A fusion adeno-associated virus capsid protein, adeno-associated virus and its application

Abstract

The present application relates to the field of biomedicine, and specifically to a fusion adeno-associated virus capsid protein, adeno-associated virus and its application. The present application provides a fusion adeno-associated virus AAV-WM14 capsid protein, comprising amino acid fragments of serotypes AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV10, and AAV13, and the capsid protein includes the amino acid sequence shown in SEQ ID NO.1. The fusion adeno-associated virus provided in the present application can be administered by intravitreal injection, efficiently infecting retinal photoreceptor cells in the eye, and can be delivered to the cochlea to efficiently infect inner hair cells and supporting cells. The fusion adeno-associated virus provided in the present application can be widely used in gene therapy for related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a fusion adeno-associated virus capsid protein, an adeno-associated virus and applications thereof. Background Art

[0002] Inherited eye diseases are caused by gene defects and often manifest as familial diseases. Blinding eye diseases seriously affect people's quality of life. Among them, inherited retinal diseases (IRDs) are relatively rare blinding eye diseases caused by genetic mutations. They are also the leading cause of legal blindness in China and the world, and there is no effective treatment. With the development of science and technology, gene therapy has provided a new treatment idea for blinding eye diseases, which is expected to bring a bright future to patients with blinding eye diseases.

[0003] The retina is a multi-layered sensory tissue that lines the back of the eye. Its main function is to convert light energy into changes in electrical potential through a process called phototransduction. The light-sensing elements of the retina are photoreceptors. The retina contains two types of photoreceptors: rods and cones. Rods (about 120 million in the human eye) are responsible for night vision, while cones (about 6 to 7 million in the human eye) are responsible for visual acuity and color vision. Photoreceptors are cells that convert optical stimuli into nerve impulses. Improper development, dysfunction or premature death of retinal photoreceptor cells can lead to vision loss. Examples of inherited retinal diseases (IRDs) include retinitis pigmentosa (RP) (also known as rod dystrophy), cone / rod dystrophy (CD / CRD), Leber congenital amaurosis (LCA), macular dystrophy (MD) and achromatopsia (rod monochromacy). Currently, more than 270 different genes are known to cause IRDs, and in some ways, gene therapy is currently the only effective treatment and intervention measure.

[0004] A key to gene therapy is delivery, and the effectiveness and safety of the vector are crucial. Among them, adeno-associated virus (AAV) has been successful in the field of gene therapy as a safe gene delivery vector. Inherited retinal diseases are a type of blinding eye disease caused by genetics, and there is currently no effective treatment. In recent years, researchers have used AAV as a vector to conduct some clinical trials in the field of gene therapy to treat such diseases by delivering normal genes. However, the current injection method is mainly to directly inject the viral vector into the retina. This surgical technique requires high requirements and there is a risk of damaging retinal tissue. In addition, the virus has a weak ability to spread laterally, and each injection of the virus can only infect a small number of cells near the injection point. Therefore, it is necessary to develop better new AAV viral vectors suitable for gene therapy of eye diseases to more effectively treat similar inherited ophthalmic diseases. In addition to IRDs, diseases such as retinal degenerative diseases and congenital glaucoma are also gene-related inherited eye diseases, and some gene therapy trials are currently underway. As an emerging treatment method, gene therapy has important application prospects in the field of ophthalmology.

[0005] In ophthalmology, so far, gene therapy for eye diseases based on AAV viral vectors requires the viral vector to be injected directly under the retina. This technology can only be performed in specialized hospitals with high-level experts and equipment, and this injection method has the risk of damaging fragile retinal tissue. Another disadvantage of this method is that due to the weak lateral diffusion ability of AAV virus, each injection of the virus can only target a small number of cells near the injection point. Therefore, it is necessary to develop AAV vectors that can efficiently reach the inner part of the retina through vitreous cavity injection, reduce the difficulty of surgery, and improve the treatment effect.

[0006] Gene therapy for ophthalmic diseases such as RP still relies on intraretinal delivery, which can cause retinal detachment, has serious collateral risks, and generally cannot achieve widespread photoreceptor transduction. Existing AAV vectors are difficult to efficiently infect photoreceptor cells when injected into the vitreous cavity. Summary of the invention

[0007] In order to solve the defects of the above-mentioned prior art, the present application provides a new adeno-associated virus with a variant capsid protein, which can be administered by intravitreal injection and efficiently infect retinal photoreceptor cells in the eye. In addition, the new adeno-associated virus of the present application is delivered to the cochlea, where it can efficiently infect inner hair cells and supporting cells, and can be widely used in gene therapy for related diseases.

[0008] In a first aspect, the present application provides a fusion adeno-associated virus AAV-WM14 capsid protein, which comprises amino acid fragments of serotypes AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV10, and AAV13; the capsid protein comprises the amino acid sequence shown in SEQ ID NO.1.

[0009] The second aspect of the present application provides a nucleic acid, which encodes the fusion adeno-associated virus AAV-WM14 capsid protein of the above-mentioned first aspect; the nucleic acid includes the nucleotide sequence shown in SEQ ID NO.2.

[0010] The third aspect of the present application provides a construct, which comprises the nucleic acid of the second aspect.

[0011] The fourth aspect of the present application provides a host cell, which comprises the construct of the third aspect, or the nucleic acid of the second aspect is integrated into the genome of the host cell, or the host cell comprises the fusion adeno-associated virus AAV-WM14 capsid protein of the first aspect.

[0012] The fifth aspect of the present application provides a fusion adeno-associated virus AAV-WM14, the capsid structure of which contains the capsid protein of the fusion adeno-associated virus AAV-WM14 of the first aspect mentioned above.

[0013] The sixth aspect of the present application provides a cell, which is obtained by transformation using the fusion adeno-associated virus AAV-WM14 of the fifth aspect.

[0014] The seventh aspect of the present application provides a fusion adeno-associated virus vector system, which comprises a packaging plasmid, and the packaging plasmid comprises the nucleic acid of the second aspect.

[0015] The eighth aspect of the present application provides a drug or a conjugate, wherein the drug or the conjugate comprises the fusion-type adeno-associated virus AAV-WM14 of the fifth aspect.

[0016] In the ninth aspect of the present application, 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 described in the sixth aspect, or the fusion adeno-associated virus vector system described in the seventh aspect, or the drug or conjugate described in the eighth aspect are provided for use in the preparation of gene therapy drugs.

[0017] The beneficial effects of this application are:

[0018] The new adeno-associated vector AAV-WM14 is more flexible and efficient than traditional transgenic methods in mediating target gene expression, and is more convenient to use and has lower costs;

[0019] The present application provides a novel adeno-associated vector AAV-WM14, which can mediate the efficient expression of target genes in retinal photoreceptor cells of adult mice under the condition of intravitreal injection;

[0020] The AAV-WM14 of the present application has a higher infection efficiency for the inner ear supporting cells of adult mice, and its infection efficiency is higher than that of AAV2. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown is a flow chart for in vivo screening of DNA family shuffling libraries.

[0022] Figure 2 Shown is the plasmid map of RC-WM14.

[0023] Figure 3 Shown is the comparison of the Cap amino acid sequence of AAV-WM14 with the amino acid sequences of 13 parental serotypes of DNA shuffling.

[0024] Figure 4 The figure shows the infection of AAV-WM14-CAG-EGFP on photoreceptor cells. This figure shows a frozen section of eyeball tissue injected with rAAV-WM14-CAG-EGFP virus. Green shows the fluorescent protein expressed by virus infection, and blue shows the cell nucleus staining. The results show that rAAV-WM14-CAG-EGFP virus has a high infection rate on retinal photoreceptor cells and can be used for ophthalmic gene therapy.

[0025] Figure 5 The image shows the infection of AAV-WM14, AAV2, and AAV2.7m8 in the eyeball tissue. The image shows the frozen sections of the eyeball tissue injected with rAAV-WM14-CAG-EGFP, AAV2-CAG-EGFP, and AAV2.7m8-CAG-EGFP viruses. The green color shows the fluorescent protein expressed by the virus infection, and the blue color shows the cell nucleus staining.

[0026] Figure 6 The figure shows the infection efficiency of photoreceptor cells by AAV-WM14-CAG-EGFP, AAV2-CAG-EGFP, and AAV2.7m8-CAG-EGFP. The results show that AAV-WM14-CAG-EGFP has a high infection efficiency for photoreceptor cells.

[0027] Figure 7 The figure shows the infection of AAV-WM14 and AAV2 in the cochlear hair cells of adult mice. The figure shows the staining results of cochlear tissue flat sections injected with rAAV-WM14-CAG-EGFP and AAV2 viruses. The green shows the EGFP fluorescent protein expressed by viral infection, and the red shows Myo7a staining, i.e. hair cell staining. The results show that rAAV-WM14-CAG-EGFP efficiently infects inner hair cells after injection into adult mice.

[0028] Figure 8 The figure shows the infection of AAV-WM14 and AAV2 on the supporting cells of the cochlea of ​​adult mice. The figure shows the staining results of cochlear tissue flat sections injected with rAAV-WM14-CAG-EGFP and AAV2 viruses. The green shows the EGFP fluorescent protein expressed by viral infection, and the magenta shows SOX2 staining, i.e. supporting cell staining. The results show that rAAV-WM14-CAG-EGFP can infect supporting cells after being injected into adult mice.

[0029] Fig. 9 Shown are statistics on the infection of AAV-WM14 and AAV2 on inner hair cells and supporting cells in the cochlea of ​​adult mice. Figure A shows the statistics of cochlear inner hair cells infected by rAAV-WM14-CAG-EGFP and AAV2 viruses. Figure B shows the statistics of cochlear supporting cells infected by rAAV-WM14-CAG-EGFP and AAV2 viruses. The results show that rAAV-WM14-CAG-EGFP efficiently infects inner hair cells after injection into adult mice, and also has the ability to infect supporting cells. DETAILED DESCRIPTION

[0030] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0031] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0032] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0033] The present application modifies the existing natural serotypes, uses the capsid sequences of 13 AAV serotypes (AAV1-13) found in humans and non-human primates as parents to perform DNA family shuffling, obtains DNA family shuffling library plasmids, packages the library plasmids into viruses, and obtains the Cap sequence with the best infection tropism after in vivo screening in animals, thereby providing a new adeno-associated virus with a variant capsid protein, which can be administered by intravitreal injection and efficiently infect retinal photoreceptor cells in the eye. In addition, the novel adeno-associated virus of the present application is delivered to the cochlea, can efficiently infect inner hair cells and supporting cells, and can be widely used in gene therapy for related diseases.

[0034] In this application, AAV is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or its derivatives.

[0035] Under an electron microscope, the nucleocapsid of adeno-associated virus is generally nearly circular. This nearly circular capsid is actually a closed icosahedral symmetrical hollow capsid composed of multiple protein capsomers, in which the genomic nucleic acid is wrapped. An icosahedral symmetric structure contains three types of rotational symmetry: 3-fold, 2-fold, and 5-fold symmetry (3-, 2-, 5-fold symmetry). That is, this symmetrical three-dimensional structure has a 3-fold symmetry axis passing through the center points of the two opposite faces of the virus particle, and the capsomers rotate 120° around the 3-fold symmetry axis three times to reset, forming a triangular face; there is a 2-fold symmetry axis (edge), and the capsomers rotate 180° around the 2-fold symmetry axis and reset twice, forming two intersecting triangular faces; there is also a 5-fold symmetry axis passing through two opposite vertices, and the capsomers rotate 72° around the 5-fold symmetry axis five times to reset, forming a pentamer. Therefore, the icosahedral symmetrical capsid is composed of 20 equilateral triangular faces, where every 2 triangular faces intersect to form edges, for a total of 30 edges; every 5 triangular faces are connected to form 12 vertices.

[0036] The genomic sequences of various serotypes of AAV, as well as the sequences of native ITRs, 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 disclosures thereof are incorporated herein by reference for AAV nucleic acid and amino acid sequences.

[0037] The present application first provides a fusion adeno-associated virus AAV-WM14 capsid protein, which contains amino acid fragments of serotypes AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV10, and AAV13.

[0038] In the present application, the amino acid fragment refers to a sequence consisting of one or more amino acids.

[0039] In a specific embodiment of the present application, the capsid protein comprises the first to fourteenth fragments 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 AAV1 or AAV6, 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 AAV10, 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 AAV13.

[0040] The first fragment is amino acids 1-197 of the capsid protein, including the amino acid sequence shown in SEQ ID NO.3.

[0041] SEQ ID NO.3:

[0042] MAADGYLPDWLEDNLSEGIREWWDLKPGAPPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPAKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPS

[0043] The second fragment is amino acids 198-223 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.4.

[0044] SEQ ID NO.4:

[0045] GVGPNTMAAGGGAPMADNNEGADGVG

[0046] The third fragment is the amino acids 224-272 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.5.

[0047] SEQ ID NO.5:

[0048] NASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDN

[0049] The fourth fragment is the amino acids 273-313 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.6.

[0050] SEQ ID NO.6:

[0051] TYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRL

[0052] The fifth fragment is the amino acids 314-411 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.7.

[0053] SEQ ID NO.7:

[0054] NFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNF

[0055] The sixth fragment is the amino acids 412-451 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.8.

[0056] SEQ ID NO.8:

[0057] QFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQ

[0058] The seventh fragment is the amino acids 452-456 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.9.

[0059] SEQ ID NO.9:

[0060] STG

[0061] The eighth fragment is the amino acids 457-459 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.10.

[0062] SEQ ID NO.10:

[0063] AGT

[0064] The ninth fragment is the amino acids 460-469 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.11.

[0065] SEQ ID NO.11:

[0066] QQLLFSQAGP

[0067] The tenth fragment is the 470th amino acid of the capsid protein, including the amino acid sequence as described in SEQ ID NO.12.

[0068] SEQ ID NO.12:

[0069] N

[0070] The eleventh fragment is the amino acids 471-491 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.13.

[0071] SEQ ID NO.13:

[0072] NMSAQAKNWLPGPCYRQQRVS

[0073] The twelfth fragment is the amino acids 492-628 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.14.

[0074] SEQ ID NO.14:

[0075] KTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDG

[0076] The thirteenth fragment is located at amino acids 629-705 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.15.

[0077] SEQ ID NO.15:

[0078] NFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSN

[0079] The fourteenth fragment is the amino acids 706-737 of the capsid protein, including the amino acid sequence as described in SEQ ID NO.16.

[0080] SEQ ID NO.16:

[0081] YNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL*

[0082] The fusion methods between amino acid fragments include but are not limited to direct fusion, use of short peptide linkers, use of flexible linkers, use of rigid linkers, fusion to structural domains, fusion peptide tags, use of molecular chaperones, and use of fusion enzymes. In a specific embodiment of the present application, the fragments can be directly fused.

[0083] In a specific embodiment of the present application, the capsid protein comprises the amino acid sequence shown in SEQ ID NO.1.

[0084] SEQ ID NO.1:

[0085] *

[0086] The * indicates the termination of the amino acid sequence, corresponding to the termination of the code.

[0087] A stop codon refers to three consecutive nucleotide sequences on messenger RNA (mRNA) that instruct the ribosome to stop translating during protein synthesis. In the standard genetic code, there are three stop codons: UAA (amber stop codon), UGA (opium stop codon), and UAG (amber stop codon). Stop codons do not encode any amino acids, but serve as signals in the protein synthesis process to allow the ribosome to recognize and terminate the extension of the polypeptide chain. When the ribosome moves along the mRNA and encounters these specific triplets, they trigger the binding of release factors, which promote the release of the polypeptide chain and the disassembly of the ribosome.

[0088] The present application also provides a nucleic acid, which encodes the above-mentioned fusion adeno-associated virus AAV-WM14 capsid protein.

[0089] The nucleic acid refers to the molecule that carries genetic information in organisms, and mainly includes two types: deoxyribonucleic acid and ribonucleic acid.

[0090] Those skilled in the art will appreciate that codons are degenerate, and therefore the amino acid sequence of the above-mentioned fusion adeno-associated virus AAV-WM14 capsid protein can correspond to a variety of nucleotide sequences, all of which can be translated into the fusion adeno-associated virus AAV-WM14 capsid protein of the present application.

[0091] In one embodiment of the present application, the nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO.2.

[0092] SEQ ID NO.2:

[0093]

[0094] Therefore, based on the amino acid sequence of the fusion adeno-associated virus AAV-WM14 capsid protein provided in the present application, any nucleotide sequence that can be translated into the fusion adeno-associated virus AAV-WM14 capsid protein that can be obtained using the prior art can realize the present application and is within the protection scope of the present application.

[0095] The present application also provides a construct, which comprises the above-mentioned nucleic acid.

[0096] The construct is constructed by inserting the above-mentioned nucleotide sequence into a suitable expression vector for expressing a specific gene or protein in a host cell or organism. The expression vector can be selected based on the existing technology, for example, it can be an Escherichia coli expression vector, a mammalian expression vector, a yeast expression vector, a plant expression vector, etc.

[0097] The present application also provides a host cell, wherein the host cell comprises the above-mentioned construct, or the above-mentioned nucleic acid is integrated into the genome of the host cell, or the host cell comprises the above-mentioned fusion adeno-associated virus AAV-WM14 capsid protein.

[0098] The host cell refers to a cell used to express or produce foreign (heterologous) proteins, virus particles or other genetic materials. For example, the host cell can be selected from bacterial cells, fungal cells, insect cells, plant cells, mammalian cells, etc., and specifically, can be selected 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. For example, in a specific embodiment of the present application, the host cell is a mammalian HEK293T. The host cell can be a passage cell or a primary cell, that is, a cell directly isolated from an organism (such as a human). The host cell can be an adherent cell or a suspended cell, that is, a cell growing in the form of a suspension.

[0099] The present application also provides a fusion-type adeno-associated virus AAV-WM14, the capsid structure of which contains the above-mentioned fusion-type adeno-associated virus AAV-WM14 capsid protein.

[0100] In one embodiment of the present application, the fusion adeno-associated virus is rAAV-WM14, that is, a recombinant adeno-associated virus that has been genetically engineered. It can be obtained by co-transfecting the Rep-Cap plasmid RC-WM14 of WM14 with the genomic plasmid pAAV-CAG-EGFP expressing a green fluorescent protein EGFP and the pHelper plasmid in appropriate amounts into HEK-293T cells and purifying them by iodixanol gradient ultracentrifugation.

[0101] In a specific embodiment of the present application, the fusion adeno-associated virus AAV-WM14 also includes a nucleotide sequence encoding a target product, and the nucleotide sequence encoding the target product can be wrapped and carried by various capsid proteins. For example, the nucleotide sequence encoding the target product can be a construct, and the construct contains a nucleic acid encoding the target product. The construct is constructed by inserting the nucleic acid encoding the target product into a suitable expression vector. Those skilled in the art can select a suitable expression vector. For example, the above expression vector is selected from any one of pAAV-CAG, pAAV-TRE, pAAV-EF1a, pAAV-GFAP, pAAV-Lgr5, pAAV-Sox2, pAAV-Syn or pAAV-CMV expression vectors.

[0102] In a specific embodiment of the present application, the target product may be a nucleic acid or a protein, and the nucleic acid may be an RNA interference molecule (RNAi), such as siRNA, shRNA, a small guide RNA (sgRNA), a micro RNA (miRNA), or other therapeutic genes, etc. The protein may be green fluorescent protein (GFP), luciferase, surfactant protein subtype D (SFTPD), surfactant protein subtype B (SFTPB), surfactant protein subtype A1 (SFTPA1), interferon gamma (IFN-γ), cystic fibrosis transmembrane regulator (CFTR), aspartic protease (Napsin A), transcription factors that regulate downstream gene expression, signal peptides or cell penetrating peptides, therapeutic antibodies or antibody fragments, etc.

[0103] The present application also provides a cell, which is obtained by transformation using the above-mentioned fusion adeno-associated virus AAV-WM14. The cell can be an engineered cell. After the fusion adeno-associated virus AAV-WM14 is transformed into the cell, it can be used for example for viral vector production. By transforming the cell with AAV, AAV vectors for research or treatment can be mass-produced; gene function research, AAV can be used to overexpress or knock out specific genes in cells to study the functions of these genes and their role in diseases; protein production, using AAV to transform cells, recombinant proteins can be efficiently expressed and produced for drug development, biological preparations or scientific research reagents; cell model establishment: AAV-transformed cells can be used to establish disease-related cell models, such as certain genetic diseases or tumor cell models, for drug screening and disease mechanism research; gene therapy research, AAV-transformed cells can be used to test the expression and function of therapeutic genes and optimize gene therapy strategies; gene silencing and editing, AAV vectors can carry siRNA or CRISPR-Cas9 systems for silencing or editing specific genes in cells.

[0104] In a specific embodiment of the present application, the cell may be a eukaryotic cell and / or a prokaryotic cell. For example, it may be selected from bacterial cells, fungal cells, insect cells, plant cells, mammalian cells, etc. Specifically, it may be selected 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. For example, in a specific embodiment of the present application, the host cell is a mammalian HEK293T. The host cell may be a passage cell or a primary cell, i.e., a cell directly isolated from an organism (such as a human). The host cell may be an adhesive cell or a suspended cell, i.e., a cell grown in the form of a suspension.

[0105] The present application also provides a fusion adeno-associated virus vector system, which comprises a packaging plasmid, wherein the packaging plasmid comprises the above-mentioned nucleic acid, and the nucleic acid encodes the capsid protein of the fusion adeno-associated virus AAV-WM14.

[0106] In a specific embodiment of the present application, the packaging plasmid further comprises a rep gene fragment of an adeno-associated virus; wherein the rep gene fragment comprises an intron, and the intron comprises a transcription termination sequence.

[0107] In a specific embodiment of the present application, an expression plasmid is also included, and the expression plasmid contains nucleotides responsible for encoding the target product; 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, shRNA, a small guide RNA (sgRNA), a micro RNA (miRNA), or other therapeutic genes, etc. The protein can be green fluorescent protein (GFP), luciferase (Luciferase), D subtype surfactant protein (SFTPD), B subtype surfactant protein (SFTPB), A1 subtype surfactant protein (SFTPA1), interferon gamma (IFN-γ), cystic fibrosis transmembrane regulator (CFTR), aspartic protease (Napsin A), transcription factors that regulate downstream gene expression, signal peptides or cell penetrating peptides, therapeutic antibodies or antibody fragments, etc.

[0108] For example, in one embodiment of the present application, a genomic plasmid pAAV-CAG-EGFP expressing a green fluorescent protein EGFP is used, and its nucleotide sequence is SEQ ID NO.19.

[0109] SEQ ID NO.19:

[0110]

[0111] In a specific embodiment of the present application, the adeno-associated virus vector system also includes a helper virus plasmid or a helper virus, and the helper virus refers to a virus that enables AAV to be replicated and packaged by mammalian cells, such as adenovirus, herpes virus and poxvirus (such as cowpox), etc. More specifically, it can be a pHelper plasmid.

[0112] In a specific embodiment of the present application, the fusion adeno-associated virus vector system also includes a host cell.

[0113] In a specific embodiment of the present application, the packaging plasmid, expression plasmid, and helper virus plasmid are transferred into a host cell, and the nucleic acid sequences therein are all integrated into the host cell to produce the fusion-type adeno-associated virus. In certain embodiments, the nucleic acid sequences are all integrated together at a single locus in the host cell genome. In other embodiments, the nucleic acid sequences encoding various genes are present as separate expression cassettes, which prevent any risk of recombination to form a virus capable of replication; the nucleic acid sequences encoding the rep and cap genes are present in the same expression cassette.

[0114] The present application also provides a fusion adeno-associated virus AAV-WM14, which is obtained by viral packaging of the fusion adeno-associated virus vector system as described above.

[0115] Virus packaging refers to the process of integrating genetic material (DNA or RNA) into virus particles to form infectious virus particles. It usually includes the following steps: plasmid construction, preparation of plasmid DNA containing viral genome and exogenous genes (such as therapeutic genes or reporter genes); transfection, transfection of plasmid DNA into host cells through physical, chemical or biological methods (such as liposome-mediated, electroporation or virus-mediated); viral replication, in which the viral genome is replicated in the host cell to form a large number of viral DNA or RNA copies; viral protein synthesis, in which the host cell's transcription and translation machinery is used to synthesize viral capsid proteins and other structural proteins; viral genome packaging, in which the newly synthesized viral genome is selectively packaged into the viral capsid to form Virus particles; virus assembly, virus particles are assembled in host cells, including the assembly of capsid proteins and the packaging of viral genomes; virus release, the assembled virus particles are released from host cells by cell lysis or budding; virus purification, virus particles are collected from host cell culture fluid and purified by centrifugation, filtration and chromatography; virus titration and quality control, the purified virus particles are titrated to determine the concentration and infectivity of the virus, and quality control is performed; virus storage, the purified virus particles are stored under appropriate conditions (such as -80°C) to maintain their stability and activity.

[0116] The present application also provides a drug or a conjugate, wherein the drug or the conjugate comprises the above-mentioned fusion adeno-associated virus AAV-WM14.

[0117] In a specific embodiment of the present application, the drug contains an effective amount of adeno-associated virus AAV-WM14. An effective amount refers to a drug dose that can produce the expected therapeutic effect. The drug also includes a pharmaceutically acceptable excipient or carrier. 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.), adhesives, etc. In addition, it may also contain other low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine and lysine; sugars or carbohydrates such as polysaccharides and monosaccharides; sugar alcohols such as mannitol or sorbitol. When preparing an aqueous solution for injection, such as physiological saline, isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, appropriate solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), non-ionic surfactants (Tween 80, HCO-50), etc. can be used in combination.

[0118] In a specific embodiment of the present application, in the drug, the fusion adeno-associated virus AAV-WM14 can be a single active ingredient, or it can be combined with one or more other active ingredients useful for the treatment of hereditary eye diseases or deafness diseases to form a combined preparation. Other active ingredients can be various other drugs that can be used to treat hereditary eye diseases or deafness diseases. The content of each component in the combined preparation is usually a safe and effective amount, which can be adjusted based on actual usage. The dosage of the drug containing the fusion adeno-associated virus AAV-WM14 and other active ingredients can generally depend on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the drug containing the fusion adeno-associated virus AAV-WM14 and other active ingredients can 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 more than 500 mg / kg / day.

[0119] In a specific embodiment of the present application, the conjugate comprises an effective amount of a fusion adeno-associated virus AAV-WM14, wherein the fusion adeno-associated virus AAV-WM14 is connected to a biologically active polypeptide. The biologically active polypeptide can 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, or an immunomodulatory peptide.

[0120] The drug or conjugate can be used alone or in combination with other active ingredients useful for treating visual system diseases or auditory system diseases, and the combination can be administered simultaneously in the same preparation or in two different preparations via the same or different routes, or administered sequentially via the same or different routes. Sequential administration means that there is a time difference of seconds, minutes, hours or days between the administration of two or more different compounds.

[0121] The subject of administration of the drug or conjugate is preferably a mammal, such as but not limited to humans, primates, livestock (such as sheep, cattle, horses, donkeys, pigs), pets (such as dogs, cats), laboratory test animals (such as mice, rabbits, rats, guinea pigs, hamsters) or captured wild animals (such as foxes, deer). Preferably, the subject is a primate. More preferably, the subject is a human.

[0122] The present application also provides the use of the above-mentioned fusion adeno-associated virus AAV-WM14 capsid protein, or the above-mentioned nucleic acid, or the above-mentioned construct, or the above-mentioned host cell, or the above-mentioned fusion adeno-associated virus AAV-WM14, or the above-mentioned cell, or the above-mentioned fusion adeno-associated virus vector system, or the above-mentioned drug or conjugate in the preparation of gene therapy drugs.

[0123] Gene therapy refers to the introduction of exogenous genes into target cells to correct or compensate for diseases caused by defective and abnormal genes to achieve the purpose of treatment; it also includes inserting exogenous genes into appropriate recipient cells of patients through gene transfer technology so that the products produced by exogenous genes can treat certain diseases. The gene therapy drugs may include gene therapy drugs for hereditary eye diseases, deafness diseases, other hereditary diseases, malignant tumors, cardiovascular diseases, infectious diseases, autoimmune diseases, neurodegenerative diseases, blood diseases, lung diseases, inflammation or metabolic diseases.

[0124] Among them, hereditary eye diseases include diseases related to retinal photoreceptor cells, including inherited retinal diseases (IRDs), including but not limited to retinitis pigmentosa (RP) (also known as rod dystrophy), cone / rod dystrophy (CD / CRD), Leber congenital amaurosis (LCA), macular dystrophy (MD) and achromatopsia (rod monochromacy), etc.

[0125] Deafness diseases include, but are not limited to, deafness diseases related to the inner hair cells or supporting cells of the cochlea, and may also include autosomal dominant hereditary deafness, autosomal recessive hereditary deafness, X-linked hereditary deafness, syndromic hearing impairment (SHL), non-syndromic hearing impairment (NSHL), auditory neuropathy, deafness caused by OTOF gene mutation, etc.

[0126] Other genetic 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), choroideremia (CHM), hereditary deafness, etc.

[0127] Malignant tumors include, but are not limited to, lymphomas, hematological tumors or solid tumors; specifically selected from adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, lymphoid tumors, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic cancer, endometrial carcinoma, uterine sarcoma, uveal melanoma, multiple myeloma, acute lymphoid leukemia, chronic lymphoid leukemia, chronic myeloid leukemia, T-cell lymphoma, B-cell lymphoma tumors, etc.

[0128] Cardiovascular diseases include but are not limited to heart failure, myocardial infarction, cardiomyopathy, hereditary arrhythmias, atherosclerosis, hypertension, calcium ion / calmodulin protein kinase IIδ (CaMKIIδ)-related heart disease, familial hypercholesterolemia, etc.

[0129] Infectious diseases include but are not limited to HIV / AIDS, hepatitis B, human papillomavirus (HPV) infection, herpes simplex virus (HSV) infection, influenza virus infection, herpes simplex virus infection, varicella virus infection, measles virus infection, etc.

[0130] 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 mellitus (T1DM), autoimmune liver disease, anti-synthetase antibody syndrome, systemic sclerosis (SSc), Sjögren's syndrome, etc.

[0131] 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, Friedreich's ataxia, etc.

[0132] Blood diseases 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, etc.

[0133] Pulmonary diseases include but are not limited to cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF), lung cancer, lung infection, alpha-1 antitrypsin deficiency, primary ciliary dyskinesia (PCD), asthma, chronic obstructive pulmonary disease (COPD), surfactant protein deficiency, pulmonary alveolar proteinosis (PAP), etc.

[0134] Inflammation includes, but is not limited to, skin inflammation, vascular inflammation, allergic reaction, autoimmune disease, fibrosis, scleroderma or transplant rejection; the autoimmune disease is selected from rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, Sjögren's syndrome, polymyositis, etc.

[0135] Metabolic diseases include but are not limited to type I diabetes, type II diabetes, phenylketonuria, glycogen storage disease, mucopolysaccharidosis (MPS), Wilson disease, Crigler-Najjar syndrome, transthyretin amyloidosis (ATTR), inherited metabolic diseases of the liver, glutaric acidemia type I (GA-I), neuropathic Gaucher disease (nGD), etc.

[0136] In a specific embodiment of the present application, when the gene therapy drug is a gene therapy drug for a genetic eye disease, the drug, under the condition of vitreous injection, mediates the expression of the target gene specifically in the retina and infects the retinal photoreceptor cells, and delivers the target product to the retinal photoreceptor cells to achieve the treatment of the genetic eye disease; when the gene therapy drug is a gene therapy drug for a deafness disease, the drug can infect the inner hair cells and supporting cells of the cochlea, and deliver the target product to the inner hair cells and supporting cells of the cochlea to achieve the treatment of the deafness disease.

[0137] The present application also provides the use of the above-mentioned fusion adeno-associated virus AAV-WM14 capsid protein, or the above-mentioned nucleic acid, or the above-mentioned construct, or the above-mentioned host cell, or the above-mentioned fusion adeno-associated virus AAV-WM14, or the above-mentioned cell, or the above-mentioned fusion adeno-associated virus vector system, or the above-mentioned drug or conjugate for delivering the target product to the retinal photoreceptor cells of an individual. The delivery of the target product can be for non-diagnostic and therapeutic purposes, for example, it can be in vitro, and the target product is delivered to the isolated retinal photoreceptor cells.

[0138] The present application also provides the use of the above-mentioned fusion adeno-associated virus AAV-WM14 capsid protein, or the above-mentioned nucleic acid, or the above-mentioned construct, or the above-mentioned host cell, or the above-mentioned fusion adeno-associated virus AAV-WM14, or the above-mentioned cell, or the above-mentioned fusion adeno-associated virus vector system, or the above-mentioned drug or conjugate for delivering the target product to the hair cells and / or supporting cells of an individual. The target product delivery can be for non-diagnostic and therapeutic purposes, for example, it can be in vitro, and the target product is delivered to isolated hair cells and / or supporting cells. The hair cells generally include outer hair cells and / or inner hair cells.

[0139] The target product can be a nucleic acid or a protein. The nucleic acid can be an RNA interference molecule (RNAi), and the protein can be a green fluorescent protein (GFP), etc.

[0140] The present application also provides a gene therapy method, comprising administering an effective amount of the above-mentioned fusion adeno-associated virus AAV-WM14, or the above-mentioned host cell, or the above-mentioned fusion adeno-associated virus vector system, or the above-mentioned drug or conjugate to a subject, and adjusting the actual dose according to the age, weight and response of the subject. The fusion adeno-associated virus AAV-WM14 is used as a vector for gene therapy.

[0141] The present application also provides a method for treating hereditary eye diseases, comprising administering an effective amount of the above-mentioned fusion adeno-associated virus AAV-WM14, or the above-mentioned host cell, or the above-mentioned fusion adeno-associated virus vector system, or the above-mentioned drug or conjugate to a subject, and adjusting the actual dose according to the age, weight and response of the subject. The fusion adeno-associated virus AAV-WM14 is injected into the vitreous cavity and infects the photoreceptor cells of the retina, thereby playing a therapeutic role.

[0142] The present application also provides a method for treating deafness, comprising administering an effective amount of the above-mentioned fusion adeno-associated virus AAV-WM14, or the above-mentioned host cell, or the above-mentioned fusion adeno-associated virus vector system, or the above-mentioned drug or conjugate to a subject, and adjusting the actual dose according to the age, weight and response of the subject. The fusion adeno-associated virus AAV-WM14 infects the inner hair cells and / or supporting cells of the cochlea, thereby playing a therapeutic role.

[0143] The administration method in the above-mentioned treatment method can be conventional according to the actual use requirements, such as injection, oral administration, spraying, dripping and other suitable administration methods. The above-mentioned treatment method can be used alone or in combination with other treatment methods or therapeutic components. Combination refers to simultaneous administration in the same preparation or in two different preparations via the same or different routes, or sequential administration via the same or different routes. Sequential administration means that there is a time difference of seconds, minutes, hours or days between the administration of two or more different compounds.

[0144] The subject of the above treatment method is preferably a mammal, such as but not limited to humans, primates, livestock (such as sheep, cattle, horses, donkeys, pigs), pets (such as dogs, cats), laboratory test animals (such as mice, rabbits, rats, guinea pigs, hamsters) or captured wild animals (such as foxes, deer). Preferably, the subject is a primate. More preferably, the subject is a human.

[0145] The fusion adeno-associated virus, drug, conjugate, or treatment method described in the present application can be used to prevent visual system diseases and can be administered as a preventive treatment method before vision loss or after a period of time after exposure to an environment that is likely to cause vision loss.

[0146] The fusion adeno-associated virus, drug, conjugate, or treatment method described in the present application can be used to prevent auditory system diseases and can be administered as a preventive treatment method before hearing loss or after a period of time after exposure to an environment that is likely to cause hearing loss.

[0147] The fusion adeno-associated virus of the present application is used to infect cells, thereby delivering genes and / or linked (for example, but not limited to, covalently linked) biologically active polypeptides to cells. Therefore, the present invention can also provide a method for delivering a transgene to a cell, the method comprising delivering the above-mentioned fusion adeno-associated virus, or the above-mentioned drug or conjugate to a cell to infect the cell, wherein the fusion adeno-associated virus, or the drug or conjugate comprises one or more transgenes.

[0148] The present application also provides a method for producing a stable fusion adeno-associated virus vector production cell line, which comprises:

[0149] (a) introducing the fusion adeno-associated virus vector of the present application into a culture of mammalian host cells;

[0150] (b) selecting mammalian host cells in the culture medium that have integrated into their endogenous chromosomes a nucleic acid sequence encoding a fusion-type adeno-associated viral vector, i.e., producer cells that produce a stable fusion-type adeno-associated viral vector.

[0151] The fusion adeno-associated virus vector includes AAV packaging genes (rep and cap genes), required helper virus genes and a gene encoding a target product, for example, a target transgene flanked by two AAV inverted terminal repeats (ITRs).

[0152] The production cell of the fusion adeno-associated virus vector can 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.

[0153] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as those familiar to one skilled in the art.

[0154] The term "vector" refers to a macromolecule or combination of macromolecules that contains or is associated with a polypeptide and 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.

[0155] The term "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or its derivatives.

[0156] The term "recombinant AAV vector" refers to an AAV vector containing a heterologous polynucleotide sequence, usually a sequence of interest for genetic transformation of cells. Generally, the heterologous polynucleotide is flanked by at least one, usually two, AAV inverted terminal repeats (ITRs).

[0157] 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 packaged polynucleotide.

[0158] The term "packaging" refers to the series of intracellular processes that lead to the assembly and encapsulation of AAV particles.

[0159] The terms AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and packaging proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes".

[0160] The term "helper virus" of AAV refers to a virus that enables AAV to be replicated and packaged by mammalian cells. Various such AAV helper viruses are known in the art, including adenovirus, herpes virus, and pox virus (eg, vaccinia).

[0161] The term "infectious" virus or viral particle is one that contains a polynucleotide component capable of delivering the virus to cells tropistic for that virus species. The term does not necessarily imply that the virus has any ability to replicate.

[0162] The term "producer cell" refers to a cell line that has the AAV packaging genes (rep and cap genes), required helper viral genes, and the DNA genome of the recombinant AAV vector (e.g., the transgene of interest flanked by two AAV inverted terminal repeats (ITRs)) stably integrated into the host cell genome.

[0163] The terms "include", "including", etc. should be understood as having an inclusive meaning rather than an exclusive or exhaustive meaning; that is, the meaning is "including but not limited to".

[0164] The term "subject" generally includes humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cows, etc., who can benefit from treatment using the formulation, kit or combination formulation.

[0165] The term "therapeutically effective amount" generally refers to an amount that can achieve the effect of treating the diseases listed above after an appropriate administration period.

[0166] 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 full recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not eventually contract a disease condition. Thus, treatment and prevention include alleviating the symptoms of a particular condition or preventing or reducing the risk of a particular condition developing. The term "prevention" may be understood as reducing the severity of an attack of a particular condition. Treatment may also reduce the severity of an existing condition or the frequency of acute attacks.

[0167] The present application is further described below by way of examples, but the scope of the present application is not limited thereby. Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. These techniques have been fully described in the existing literature. The instruments and reagents used in the examples, unless otherwise specified, can be obtained by conventional means.

[0168] Example 1 Obtaining a novel adeno-associated virus AAV-WM14

[0169] (1) Construction of DNA family shuffling library

[0170] The serotype of AAV is determined by the sequence of the Cap protein in the Rep-Cap plasmid required for AAV packaging. Therefore, AAVs packaged with different Cap sequences have different infection tropisms. We modified the existing natural serotypes and used the capsid sequences of 13 AAV serotypes (AAV1-13) found in humans and non-human primates as parents for DNA family shuffling. The parental capsid sequences were mixed in an equimolar ratio for a total of 4 μg for DNA family shuffling. The complete parental capsid sequences were randomly interrupted into fragments of different lengths by treating them with 0.04 U of DNaseI at 25 ℃ for 30 s. DNA fragments of 100-500 bp in size were recovered and purified, and 500 ng were used for primer-free PCR to extend them into complete chimeric capsid sequences. After amplifying enough chimeric capsid sequences, the chimeric capsid sequences were recombined into a library vector containing the AAV2 Rep gene and terminal repeat sequences. The recombinant product was transformed into competent cells by electroporation, and part of the bacterial solution was plated. The colonies grown on the plate were counted and identified by sequencing to determine their diversity. The remaining bacterial solution was inoculated into 500 mL of culture medium and cultured at 37°C overnight. The plasmid extracted the next day was the DNA family shuffling library plasmid. The in vivo screening process of the DNA family shuffling library is as follows: Figure 1 As shown, first the plasmid library is constructed, followed by library virus packaging, and then three rounds of screening are carried out in the mouse eye to recover the retinal tissue. The viral DNA of the retinal tissue is recovered in the third round of screening and NGS sequencing is performed. Through analysis of the sequencing results, the sequences with the highest read rankings are selected as the preferred verification sequences.

[0171] The serotype of AAV is determined by the sequence of the Cap protein in the Rep-Cap plasmid required for AAV packaging. Therefore, AAVs produced by packaging with different Cap sequences have different infection tropisms. The existing 13 natural serotype capsid sequences were used as parents for DNA family shuffling, and the obtained DNA family shuffling library plasmid and adenovirus helper plasmid pHelper were co-transfected into HEK-293T cells in appropriate amounts, pHelper plasmid (the full sequence of the plasmid is the same as that of AAV-ie patent document CN110437317A as shown in SEQ ID NO. 12). Each 15 cm dish of cells was transfected with only 20 ng of the DNA family shuffling library plasmid. The culture medium was collected 72 hours after transfection, and the cells and culture medium were collected 48 hours later. AAV was released by lysing cells with 110 mM citrate buffer (pH 4.2), and the supernatant containing the virus was neutralized with 1 / 5 volume of 2M HEPES after centrifugation, and then polyethylene glycol 8000 and 500 mM sodium chloride were added at a final concentration of 8%, and the virus was precipitated overnight at 4°C. After centrifugation, the pellet was resuspended in PBS containing 2 mM Mg2+, and benzonase was added at a final concentration of 100 U / mL to digest the nucleic acid at 37°C for at least 1 hour. The virus suspension was purified by ultracentrifugation using iodixanol density gradient solution (15%, 25%, 40% and 60%), and then the virus titer was determined by qPCR using primers specific for the WPRE gene of AAV2 (WPRE-F: GTCAGGCAACGTGGCGTGGTGTG (SEQ ID NO.17); WPRE-R: GGCGATGAGTTCCGCCGTGGC (SEQ ID NO.18)). After three rounds of in vivo animal screening and NGS sequencing, the Cap sequence with the highest sequencing reads was obtained and named WM14-Cap, with an amino acid sequence of SEQ ID NO.1 and a nucleotide sequence of SEQ ID NO.2.

[0172] Wild-type AAVs have a high degree of homology, so the amino acid sequences of different serotypes of AAVs are highly similar. Therefore, the Cap amino acid sequence of AAV-WM14 was compared with the amino acid sequences of the 13 parental serotypes of DNA shuffling. The results are as follows: Figure 3 shown.

[0173] (2) Preparation and purification of rAAV-WM14 virus

[0174] The gene sequence encoded by WM14-Cap obtained in the above sequencing results was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and the synthetic sequence was constructed on the AAV2-Rep backbone to obtain the Rep-Cap plasmid of WM14, namely RC-WM14. Figure 2As shown, the sequence was identified as correct. The obtained plasmid RC-WM14 was co-transfected with a genomic plasmid pAAV-CAG-EGFP expressing a green fluorescent protein EGFP, whose nucleotide sequence is SEQ ID NO.19, and the pHelper plasmid was co-transfected into HEK-293T cells in an appropriate amount, and the AAV virus was purified by iodixanol gradient ultracentrifugation. The final product was rAAV-WM14, i.e. AAV-WM14-CAG-EGFP. Then, the WPRE gene-specific primers of AAV2 (SEQ ID NO.17 and SEQ ID NO.18) were used to determine the virus titer by qPCR to be 2E+13 GC / mL, and placed at -80 °C for use.

[0175] Example 2 In vivo verification of the novel adeno-associated virus rAAV-WM14 in the visual system

[0176] Place an anesthetized adult mouse under a stereo microscope, drip 1-2 drops of tropicamide eye drops on the eyeball to dilate the pupil, and apply ofloxacin eye ointment. Hold the eyeball from the optic disc with elbow forceps in the left hand and fix it. Hold a 1mL syringe or glass needle in the right hand to puncture the cornea to reduce intraocular pressure. Gently wipe away the periocular fluid with a tissue. Hold the eyeball with forceps in the left hand, hold the glass needle with 1.1 μL of virus in the right hand, insert the needle at 50° between the posterior edge of the cornea and the iris plane, and slowly inject rAAV-WM14 with a titer of 1E13 gc / mL. After the injection, the glass needle stays for 30 s and then slowly withdraws. After applying ofloxacin eye ointment on the wound, the mouse is placed in a mouse cage in a 41 ℃ water bath to keep warm. After the mouse wakes up, it is moved to the mouse room for breeding. After 3 weeks, the eyeball tissue is taken. The mouse is killed by spinal dislocation, and the cornea is punctured with a 1 mL syringe after the eyeball is removed to release the aqueous humor. The eyeball was placed in a small culture dish filled with PBS solution and dissected under a microscope. The cornea was clamped with pointed forceps, and ophthalmic scissors were inserted from the opening of the cornea to cut the cornea along the circumference. The lens was removed with forceps, and the optic nerve was retained for 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 sectioning, the sections with good effect and complete tissue were selected for staining under a fluorescent microscope.

[0177] Immunofluorescence results showed that rAAV-WM14 could efficiently infect photoreceptor cells (cones and rods). Figure 4 At the same time, this example also analyzed the infection of retinal cells by rAAV-WM14, AAV2, and AAV2.7m8 viruses. The results are as follows Figure 5As shown in Figure 2, AAV-WM14 efficiently targets photoreceptor cells and RPE cells, AAV2 mainly infects retinal ganglion cells, and AAV2.7m8 can infect a small amount of photoreceptor cells. The number of EGFP-positive photoreceptor cells was counted, and the results are shown in Figure 2. Figure 6 As shown, the infection efficiency of rAAV-WM14-CAG-EGFP virus on photoreceptor cells is as high as 97.7%, which is significantly higher than AAV2 and AAV2.7m8 viruses. The results show that rAAV-WM14-CAG-EGFP virus has a high infection rate on retinal photoreceptor cells and can be used for ophthalmic gene therapy.

[0178] Example 3 In vivo validation of the novel adeno-associated virus rAAV-WM14 in the auditory system

[0179] The adeno-associated virus used in the experiment was delivered into the cochlea of ​​adult mice using a glass electrode by microinjection of the posterior semicircular canal. The adeno-associated viruses selected in the experiment were AAV2 and AAV-WM14-CAG-EGFP. The specific experimental method is as follows: a. Weigh the weight of P30 mice, anesthetize the mice with an appropriate amount of sodium pentobarbital intraperitoneally, and remove the hair behind the right ear of the mice after the mice completely lose consciousness and mobility. Use depilatory cream and a shaver to remove the hair behind the right ear of the mice to expose the skin behind the ear. b. Use 75% alcohol to thoroughly disinfect the exposed skin, carefully cut the subcutaneous tissue behind the ear with Venus scissors, and then tear the subcutaneous fat tissue with surgical forceps disinfected with 75% alcohol, carefully search deep to avoid damaging the surrounding blood vessels and nerves until the posterior semicircular canal is exposed. c. Carefully tear open the muscle tissue and membrane structure on the posterior semicircular canal, and use pointed surgical forceps to chisel an opening on the posterior semicircular canal that is equivalent to the diameter of the glass tube for injection. Place the tip of the glass tube into the opening, slowly inject 1 uL of virus in the tube, and slowly remove the glass tube after one minute of rest. d. After injection, first use veterinary tissue glue to glue the opening of the posterior semicircular canal, and then use veterinary tissue glue to glue the wounds of the skin and muscles. Subsequently, place the postoperative mice on a 37°C constant temperature heating pad and return them to the animal room for breeding after they wake up naturally. Any damaged cochlea will not be used for subsequent research. 14 days after the virus injection, the cochlear tissue was collected and immunofluorescence staining was performed to analyze the virus infection.

[0180] Immunostaining results Figure 7 As shown, the results showed that rAAV-WM14-CAG-EGFP efficiently infected inner hair cells after injection into adult mice; Figure 8 As shown, rAAV-WM14-CAG-EGFP was able to infect Sertoli cells after injection into adult mice. Fig. 9The statistical results of WM14 and AAV2 infection of cochlear inner hair cells and supporting cells show that rAAV-WM14-CAG-EGFP can efficiently infect inner hair cells; it also has the ability to infect supporting cells, with an efficiency of up to 35%, which is significantly higher than AAV2. Therefore, WM14 can be used for gene therapy of deafness.

[0181] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

Claims

1. A fusion adeno-associated virus AAV-WM14 capsid protein, the fusion 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 in SEQ ID NO.

1.

2. The fusion adeno-associated virus AAV-WM14 capsid protein according to claim 1, characterized in that The capsid protein comprises the first to fourteenth fragments 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 AAV13.

3. The fusion adeno-associated virus AAV-WM14 capsid protein according to claim 2, characterized in that: The first fragment includes the amino acid sequence shown in SEQ ID NO.3, the second fragment includes the amino acid sequence shown in SEQ ID NO.4, the third fragment includes the amino acid sequence shown in SEQ ID NO.5, the fourth fragment includes the amino acid sequence shown in SEQ ID NO.6, the fifth fragment includes the amino acid sequence shown in SEQ ID NO.7, the sixth fragment includes the amino acid sequence shown in SEQ ID NO.8, the seventh fragment includes the amino acid sequence shown in SEQ ID NO.9, the eighth fragment includes the amino acid sequence shown in SEQ ID NO.10, the ninth fragment includes the amino acid sequence shown in SEQ ID NO.11, the tenth fragment includes the amino acid sequence shown in SEQ ID NO.12, the eleventh fragment includes the amino acid sequence shown in SEQ ID NO.13, the twelfth fragment includes the amino acid sequence shown in SEQ ID NO.14, the thirteenth fragment includes the amino acid sequence shown in SEQ ID NO.15, and the fourteenth fragment includes the amino acid sequence shown in SEQ ID NO.

16.

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

5. The nucleic acid according to claim 4, characterized in that The nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.

2.

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

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

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

9. The fusion adeno-associated virus AAV-WM14 according to claim 8, characterized in that: The fusion adeno-associated virus AAV-WM14 also includes a nucleotide sequence encoding a target product; the target product is a nucleic acid or a protein.

10. A cell, wherein the cell is transformed using the fusion-type adeno-associated virus AAV-WM14 according to any one of claims 8 to 9.

11. A fusion adeno-associated virus vector system, comprising a packaging plasmid, wherein the packaging plasmid comprises the nucleic acid according to any one of claims 4 to 5.

12. The fusion adeno-associated virus vector system according to claim 11, characterized in that: The fusion adeno-associated virus vector system also has any one or more of the following features: 1) The packaging plasmid also contains a rep gene fragment of an adeno-associated virus; 2) The adeno-associated virus vector system further comprises an expression plasmid, wherein the expression plasmid comprises nucleotides responsible for encoding a target product; 3) The adeno-associated virus vector system further includes a helper virus plasmid or a helper virus; 4) The fusion adeno-associated virus vector system also includes a host cell.

13. Use of the fusion adeno-associated virus AAV-WM14 capsid protein as described in any one of claims 1-3, or the nucleic acid as described in any one of claims 4-5, or the construct as described in claim 6, or the host cell as described in claim 7, or the fusion adeno-associated virus AAV-WM14 as described in any one of claims 8-9, or the cell as described in claim 10, or the fusion adeno-associated virus vector system as described in any one of claims 11-12 in the preparation of a gene therapy drug, wherein the gene therapy drug is selected from the group consisting of gene therapy drugs for hereditary retinal diseases caused by retinal photoreceptor cells, and gene therapy drugs for deafness caused by cochlear inner hair cells or supporting cells, and the hereditary retinal diseases are selected from the group consisting of retinitis pigmentosa, cone / rod dystrophy, Leber congenital amaurosis, macular dystrophy, and achromatopsia.

14. The use according to claim 13, characterized in that Under the condition of vitreous injection, the drug can mediate the target gene to be specifically expressed in the retina and infect retinal photoreceptor cells, or the drug can infect the cochlear inner hair cells or supporting cells.

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