGGCTTTGGACTTAAACATCCGCCTCCTCAGATCCTGATCAAGAACACTCCCGTTCCCGCTAATCCTCCGGAGGTGTTTACTCCTGCCAAGTTTGCTTCGTTCATCACACAGTACAGCACCGGACAAGTCAGCGTGGAAATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTAGACACTAATGGTGTTTATAGTGAACCTCGCCCTATTGGAACCCGGTATCTCACACGAAACTTGTGA

Therefore, based on the amino acid sequence of the fusion adeno-associated virus AAV-WM14 capsid protein provided by the application, any nucleotide sequence which can be translated into the fusion adeno-associated virus AAV-WM14 capsid protein obtained by the prior art can realize the application, and the application is within the protection scope of the application.

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

The construct is constructed by inserting the above nucleotide sequence into a suitable expression vector for expressing a specific gene or protein in a host cell or organism, and the expression vector may be selected from suitable expression vectors based on the prior art, for example, an E.coli expression vector, a mammalian expression vector, a yeast expression vector, a plant expression vector, etc.

The application also provides a host cell comprising the construct, or the nucleic acid integrated in the genome of the host cell, or the host cell comprising the fusion adeno-associated virus AAV-WM14 capsid protein.

By host cell is meant a cell that is used to express or produce foreign (heterologous) proteins, viral particles, or other genetic material. For example, the host cell may be selected from bacterial cells, fungal cells, insect cells, plant cells, mammalian cells, etc., specifically from escherichia coli, streptomyces, salmonella typhimurium, yeast, filamentous fungi, drosophila S2 or Sf9 cells, CHO cells, COS cells, HEK293F cells, bowes melanoma cells, NS0 cells, BHK cells, per.c6 cells, etc., such as in particular embodiments of the application, the host cell is mammalian HEK293T. 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). The host cell may be an adherent cell or a cell grown in suspension, i.e., a suspension.

The application also provides a fusion type adeno-associated virus AAV-WM14, wherein the capsid structure of the fusion type adeno-associated virus AAV-WM14 contains the fusion type adeno-associated virus AAV-WM14 capsid protein.

In one embodiment of the application, the fusion adeno-associated virus is rAAV-WM14, namely, the recombinant adeno-associated virus subjected to genetic engineering can be obtained by co-transferring Rep-Cap plasmid RC-WM14 of WM14 and genome plasmid pAAV-CAG-EGFP expressing a green fluorescent protein EGFP, and pHelper plasmid into HEK-293T cells in a proper amount and adopting iodixanol gradient ultra-high speed centrifugation and purification.

In a specific embodiment of the application, the fusion adeno-associated virus AAV-WM14 further comprises a nucleotide sequence encoding a product of interest, which may be carried in a package for a variety of capsid proteins, e.g., the nucleotide sequence encoding the product of interest may be a construct comprising a nucleic acid encoding the product of interest. The construct is obtained 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, any one 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 specific embodiments of the application, the product of interest may be a nucleic acid or a protein, the nucleic acid may be an RNA interference molecule (RNAi), e.g., siRNA, shRNA, small guide RNA (sgRNA), micro RNA (miRNA), other therapeutic genes, etc. The protein may be Green Fluorescent Protein (GFP), luciferase (Luciferase), subtype D surface active protein (SFTPD), subtype B surface active protein (SFTPB), A1 subtype surface active protein (SFTPA 1), interferon gamma (IFN- γ), cystic Fibrosis Transmembrane Regulator (CFTR), aspartic protease (Napsin a), transcription factors regulating expression of downstream genes, signal peptides or cell penetrating peptides, therapeutic antibodies or antibody fragments, and the like.

The application also provides a cell obtained by transformation of the fusion adeno-associated virus AAV-WM 14. The cell may be an engineered cell. The AAV-WM14 fusion type adeno-associated virus can be used for producing virus vectors, AAV-related virus AAV-WM14 fusion type cells can be used for producing AAV vectors for research or treatment in large scale through AAV transformation cells, gene function research can be used for over-expressing or knocking out specific genes in the cells to research functions of the genes and functions of the genes in diseases, protein production can be used for efficiently expressing and producing recombinant proteins by using AAV transformation cells for drug development, biological agents or research reagents, cell model establishment can be used for establishing disease-related cell models, such as certain genetic diseases or tumor cell models, for drug screening and disease mechanism research, gene therapy research can be used for testing expression and functions of therapeutic genes and optimizing gene therapy strategies, AAV vectors can carry siRNA or CRISPR-Cas9 systems for achieving silencing or editing of specific genes in the cells.

In particular embodiments of the application, the cells may be eukaryotic and/or prokaryotic cells. For example, from bacterial cells, fungal cells, insect cells, plant cells, mammalian cells, etc., specifically from E.coli, streptomyces, salmonella typhimurium, yeast, filamentous fungi, drosophila S2 or Sf9 cells, CHO cells, COS cells, HEK293F cells, bowes melanoma cells, NS0 cells, BHK cells, PER.C6 cells, etc., such as in particular embodiments of the application, the host cell is mammalian HEK293T. 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). The host cell may be an adherent cell or a cell grown in suspension, i.e., a suspension.

The application also provides a fusion type adeno-associated virus vector system. The fusion type adeno-associated virus vector system comprises a packaging plasmid, wherein the packaging plasmid comprises the nucleic acid, and the nucleic acid codes for AAV-WM14 capsid protein of the fusion type adeno-associated virus.

In a specific embodiment of the application, the packaging plasmid further comprises a rep gene fragment of an adeno-associated virus, wherein the rep gene fragment comprises an intron comprising a transcription termination sequence.

In a specific embodiment of the application, the expression plasmid further comprises an expression plasmid containing a nucleotide responsible for encoding a target product, wherein the target product can be a nucleic acid or a protein, and the nucleic acid can be an RNA interference molecule (RNAi), such as siRNA and shRNA, small guide RNA (sgRNA), micro RNA (miRNA), other therapeutic genes and the like. The protein may be Green Fluorescent Protein (GFP), luciferase (Luciferase), subtype D surface active protein (SFTPD), subtype B surface active protein (SFTPB), A1 subtype surface active protein (SFTPA 1), interferon gamma (IFN- γ), cystic Fibrosis Transmembrane Regulator (CFTR), aspartic protease (Napsin a), transcription factors regulating expression of downstream genes, signal peptides or cell penetrating peptides, therapeutic antibodies or antibody fragments, and the like.

For example, in one embodiment of the present application, a genomic plasmid pAAV-CAG-EGFP expressing a green fluorescent protein EGFP having the nucleotide sequence of SEQ ID NO.19 is used.

SEQ ID NO.19:

CGCTATTACGCCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGAGTACCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCGGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGGATCCCCGGGTACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGCAGCGGATACCCGTATGACGTCCCAGATTACGCTTGAGAATTCGATATCAAGCTTATCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACCCGGGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCTTCCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTT

In a specific embodiment of the application, the adeno-associated viral vector system further comprises a helper virus or helper virus, which is a virus that allows replication and packaging of AAV by mammalian cells, such as adenovirus, herpes virus, and poxvirus (e.g., vaccinia), and the like, and more specifically, a pHelper plasmid.

In a specific embodiment of the application, the fusion adeno-associated viral vector system further comprises a host cell.

In a specific embodiment of the present application, the packaging plasmid, expression plasmid, helper virus plasmid are transformed into a host cell, wherein the nucleic acid sequence is integrated into the host cell in its entirety to produce the fusion adeno-associated virus. In certain embodiments, the nucleic acid sequences are all integrated together at a single locus within the host cell genome. In other embodiments, the nucleic acid sequences encoding the various genes are present as separate expression cassettes that prevent any risk of recombination to form a virus that is capable of replication, and the nucleic acid sequences encoding the rep and cap genes are present in the same expression cassette.

The application also provides a fusion adeno-associated virus AAV-WM14 which is obtained by virus packaging of the fusion adeno-associated virus vector system.

Viral packaging refers to the process of integrating genetic material (DNA or RNA) into viral particles to form viral particles with infectious capacity. Generally comprising the steps of plasmid construction, preparation of plasmid DNA containing viral genome and foreign gene (e.g., therapeutic gene or reporter gene), transfection of plasmid DNA into host cells by physical, chemical or biological means (e.g., liposome-mediated, electroporation or virus-mediated), viral replication, replication of viral genome within host cells to form a large number of copies of viral DNA or RNA, viral protein synthesis, transcription and translation mechanisms of host cells are used to synthesize capsid proteins and other structural proteins of the virus, viral genome packaging, the newly synthesized viral genome is selectively packaged into viral capsids to form viral particles, viral assembly, viral particle assembly within host cells including assembly of capsid proteins and encapsulation of viral genomes, viral release, release of assembled viral particles from host cells by cell lysis or budding, viral purification, collection of viral particles from host cell culture broth and purification by centrifugation, filtration and chromatography, viral titration and quality control, titration of the purified viral particles, determination of viral concentration and infection, and storage of the viral particles, and storage stability of the viral particles, e.g., preservation of their activity at-80 ℃.

The application also provides a drug or conjugate comprising the fusion adeno-associated virus AAV-WM14.

In a specific embodiment of the application, the medicament comprises an effective amount of the fusion adeno-associated virus AAV-WM14. An effective amount refers to a dosage of a drug that is capable of producing the desired therapeutic effect. The medicament also comprises pharmaceutically acceptable auxiliary materials or carriers. Specific examples of some substances of pharmaceutically acceptable excipients or carriers are sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, tween, etc.), chelating agents (EDTA, etc.), binders, etc. In addition, other low molecular weight polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, amino acids such as glycine, glutamine, asparagine, arginine, and lysine, saccharides such as polysaccharides and monosaccharides, or carbohydrates, and sugar alcohols such as mannitol or sorbitol may be contained. When preparing an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and the like, an appropriate solubilizing agent such as alcohol (ethanol or the like), polyol (propylene glycol, PEG or the like), nonionic surfactant (Tween 80, HCO-50) or the like may be used in combination.

In a specific embodiment of the present application, the fusion adeno-associated virus AAV-WM14 may be a single active ingredient in the medicament, or may be combined with one or more other active ingredients useful for the treatment of hereditary eye disease or deafness disease to form a combined preparation. The other active ingredients may be other various drugs that may be used in the treatment of hereditary eye disease or deafness disease. The content of each component in the combined preparation is usually a safe and effective amount, which can be adjusted based on the actual use. The amount of drug administered containing the fusion adeno-associated virus AAV-WM14 and other active ingredients may generally depend on the weight of the patient, the type of application, the condition and severity of the disease, for example, the amount of drug administered containing the fusion adeno-associated virus AAV-WM14 and other active ingredients may generally be 1~1000 mg/kg/day、1~3 mg/kg/day、3~5 mg/kg/day、5~10 mg/kg/day、10~20 mg/kg/day、20~30 mg/kg/day、30~40 mg/kg/day、40~60 mg/kg/day、60~80 mg/kg/day、80~100 mg/kg/day、100~200 mg/kg/day、200~500 mg/kg/day、 or greater than 500mg/kg/day.

In a specific embodiment of the application, the conjugate comprises an effective amount of a fusion adeno-associated virus AAV-WM14, said fusion adeno-associated virus AAV-WM14 having a biologically active polypeptide attached thereto. The biologically active polypeptide may be a cell penetrating peptide, a tumor targeting peptide, a pH sensitive peptide, an antiviral peptide, a therapeutic protein fragment, an affinity chromatography purified peptide ligand, a polypeptide in gene therapy, an immunomodulatory peptide.

The drug or conjugate may be used alone or in combination with other active ingredients useful for the treatment of vision or auditory system disorders, either in the same formulation or in two different formulations, via the same or different routes, or sequentially via the same or different routes. Sequential administration means that there is a time difference in seconds, minutes, hours or days between the administration of two or more different compounds.

The subject to which the drug or conjugate is administered is preferably a mammal, such as, but not limited to, a human, primate, livestock (e.g., sheep, cow, horse, donkey, pig), companion animal (e.g., dog, cat), laboratory test animal (e.g., mouse, rabbit, rat, guinea pig, hamster) or captured wild animal (e.g., fox, deer). Preferably, the subject is a primate. More preferably, the subject is a human.

The application also provides application of the fusion type adeno-associated virus AAV-WM14 capsid protein, or the nucleic acid, or the construct, or the host cell, or the fusion type adeno-associated virus AAV-WM14, or the cell, or the fusion type adeno-associated virus vector system, or the medicine or conjugate in preparing gene therapy medicines.

Gene therapy refers to the introduction of exogenous genes into target cells to correct or compensate for diseases caused by defective or abnormal genes for therapeutic purposes, and also includes the insertion of exogenous genes into appropriate recipient cells of a patient by gene transfer techniques to allow the products produced by the exogenous genes to treat a disease. The gene therapy agent may include gene therapy agents for hereditary eye disease, deafness disease, other hereditary diseases, malignant tumors, cardiovascular diseases, infectious diseases, autoimmune diseases, neurodegenerative diseases, blood diseases, pulmonary diseases, inflammation or metabolic diseases.

Among them, hereditary eye diseases include retinal photoreceptor-related diseases, including hereditary retinal diseases (IRDs), including but not limited to retinal pigment degeneration (RP) (also known as rod dystrophy), cone/rod dystrophy (CD/CRD), leber Congenital Amaurosis (LCA), macular Dystrophy (MD), and achromatopsia (rod monochromatics), etc.

Deafness diseases include, but are not limited to, diseases associated with intra-cochlear hair cells or supporting cells, and may also include autosomal dominant hereditary hearing loss, autosomal recessive hereditary hearing loss, X-linked hereditary hearing loss, syndrome-type hearing disorder (SHL), non-syndrome-type hearing disorder (NSHL), auditory neuropathy, deafness caused by OTOF gene mutation, and the like.

Other hereditary diseases include, but are not limited to, cystic fibrosis, hemophilia, retinal dystrophy, duchenne Muscular Dystrophy (DMD), spinal Muscular Atrophy (SMA), sickle cell disease, beta thalassemia, leber Hereditary Optic Neuropathy (LHON), choroidal less disease (CHM), hereditary hearing loss, and the like.

Malignant tumors include, but are not limited to, lymphomas, hematological tumors, or solid tumors, particularly selected from the group consisting of adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid tumor, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myelogenous leukemia, brain low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, ovarian carcinoma, pancreatic carcinoma, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymus carcinoma, endometrial carcinoma, uterine sarcoma, uveal melanoma, multiple myeloma, acute leukemia, chronic leukemia, T-cell lymphoma, B-cell lymphoma, and the like.

Cardiovascular diseases include, but are not limited to, heart failure, myocardial infarction, cardiomyopathy, genetic arrhythmia, atherosclerosis, hypertension, calcium ion/calmodulin protein kinase II delta (CaMKII delta) -related heart diseases, familial hypercholesterolemia, and the like.

Infectious diseases include, but are not limited to, HIV/AIDS, hepatitis b, human Papilloma Virus (HPV) infection, herpes Simplex Virus (HSV) infection, influenza virus infection, herpes simplex virus infection, varicella virus infection, measles virus infection, and the like.

Autoimmune diseases include, but are not limited to, systemic Lupus Erythematosus (SLE), rheumatoid Arthritis (RA), myasthenia Gravis (MG), multiple Sclerosis (MS), inflammatory Bowel Disease (IBD), type 1 diabetes (T1 DM), autoimmune liver disease, anti-synthetase antibody syndrome, systemic sclerosis (SSc), sjogren's syndrome, and the like.

Neurodegenerative diseases include, but are not limited to, alzheimer's Disease (AD), parkinson's Disease (PD), amyotrophic Lateral Sclerosis (ALS), huntington's Disease (HD), spinal Muscular Atrophy (SMA), frontotemporal dementia (FTD), multiple Sclerosis (MS), hereditary cerebellar ataxia, friedel-crafts ataxia, and the like.

Hematological disorders include, but are not limited to, hemophilia, beta thalassemia, sickle cell disease, fanconi anemia, acute Myeloid Leukemia (AML), X-linked severe combined immunodeficiency (SCID-X1), metachromatic leukodystrophy, adrenoleukodystrophy, and the like.

Pulmonary diseases include, but are not limited to, cystic Fibrosis (CF), idiopathic Pulmonary Fibrosis (IPF), lung cancer, pulmonary infection, alpha-1 antitrypsin deficiency, primary Ciliated Dyskinesia (PCD), asthma, chronic Obstructive Pulmonary Disease (COPD), surfactant protein deficiency, alveolar protein deposition (PAP), and the like.

Inflammation includes, but is not limited to, skin inflammation, vascular inflammation, allergies, autoimmune diseases selected from the group consisting of rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, xerophthalmia, polymyositis, and the like, fibrous tissue formation, scleroderma, or graft rejection.

Metabolic diseases include, but are not limited to, type I diabetes, type II diabetes, phenylketonuria, glycogen storage Disease, mucopolysaccharidosis (MPS), wilson Disease (Wilson Disease), chrigler-naltrexon syndrome, transthyretin Amyloidosis (ATTR), liver genetic metabolic Disease, glutarate type I (GA-I), neuropathic gaucher Disease (nGD), and the like.

In a specific embodiment of the present application, when the gene therapy drug is a gene therapy drug for hereditary eye disease, the drug mediates the specific expression of a target gene in the retina and the infection of retinal photoreceptor cells under the condition of vitreous injection and delivers a target product to the retinal photoreceptor cells to achieve the treatment of hereditary eye disease, and when the gene therapy drug is a gene therapy drug for deafness disease, the drug can infect intra-cochlear hair cells and supporting cells and deliver a target product to intra-cochlear hair cells and supporting cells of the cochlea to achieve the treatment of deafness disease.

The application also provides the use of the fusion adeno-associated virus AAV-WM14 capsid protein, or the nucleic acid, or the construct, or the host cell, or the fusion adeno-associated virus AAV-WM14, or the cell, or the fusion adeno-associated virus vector system, or the drug or conjugate, as described above, for delivering a product of interest to a retinal photoreceptor cell of an individual. The delivery of the product of interest may be for non-diagnostic therapeutic purposes, e.g., may be in vitro, with delivery of the product of interest to retinal photoreceptor cells ex vivo.

The application also provides the use of the fusion adeno-associated virus AAV-WM14 capsid protein, or the nucleic acid, or the construct, or the host cell, or the fusion adeno-associated virus AAV-WM14, or the cell, or the fusion adeno-associated virus vector system, or the drug or conjugate, as described above, for delivering a product of interest to a hair cell and/or a support cell of an individual. The delivery of the product of interest may be for non-diagnostic therapeutic purposes, e.g., may be in vitro, with delivery of the product of interest to hair cells and/or support cells ex vivo. The hair cells typically comprise outer hair cells and/or inner hair cells.

The target product may be a nucleic acid or a protein, the nucleic acid may be an RNA interference molecule (RNAi), the protein may be Green Fluorescent Protein (GFP), etc.

The present application also provides a gene therapy method comprising administering to a subject an effective amount of the above-described fusion adeno-associated virus AAV-WM14, or the above-described host cell, or the above-described fusion adeno-associated virus vector system, or the above-described drug or conjugate, and adjusting the actual dosage according to the age, weight, and response of the subject. The fusion type adeno-associated virus AAV-WM14 is used as a vector for gene therapy.

The present application also provides a method of treating a hereditary eye disease, comprising administering to a subject an effective amount of the above-described fusion adeno-associated virus AAV-WM14, or the above-described host cell, or the above-described fusion adeno-associated virus vector system, or the above-described drug or conjugate, and adjusting the actual dosage according to the age, weight, and response of the subject to be administered. The fusion adeno-associated virus AAV-WM14 is injected through a vitreous cavity and infects photoreceptor cells of retina, thereby playing a therapeutic role.

The present application also provides a method for treating a deafness disease, comprising administering to a subject an effective amount of the above-described fusion adeno-associated virus AAV-WM14, or the above-described host cell, or the above-described fusion adeno-associated virus vector system, or the above-described drug or conjugate, and adjusting the actual dosage according to the age, weight, and response of the subject. The fusion adeno-associated virus AAV-WM14 infects intra-cochlear hair cells and/or supporting cells, thereby playing a therapeutic role.

The administration mode in the above therapeutic method can be conventional according to the requirement in practical use, such as injection, oral administration, spray, dripping, etc. The above-described methods of treatment may be used alone or in combination with other methods of treatment or therapeutic components. By combined 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. Sequential administration means that there is a time difference in seconds, minutes, hours or days between the administration of two or more different compounds.

The subject of the above treatment method is preferably a mammal, such as, but not limited to, a human, primate, livestock (e.g., sheep, cow, horse, donkey, pig), companion animal (e.g., dog, cat), laboratory test animal (e.g., mouse, rabbit, rat, guinea pig, hamster) or captured wild animal (e.g., fox, deer). Preferably, the subject is a primate. More preferably, the subject is a human.

The fusion adeno-associated virus, or the drug or the conjugate, or the treatment method of the application can be used for preventing vision system diseases, and can be used as a preventive treatment method to be applied before vision is reduced or after a period of time after the fusion adeno-associated virus, or the drug or the conjugate, or the treatment method is contacted with an environment which is liable to cause vision reduction.

The fusion adeno-associated virus, or the drug or the conjugate, or the treatment method of the application can be used for preventing auditory system diseases, and can be applied before hearing injury or after a period of time after contacting an environment which is liable to cause hearing injury as a preventive treatment method.

The fusion 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. Thus, the present application also provides a method of delivering a transgene to a cell by delivering a fusion adeno-associated virus as described above, or a drug or conjugate as described above, to the cell to infect the cell, wherein the fusion adeno-associated virus, or the drug or conjugate, comprises one or more transgenes.

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

(a) Introducing the fusion adeno-associated viral vector of the application into a culture of mammalian host cells;

(b) Screening the culture for mammalian host cells having integrated in their endogenous chromosomes a nucleic acid sequence encoding a fusion adeno-associated viral vector, i.e., producer cells producing a stable fusion adeno-associated viral vector.

The fusion adeno-associated viral vectors include AAV packaging genes (rep and cap genes), desired helper viral genes, and genes encoding products of interest, e.g., transgenes of interest flanked by two AAV Inverted Terminal Repeats (ITRs).

The producer cell of the fusion adeno-associated viral vector may be a mammalian cell. 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.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The term "vector" refers to a macromolecule or combination of macromolecules that contain or bind to a polypeptide and that can be used to mediate the delivery of the polypeptide to a cell. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, or other gene delivery vectors.

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

The term "recombinant AAV vector" refers to an AAV vector containing a heterologous polynucleotide sequence, typically a sequence of interest for genetically transforming a cell. Generally, a heterologous polynucleotide is flanked by at least one, and typically two, AAV Inverted Terminal Repeats (ITRs).

The term "AAV virus" or "AAV viral particle" or "AAV vector particle" refers to a viral particle of an AAV vector comprising at least one AAV capsid protein and one encapsulated polynucleotide.

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

The terms AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and packaging proteins of adeno-associated viruses. AAV rep and cap herein refer to AAV "packaging genes".

The term "helper virus" of an AAV refers to a virus that enables the AAV to be replicated and packaged by mammalian cells. Various such AAV helper viruses are known in the art, including adenoviruses, herpesviruses, and poxviruses (e.g., vaccinia).

The term "infectious" virus or viral particle is a cell containing a substance capable of delivering a polynucleotide component into the virus species with tropism. The term does not necessarily imply that the virus has any replication capacity.

The term "producer cell" refers to a cell line having AAV packaging genes (rep and cap genes), desired helper viral genes, and the DNA genome of a recombinant AAV vector stably integrated into the host cell genome (e.g., a transgene of interest flanked by two AAV Inverted Terminal Repeats (ITRs)).

The terms "comprising," including, "and the like are to be construed as inclusive and not exclusive or exhaustive, i.e., as" including but not limited to.

The term "individual" generally includes humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., which may benefit from treatment with the formulation, kit or combination.

The term "therapeutically effective amount" generally means an amount which, after a suitable period of administration, achieves the effect of treating the diseases as set forth above.

The terms "therapeutic" and "prophylactic" are to be understood in their broadest sense. The term "therapeutic" does not necessarily imply that the mammal is treated until complete recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not ultimately be infected with a disease condition. Thus, treatment and prevention includes alleviation of symptoms of a particular disorder or prevention or reduction of risk of developing a particular disorder. The term "preventing" is understood to mean reducing the severity of the onset of a particular disorder. Treatment may also reduce the severity of existing conditions or the frequency of episodes.

The application is further illustrated by the following examples, which are not intended to limit the scope of the application. Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present application employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art. The apparatus, reagents used in the examples, unless otherwise specified, were all obtained by conventional means.

Example 1 acquisition of novel adeno-associated Virus AAV-WM14

(1) Construction of DNA family shuffling library

The serotype of AAV is determined by the sequence of Cap proteins in the Rep-Cap plasmid required for AAV packaging, and therefore AAV produced by different Cap sequence packaging has different infectivity, we have engineered the existing natural serotypes to DNA family shuffling with capsid sequences of 13 AAV serotypes (AAV 1-13) found in humans and non-human primates as parents. The parental capsid sequences were mixed in equimolar ratio for DNA family shuffling at 4 μg, 30 s treated with DNase I at 0.04U at 25 ℃ to randomly break the complete parental capsid sequences into fragments of different lengths. The DNA fragment of 100-500 bp size was recovered and purified, and 500 ng was subjected to primer-free PCR to extend it into the complete chimeric capsid sequence. After amplification to obtain sufficient chimeric capsid sequences, the chimeric capsid sequences are recombined onto a library vector containing the AAV2 Rep gene and terminal repeats. The recombinant product is converted into competent cells by electric shock, part of bacterial liquid is coated on a plate, colonies growing on the plate are counted, and the colonies are identified by sequencing to determine the diversity of the colonies. The remaining bacterial liquid is inoculated into a 500 mL culture medium for culture at 37 ℃ overnight, and the plasmid extracted the next day is the DNA family reorganization library plasmid. The in vivo screening flow of DNA family reorganization library is shown in figure 1, firstly, constructing plasmid library, packaging library virus, then screening three rounds in mouse eyes, recovering retina tissue, screening three rounds, recovering virus DNA of retina tissue, performing NGS sequencing, analyzing by sequencing result, selecting the sequence with the top reading rank as the preferred verification sequence.

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. DNA family shuffling was performed on the existing 13 native serotype capsid sequences as parents, and the resulting DNA family shuffling library plasmid and adenovirus helper plasmid pHelper were co-transferred in appropriate amounts into HEK-293T cells, pHelper plasmid (plasmid full sequence is shown as SEQ ID NO. 12 of AAV-ie patent document CN 110437317A). Cells of 15cm dishes each were transfected with only 20ng of DNA family shuffling library plasmid. The medium was collected 72 hours after transfection and cells and medium were collected 48 more hours after transfection. AAV was released by lysing the cells with 110mM citrate buffer (pH 4.2), and after centrifugation the virus-containing supernatant was neutralized with 1/5 volume of 2M HEPES, followed by addition of polyethylene glycol 8000 at a final concentration of 8% and 500mM sodium chloride, and precipitation of the virus was performed overnight at 4 ℃. After centrifugation, the pellet was resuspended in PBS containing 2mM Mg2+ and benzonase was added to a final concentration of 100U/mL and the nucleic acid digested at 37℃for at least 1 hour. Virus suspension was purified by ultracentrifugation with iodixanol density gradient solutions (15%, 25%, 40% and 60%) and then virus titers were determined by qPCR using WPRE gene specific primers for AAV2 (WPRE-F: GTCAGGCAACGTGGCGTGGTGTG (SEQ ID No. 17); WPRE-R: GGCGATGAGTTCCGCCGTGGC (SEQ ID No. 18)). Three rounds of animals are screened in vivo, NGS is sequenced, and the Cap sequence with the first sequencing reading rank is obtained through analysis, and is named WM14-Cap, the amino acid sequence of the Cap is SEQ ID NO.1, and the nucleotide sequence of the Cap is SEQ ID NO.2.

The wild-type AAV has relatively high homology, so that the amino acid sequences of AAV of different serotypes are highly similar, and therefore, the Cap amino acid sequence of AAV-WM14 is compared with the amino acid sequences of the 13 parental serotypes of DNA shuffling, and the results are shown in FIG. 3.

(2) Preparation and purification of rAAV-WM14 Virus

The WM14-Cap coded gene sequence obtained in the sequencing result is synthesized by the Souzhou Jin Weizhi biotechnology limited company, and the synthesized sequence is constructed on an AAV2-Rep skeleton to obtain a WM14 Rep-Cap plasmid, namely RC-WM14, which is shown in figure 2, and the sequence identification is correct. And co-transferring the obtained plasmid RC-WM14 and a genome plasmid pAAV-CAG-EGFP expressing a green fluorescent protein EGFP, wherein the nucleotide sequence of the plasmid pAAV-CAG-EGFP is SEQ ID NO.19, the pHelper plasmid is co-transferred into HEK-293T cells in proper quantity, and the AAV virus is purified by adopting iodixanol gradient ultra-high speed centrifugation, so that the final product is rAAV-WM14, namely AAV-WM14-CAG-EGFP. The virus titer was then determined by qPCR to 2E+13 GC/mL using WPRE gene-specific primers of AAV2 (SEQ ID NO.17 and SEQ ID NO. 18) and placed at-80℃for use.

Example 2 live experience of novel adeno-associated Virus rAAV-WM14 in the visual System

Anesthetized adult mice were placed under a microscope and 1-2 drops of topiramate eye drops were dropped onto the eye to dilate the pupil and applied ofloxacin eye cream. Left hand elbow forceps hold up and secure the eye from the optic disc and right hand 1mL syringe or glass needle punctures the cornea to reduce intraocular pressure. The periocular fluid was gently wiped off with a paper towel. The eyeball is fixed by holding forceps left, a glass needle for sucking 1.1 mu L of virus is held by holding the forceps right, the needle is inserted at an angle of 50 DEG between the posterior edge of the corneosclera and the plane of the iris, rAAV-WM14 is slowly injected, and the titer is 1E13 gc/mL. After the injection, the glass needle stays at 30 s and is slowly pulled out. After the ofloxacin eye ointment is smeared on the wound, the mice are placed 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 3 weeks, eye tissue was taken. Mice were sacrificed by spinal dislocation, the eyes were removed, the cornea was punctured with a 1mL syringe, and aqueous humor was discharged. The eyeballs were placed in a small petri dish containing a PBS solution and dissected under a microscope. The forceps are used for clamping the cornea, the ophthalmic scissors enter from the opening of the cornea, the cornea is cut off circumferentially, the lens is taken out by the forceps, and the optic nerve remains about 2 mm. The eye cup was fixed in 4% PFA for 12 h at 4 ℃. The fixed eye cup was dehydrated in 30% sucrose for 6-8 h at 4 ℃. After frozen sections, sections with better effect and complete tissue are picked under a fluorescence microscope for staining.

Immunofluorescence results indicate that rAAV-WM14 can efficiently infect photoreceptor cells (cone rod cells), and the results are shown in FIG. 4. Meanwhile, the embodiment also analyzes the condition of the rAAV-WM14, AAV2, AAV2.7m8 virus infection of retina cells, the result is shown in figure 5, AAV-WM14 targets photoreceptor cells and RPE cells efficiently, AAV2.7m8 mainly infects retinal ganglion cells, AAV2.7m8 can infect photoreceptor cells in small quantity, statistics on the number of EGFP positive photoreceptor cells is shown in figure 6, and the result shows that the infection efficiency of the rAAV-WM14-CAG-EGFP virus on photoreceptor cells is up to 97.7% and is obviously higher than that of AAV2, AAV2.7m8 virus. The result shows that the rAAV-WM14-CAG-EGFP virus has higher infection to retina photoreceptor cells and can be used for ophthalmic gene therapy.

Example 3 live experience of novel adeno-associated Virus rAAV-WM14 in auditory System

Experimental adeno-associated virus was delivered into the cochlea of adult mice using a glass electrode by means of posterior semicircular canal microinjection. Adeno-associated viruses selected in the experiments were AAV2 and AAV-WM14-CAG-EGFP. The specific experimental method is that a, weighing the weight of the P30 mouse, injecting a proper amount of pentobarbital sodium into the abdominal cavity to anesthetize the mouse, and removing the hair behind the right ear and the back ear of the mouse by using depilatory cream and a shaver after the mouse completely loses consciousness and activity ability, and exposing the skin behind the ear. b. Thoroughly sterilizing the exposed skin with 75% alcohol, carefully cutting subcutaneous tissue behind the ear with Venus scissors, tearing off subcutaneous adipose tissue with 75% alcohol sterilized forceps, carefully looking deep, and avoiding damaging surrounding blood vessels and nerves until the posterior semicircular canal is exposed. c. The muscular tissue and membrane structure on the posterior semicircular canal was carefully torn, and an opening corresponding to the diameter of the glass tube for injection was chiseled in the posterior semicircular canal using pointed surgical forceps. The tip of the glass tube was placed in the opening, 1 uL virus in the tube was slowly injected, and after one minute of rest, the glass tube was slowly removed. d. After injection, the openings at the latter half-ducts are bonded with veterinary tissue glue, and then the wounds of the skin and the muscle are bonded with veterinary tissue glue. Subsequently, the operated mice were placed on a thermostatic heating pad at 37 ℃ and returned to the animal house for feeding after natural recovery. Any damaged cochlea is no longer used for subsequent studies. After 14 days of virus injection, cochlear tissues were harvested and immunofluorescent stained for analysis of virus infection.

The immunostaining results are shown in FIG. 7, which shows that the rAAV-WM14-CAG-EGFP can efficiently infect inner hair cells after being injected into adult mice, and in addition, the rAAV-WM14-CAG-EGFP can infect supporting cells after being injected into adult mice, as shown in FIG. 8. FIG. 9 is a statistical result of WM14 and AAV2 on intra-cochlear hair cell and supporting cell infection, and the result shows that rAAV-WM14-CAG-EGFP can efficiently infect inner hair cells, has the capability of infecting supporting cells, and has the efficiency of 35 percent and is obviously higher than AAV2. Thus, WM14 can be used for gene therapy of deafness diseases.

Claims

1. The fusion type adeno-associated virus AAV-WM14 capsid protein comprises amino acid fragments of serotypes AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV10 and AAV13, and the amino acid sequence of the capsid protein is shown as SEQ ID No. 1.

2. The fusion adeno-associated virus AAV-WM14 capsid protein according to claim 1, wherein the capsid protein comprises a first fragment to a fourteenth fragment connected in sequence, the first fragment comprises an amino acid fragment from AAV7, the second fragment comprises an amino acid fragment from AAV8, the third fragment comprises an amino acid fragment from AAV1 or AAV6, the fourth fragment comprises an amino acid fragment from AAV8 or AAV10, the fifth fragment comprises an amino acid fragment from AA1 or AA6, the sixth fragment comprises an amino acid fragment from AAV8, the seventh fragment comprises an amino acid fragment from AAV10, the eighth fragment comprises an amino acid fragment from AAV4, the ninth fragment comprises an amino acid fragment from AAV10, the tenth fragment comprises an amino acid fragment from AAV8, the eleventh fragment comprises an amino acid fragment from AA10, the twelfth fragment comprises an amino acid fragment from AAV2, the thirteenth fragment comprises an amino acid fragment from AAV7, and the fourteenth fragment comprises an amino acid fragment from AAV2 or AAV3 or 13.

3. The fusion adeno-associated virus AAV-WM14 capsid protein according to claim 2, wherein the first fragment comprises an amino acid sequence as shown in SEQ ID No.3, the second fragment comprises an amino acid sequence as shown in SEQ ID No.4, the third fragment comprises an amino acid sequence as shown in SEQ ID No.5, the fourth fragment comprises an amino acid sequence as shown in SEQ ID No.6, the fifth fragment comprises an amino acid sequence as shown in SEQ ID No.7, the sixth fragment comprises an amino acid sequence as shown in SEQ ID No.8, the seventh fragment comprises an amino acid sequence as shown in SEQ ID No.9, the eighth fragment comprises an amino acid sequence as shown in SEQ ID No.10, the ninth fragment comprises an amino acid sequence as shown in SEQ ID No.11, the tenth fragment comprises an amino acid sequence as shown in SEQ ID No.12, the eleventh fragment comprises an amino acid sequence as shown in SEQ ID No.13, the twelfth fragment comprises an amino acid sequence as shown in SEQ ID No.7, the seventh fragment comprises an amino acid sequence as shown in SEQ ID No.8, the thirteenth fragment comprises an amino acid sequence as shown in SEQ ID No. 16.

4. A nucleic acid encoding the fusion adeno-associated virus AAV-WM14 capsid protein of any one of claims 1-3.

5. The nucleic acid of claim 4, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO. 2.

6. A construct comprising the nucleic acid of any one of claims 4-5.

7. A host cell comprising the construct of claim 6, or having the nucleic acid of any one of claims 4-5 integrated into the genome of the host cell, or comprising the fusion adeno-associated virus AAV-WM14 capsid protein of any one of claims 1-3.

8. A fusion adeno-associated virus AAV-WM14, the capsid structure of which comprises the fusion adeno-associated virus AAV-WM14 capsid protein of any one of claims 1-3.

9. The fusion adeno-associated virus AAV-WM14 according to claim 8, wherein the fusion adeno-associated virus AAV-WM14 further comprises a nucleotide sequence encoding a product of interest, wherein the product of interest is a nucleic acid or a protein.

10. A cell transformed with the fusion adeno-associated virus AAV-WM14 of any one of claims 8-9.

11. A fusion adeno-associated viral vector system comprising a packaging plasmid comprising the nucleic acid of any one of claims 4-5 therein.

12. The fusion adeno-associated viral vector system of claim 11, further characterized by any one or more of the following:

1) The packaging plasmid also comprises rep gene fragments of adeno-associated viruses;

2) The adeno-associated viral vector system further comprises an expression plasmid comprising nucleotides responsible for encoding the product of interest;

3) The adeno-associated viral vector system further comprises a helper viral plasmid or helper virus;

4) The fusion adeno-associated viral vector system also includes a host cell.

13. Use of the fusion adeno-associated virus AAV-WM14 capsid protein of any one of claims 1-3, or the nucleic acid of any one of claims 4-5, or the construct of claim 6, or the host cell of claim 7, or the fusion adeno-associated virus AAV-WM14 of any one of claims 8-9, or the cell of claim 10, or the fusion adeno-associated virus vector system of any one of claims 11-12, for the preparation of a gene therapy agent selected from the group consisting of a gene therapy agent for a hereditary retinal disease caused by retinal photoreceptor cells, a gene therapy agent for deafness caused by intracochlear hair cells or supporting cells, said hereditary retinal disease selected from the group consisting of retinal pigment degeneration, cone/rod dystrophy, leber congenital amaurosis, macular dystrophy, and holocelluminar.

14. The use according to claim 13, wherein the drug is capable of mediating the expression of the gene of interest specifically in the retina and infecting retinal photoreceptor cells under vitreal injection conditions, or wherein the drug is capable of infecting intra-cochlear hair cells or supporting cells.