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Definitions

• the present invention relates to the field of biomedicine, to a fusion adeno-associated virus and its application, especially to its application in the prevention and/or treatment of ear diseases and/or ophthalmic diseases.
• Gene therapy is an emerging treatment method that delivers functional genes into patients to counteract or replace dysfunctional genes, thereby curing disease without chemical intervention, radiation therapy, or surgery.
• Gene therapy introduces genetic material into target cells through viral or non-viral vectors to treat or prevent diseases by correcting or supplementing defective genes. The effects of treatment can be long-lasting and do not require repeated intervention.
• Gene therapy can be achieved through ex vivo or in vivo strategies. Ex vivo gene therapy is to collect target cells from the patient, genetically modify them and return them to the patient; in vivo gene therapy is to deliver genetic material directly to the patient's target organ or tissue. middle.
• Various types of gene delivery strategies have been used to treat a variety of diseases including cancer, blindness, immune and neuronal diseases.
• Viral vectors are the most commonly used gene therapy delivery vehicles because they are highly efficient in entering cells and introducing genetic material. Commonly used ones include adenovirus, retrovirus, lentivirus, and adeno-associated virus (AAV).
• AAV vectors have achieved good results in clinical trials of in vivo gene therapy, and retroviral and lentiviral vectors are the preferred vectors in clinical trials of in vitro gene therapy.
• the main factors that contribute to the success of AAV as a therapeutic gene delivery vehicle are its low immunogenicity and lack of pathogenicity. Multiple marketed drug approvals and ongoing clinical trials have proven that AAV vectors have become one of the main gene delivery tools for gene therapy.
• Glybera was the first AAV therapy approved in Europe for the treatment of lipoprotein lipase deficiency.
• the launch of these drugs clearly demonstrates the safety and efficacy of AAV-based treatments.
• Luxturna for the treatment of Leber's congenital amaurosis and Zolgensma for the treatment of spinal muscular atrophy (SMA) were approved by the U.S. Food and Drug Administration (FDA) in 2017 and 2019 respectively.
• FDA U.S. Food and Drug Administration
• Current clinical research focuses on treating single-gene diseases through gene replacement, gene silencing, or gene editing.
• AAV is also expected to be used in the development of vaccines.
• AAV has the characteristics of low immunogenicity and low pathogenicity, and AAV-based gene therapy has also achieved amazing success, due to the pre-existing neutralizing antibodies of the natural AAV capsid protein in the human immune system, currently as many as 50% Patients were excluded from this treatment approach.
• engineered AAV capsids to evade pre-existing neutralizing antibodies or adopt methods to temporarily eliminate neutralizing antibodies before treatment.
• Engineering the AAV capsid can not only evade neutralizing antibodies, but also enable AAV to target the expected tissue cell types, achieving the purpose of precise treatment. Directed evolution is currently a powerful tool for transforming AAV capsids.
• directed evolution uses high-throughput technology to introduce mutations into genes and their encoded proteins, and simulates the greatly accelerated protein diversification and selection process of natural evolution.
• the method consists of three basic steps: building a sequence-diversified library of the target gene, screening a library of proteins encoded by the mutant gene library, and amplifying the gene sequence encoding the desired trait. These three steps constitute a round of directed evolution, and multiple rounds of evolutionary screening can be iteratively performed until specific genes/proteins with improved properties are obtained.
• adeno-associated virus vectors are known to infect supporting cells while infecting hair cells; Supporting cells and hair cells express different pathogenic genes respectively.
• the non-specificity of this vector infection has a negative impact on the treatment of the disease. Therefore, to achieve precise gene therapy, efficient and specific new recombinant adeno-associated virus vectors are needed.
• the purpose of the present invention is to overcome the shortcomings of the existing technology and propose to transform an emerging new adeno-associated virus vector that can efficiently and specifically deliver genes in tissues such as the cochlea and the retina of the eye for use in gene therapy.
• the modified novel adeno-associated virus vector in the present invention infects mouse cochlea in vivo, and can mediate the specific expression of target genes in cochlear supporting cells, thereby specifically labeling or controlling supporting cells.
• This new type of adeno-associated virus has broad application value and market prospects in the structural and functional analysis of cochlear supporting cells, establishment of disease models, and gene therapy.
• the viral vector has better infection characteristics than the current vectors and is also widely used in the analysis of the structure and function of ophthalmic cells, the establishment of disease models, and gene therapy. application value and market prospects.
• One object of the present invention is to provide a fusion adeno-associated virus capsid protein, which capsid protein includes fusion peptides of peptides of serotypes AAV1, AAV2, AAV6 and AAV7 or variants thereof.
• Another object of the present invention is to provide a nucleic acid encoding the fused adeno-related protein as described above. Viral capsid protein.
• Another object of the present invention is to provide a construct containing the nucleic acid as described above.
• Another object of the present invention is to provide a host cell, the host cell comprising the construct as described above or the exogenous nucleic acid as described above integrated into the genome, or the host cell comprising the fusion type as described above.
• Adeno-associated virus is provided.
• Another object of the present invention is to provide a fusion adeno-associated virus, the capsid structure of the fusion adeno-associated virus contains the fusion adeno-associated virus capsid protein as described in any one of the above.
• Host cells transformed using the fusogenic adeno-associated virus as described above are transformed using the fusogenic adeno-associated virus as described above.
• Another object of the present invention is to provide a fusion adeno-associated virus vector system, including a packaging plasmid containing the nucleic acid or nucleic acid fragment as described above.
• Another object of the present invention is to provide a fusion adeno-associated virus, which is obtained by virus packaging using the fusion adeno-associated virus vector system as described above.
• Another object of the present invention is to provide a pharmaceutical composition comprising the fusion adeno-associated virus as described in any one of the above and pharmaceutically acceptable excipients.
• Another object of the present invention is to provide fusion adeno-associated virus capsid proteins, nucleic acids, constructs, fusion adeno-associated viruses, host cells, fusion adeno-associated virus vector systems, pharmaceutical compositions or conjugates as described above.
• the present invention has the following advantages and beneficial effects: (1) It provides a new supporting cell-specific adeno-associated virus vector, which can mediate the expression of the target gene specifically in the cochlear supporting cells of newborn mice. ; (2) A new adeno-associated virus vector is provided, which can mediate the expression of the target gene specifically in the inner hair cells of the adult mouse cochlea; (3) A new adeno-associated virus vector is provided, which can mediate the target gene.
• the gene is specifically expressed in the RPE layer of adult mouse retina; (4)
• the new adeno-associated virus vector mediates the expression of target genes more flexibly, safer, more convenient to apply and lower in cost than traditional transgenic methods; (5 )
• New and specific adeno-associated virus vectors make it possible to develop gene therapies for the precise treatment of otological and ophthalmic related diseases.
• Figure 1 shows the in vivo screening process for DNA family shuffled libraries.
• Figure 2 shows a computer-simulated AAV-M9 structure diagram.
• Figure 3 shows the infection characteristics of adeno-associated virus in neonatal mice.
• Figure 3a shows AAV-ie and AAV- M9 was injected into the cochlea of newborn mice. 14 days later, the cochlear tissue was removed and dissected. Photos of immunostaining of green fluorescent EGFP and the hair cell-specific marker protein Myo7a. The picture selects the hair cell level.
• Figure 3b shows the immunostaining photos of green fluorescent EGFP and the hair cell-specific marker protein Myo7a after AAV-ie and AAV-M9 were injected into the cochlea of newborn mice 14 days later and the cochlear tissue was removed and dissected. The picture selects the supporting cell level.
• Figure 3c shows the statistical results of the hair cells and Dieters cells in Figures 3a and 3b.
• Figure 4 shows the safety of adeno-associated virus in neonatal mice.
• Figure 4a shows AAV-M9 injected into the cochlea of newborn mice, the cochlear tissue removed 14 days later, and the immunostaining photos of the hair cell-specific marker protein Myo7a after dissection of the normal mouse cochlea.
• Figure 4b shows the statistical results of the outer hair cells and inner hair cells in Figure 4a.
• Figure 5 shows the infection characteristics of adeno-associated virus in adult mice.
• Figure 5a shows the immunostaining photos of green fluorescent EGFP and the hair cell-specific marker protein Myo7a after AAV-ie and AAV-M9 were injected into the cochlea of adult mice 14 days later and the cochlear tissue was removed and dissected.
• Figure 5b shows the statistical results of the outer hair cells in Figure 5a.
• Figure 5c shows the statistical results of the inner hair cells in Figure 5a.
• Figure 6 shows the safety of adeno-associated virus in adult mice.
• Figure 6a shows AAV-M9 injected into the cochlea of adult mice, the cochlear tissue removed 14 days later, and the immunostaining photos of the hair cell-specific marker protein Myo7a after cochlea dissection in normal mice.
• Figure 6b shows the statistical results of the outer hair cells and inner hair cells in Figure 6a.
• Figure 7 shows the infection of the RPE layer of the eyeball by AAV-M9-CMV-EGFP.
• Figure 8 shows the ocular transfection characteristics of AAV-M9-CMV-EGFP in non-human primates.
• the invention provides a fusion adeno-associated virus capsid protein, which capsid protein includes a peptide segment (fusion peptide segment or chimeric peptide segment) formed by fusion of peptide segments of serotypes AAV1, AAV2, AAV6 and AAV7, or a peptide segment thereof. Variants.
• the fusion peptide segment in the fusion adeno-associated virus capsid protein, includes a first peptide segment, a second peptide segment, a third peptide segment, a fourth peptide segment and a third peptide segment connected in sequence.
• the first peptide segment includes the amino acid fragment from positions 1 to 262 of SEQ ID No: 2 (SEQ ID NO: 3), and the second peptide segment includes the amino acid fragment from positions 263 to 262 of SEQ ID No: 2.
• the peptide segments are directly fused to each other.
• the peptide segments of the serotypes AAV1 and AAV6 are assembled into the triple symmetrical axon of the capsid protein; the peptide segments of the serotypes AAV2, AAV6, and AAV7 are assembled
• the peptide segment of the serotype AAV6 forms a recess at the two-fold symmetry axis of the capsid protein.
• a schematic diagram of its computer simulation structure is shown in Figure 2.
• the nucleocapsid of adeno-associated virus is generally nearly round.
• This nearly circular capsid is actually a closed icosahedral symmetrical hollow capsid composed of multiple protein capsomeres arranged in a manner, in which the genomic nucleic acid is wrapped.
• An icosahedral symmetry structure contains three rotational symmetry modes: 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 opposite sides of the virus particle.
• the capsomere rotates 120° three times around the 3-fold symmetry axis to form a triangular surface; it has a 2-fold symmetry axis (edge), and the capsomere
• the shell granule rotates 180° around the 2-fold symmetry axis and resets twice, forming two intersecting triangular surfaces; there is also a 5-fold symmetry axis passing through two opposite vertices, and the shell granule rotates five times around the 5-fold symmetry axis 72° and resets five times. , forming a pentamer.
• the icosahedral symmetry capsid is composed of 20 equilateral triangular faces, of which every 2 triangular faces intersect to form an edge, with a total of 30 edges; every 5 triangular faces meet to form 12 vertices.
• the capsid protein includes:
• the polypeptide fragment in b) specifically refers to: the amino acid sequence shown in SEQ ID No: 2 through substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30, 1-20 , 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more (specifically, can be added to the N-terminal and/or C-terminal is obtained from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has an amino acid sequence
• the amino acid sequence in b) can be 90%, 91%, 92%, 93%, 94%, 95% similar to SEQ ID No: 2 , 96%, 97%, 98% or above sequence identity.
• the nucleic acid sequence of the gene encoding the capsid protein includes the nucleotide sequence shown in SEQ ID No: 1.
• the nucleic acid sequence of the gene encoding the capsid protein is such as SEQ ID No.1 is shown.
• the nucleic acid sequence of the gene encoding the capsid protein includes 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Nucleotide sequences with 98% or greater sequence identity.
• the present invention also provides a nucleic acid encoding the fused adeno-associated virus capsid protein as described above.
• the present invention also provides a construct containing the above-mentioned nucleic acid.
• the construct can usually be constructed by inserting the above nucleic acid into a suitable expression vector, and those skilled in the art can select a suitable expression vector.
• the present invention also provides a host cell, which contains the above-mentioned construct or the above-mentioned exogenous nucleic acid integrated into the genome, or the host cell contains the fusion adeno-associated virus as described in any one of the above.
• suitable host cells include mammalian cells (such as CHO or COS), plant cells, human cells (human embryonic kidney cells such as HEK293FT), bacterial cells (such as Escherichia coli, Streptomyces spp., Salmonella typhimurium) , fungal cells (such as yeast), insect cells (such as Sf9), etc.
• the host cell is an animal cell, and more preferably a human cell.
• Host cells can be cultured cells or primary cells, ie, isolated directly from an organism (eg, human). The host cells may be adherent cells or suspended cells, ie, cells grown in suspension.
• the present invention also provides a fused adeno-associated virus, which contains the fused adeno-associated virus capsid protein as described in any one of the above.
• the fusion adeno-associated virus also includes a heterologous nucleotide sequence encoding the target product, and the heterologous nucleotide sequence encoding the target product can be packaged and carried by various capsid proteins.
• the above-mentioned heterologous nucleotide sequence encoding the product of interest can generally be a construct, and the construct can generally contain a nucleic acid encoding the product of interest.
• the construct can usually be 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.
• the above-mentioned expression vector can include but is not limited to pAAV-CAG, pAAV- TRE, pAAV-EF1a, pAAV-GFAP promoter, pAAV-Lgr5 promoter, pAAV-Sox2 promoter expression vector, etc.
• the fusion adeno-associated virus when the fusion adeno-associated virus encodes a heterologous nucleotide sequence of a target product, the fusion adeno-associated virus contains a capsid, and the viral vector carries a transgene encoding a gene product, and the transgene Under the control of regulatory sequences that direct its expression in host cells; in some preferred embodiments, the amino acid sequence of the capsid protein is as shown in SEQ ID NO: 2.
• the target product may be a nucleic acid or a protein
• the nucleic acid includes but is not limited to small guide RNA (sgRNA), interfering RNA (RNAi), etc.
• the protein coding gene includes but is not limited to Prestin and Atoh1.
• the fusion adeno-associated virus can be used as a carrier material to introduce exogenous genes into the cells of test individuals.
• the fusion adeno-associated virus can specifically infect the cells of young individuals. Sertoli cells, which specifically infect the inner hair cells of adult individuals.
• the present invention also provides an engineered host cell transformed using the fusion adeno-associated virus as described above.
• the engineered host cell contains the fusion adeno-associated virus described above.
• the host cell may be a eukaryotic cell and/or a prokaryotic cell.
• suitable host cells include mammalian cells (such as CHO or COS), plant cells, human cells (human embryonic kidney cells such as HEK293FT), bacterial cells (such as Escherichia coli, Streptomyces spp., Salmonella typhimurium) , fungal cells (such as yeast), insect cells (such as Sf9), etc.
• the host cell is an animal cell, and more preferably a human cell.
• Host cells can be cultured cells or primary cells, ie, isolated directly from an organism (eg, human). The host cells may be adherent cells or suspended cells, ie, cells grown in suspension.
• the present invention also provides a fusion adeno-associated virus vector system, which vector system includes a packaging medium
• the packaging plasmid contains the nucleic acid or nucleic acid fragment as described above.
• the packaging plasmid also contains a rep gene fragment of adeno-associated virus.
• the rep gene fragment contains an intron
• the intron contains a transcription termination sequence.
• the adeno-associated virus vector system also includes an expression plasmid, which contains heterologous nucleotides responsible for encoding the target product.
• the target product may be a nucleic acid or a protein, the nucleic acid includes but is not limited to small guide RNA (sgRNA), interfering RNA (RNAi), etc., and the protein coding gene includes but is not limited to Prestin and Atoh1.
• the adeno-associated virus vector system also includes a helper virus plasmid or a helper virus. Further, the adeno-associated virus vector system also includes host cells.
• the packaging plasmid, expression plasmid, and helper virus plasmid are transferred into host cells, and all nucleic acid sequences therein are integrated into the host cells to produce the fusion adeno-associated virus.
• the nucleic acid sequences are all integrated together at a single locus within the cellular genome of the host cell.
• the nucleic acid sequences encoding the various genes are present as separate expression cassettes, which prevents 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.
• the present invention also provides a fusion adeno-associated virus, which is obtained by virus packaging using the fusion adeno-associated virus vector system as described above.
• the present invention also provides a pharmaceutical composition, which comprises the fusion adeno-associated virus as described above and pharmaceutically acceptable auxiliary materials.
• the fusion adeno-associated virus or pharmaceutical composition provided by the present invention can be adapted to the appropriate administration method, and can be injected into the cochlea, eyes, muscles, nervous system or blood circulation system. Those skilled in the art can select an appropriate dosage according to the mode of administration.
• the excipients may include various excipients and diluents, which are not necessary active ingredients themselves and do not have excessive toxicity after administration. Suitable excipients will be well known to those skilled in the art.
• the acceptable carriers include sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, and surfactants. (PEG, Tween, etc.), chelating agents (EDTA, etc.), adhesives, etc.
• polypeptides such as serum albumin, gelatin or immunoglobulin; amino acids such as glycine, glutamine, asparagine, arginine and lysine; sugars such as polysaccharides and monosaccharides or Carbohydrates; sugar alcohols such as mannitol or sorbitol.
• aqueous solutions for injection such as physiological saline
• isotonic solutions containing glucose or other auxiliary drugs such as D-sorbitol, D-mannose, D-mannitol, sodium chloride
• solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), nonionic surfactants (Tween 80, HCO-50), etc. can be used in combination.
• the fusion adeno-associated virus AAV-M9 can be a single active ingredient, or can be combined with one or more other active ingredients useful for the treatment of hearing loss or ophthalmic diseases, constitute a combined preparation.
• the other active ingredients can be various other drugs that can be used for the treatment of hearing impairment or the treatment of eye diseases.
• the content of active components in the composition is usually a safe and effective amount, which should be adjustable to those skilled in the art, for example, the activity of the fusion adeno-associated virus AAV-M9 and the pharmaceutical composition
• the dosage of the ingredients usually depends on the patient's weight, the type of application, the condition and severity of the disease.
• the dosage of the bifunctional compound as an active ingredient can usually be 1 to 1000 mg/kg/day, 1 to 3 mg /kg/day, 3 ⁇ 5mg/kg/day, 5 ⁇ 10mg/kg/day, 10 ⁇ 20mg/kg/day, 20 ⁇ 30mg/kg/day, 30 ⁇ 40mg/kg/day, 40 ⁇ 60mg/kg /day, 60 ⁇ 80mg/kg/day, 80 ⁇ 100mg/kg/day, 100 ⁇ 200mg/kg/day, 200 ⁇ 500mg/kg/day, or more than 500mg/kg/day.
• the present invention also provides a conjugate, which includes the fused adeno-associated virus AAV-M9 or a linked biologically active polypeptide as described above.
• the invention also provides the above-mentioned fused adeno-associated virus AAV-M9 capsid protein, nucleic acid, construct, fused adeno-associated virus AAV-M9, host cells, fused adeno-associated virus vector system, pharmaceutical composition or conjugate
• diseases include but are not limited to hearing impairment, ophthalmic diseases, inflammation, One or more of tumors, metabolic diseases, pain, neurodegenerative inflammatory diseases, etc.
• the hearing impairment disease is selected from the group consisting of hearing loss, deafness, and tinnitus.
• Indicated ophthalmic diseases include, but are not limited to, dry AMD, wet AMD, or choroidal neovascularization (CNV); for example, may be selected from age-related macular degeneration (AMD), choroidal neovascularization (CNV), choroidal neovascular membrane (CNVM), Cystoid macular edema (CME), epiretinal membrane (ERM), and macular holes; myopia-related choroidal neovascularization, vascular streaks, retinal detachment, diabetic retinopathy, diabetic macular edema (DME), retinal pigment epithelium (RPE) ), hypertrophic lesions of the retinal pigment epithelium (RPE), retinal vein occlusion, chorioretinal vein occlusion, macular edema; corneal angiogenesis due to hypoxia, pterygium conjunctiva, subretinal edema and intraretinal edema E
• the inflammation is selected from cutaneous inflammation, vascular inflammation, allergy, autoimmune disease, fibrosis, scleroderma or graft rejection; the autoimmune disease is selected from rheumatoid arthritis, systemic sclerosis, systemic One or more of lupus erythematosus, Sjogren's syndrome, polymyositis, etc.
• the cancer is selected from lymphoma, hematoma or solid tumor; specifically selected from adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, intracervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid Neoplasms, 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 brain glioma, 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,
• the metabolic disease is selected from diabetes, including type I and type II diabetes, and diseases and conditions related to diabetes; the metabolic disease includes but is not limited to atherosclerosis, cardiovascular disease, nephropathy, neuropathy, retinopathy, beta - One or more of cell dysfunction, dyslipidemia, hyperglycemia, insulin resistance, chronic obstructive pulmonary disease, etc.
• the gene therapy refers to the treatment of hearing impairment.
• the fusion adeno-associated virus or pharmaceutical composition can achieve the treatment of hearing impairment by delivering the target product to the individual's hair cells and/or supporting cells.
• the fused adeno-associated virus AAV-M9 host cells, vector systems, pharmaceutical compositions or conjugates of the present invention for delivering a product of interest to hair cells and/or supporting cells of an individual
• the delivery of the product of interest may be for non-diagnostic therapeutic purposes, for example, may be in vitro, to isolated hair cells and/or supporting cells.
• the hair cells usually include outer hair cells and/or inner hair cells; preferably, the hair cells are inner hair cells of adult individuals.
• the target product is nucleic acid or protein
• the nucleic acid can be small guide RNA (sgRNA), interfering RNA (RNAi), etc.
• the hearing impairment may be caused by cochlear damage caused by environmental factors. Therefore, the present invention also provides the use of the above-mentioned fusion adeno-associated virus in medicine for treating hearing impairment caused by environmental factors in individuals.
• the hearing impairment disease is a hair cell and/or supporting cell related disease.
• the hearing impairment disease is preferably a Sertoli cell-related disease.
• the hearing impairment disease is preferably an inner hair cell related disease.
• the hearing impairment is prevented or treated by inducing inner hair cell regeneration.
• the hearing impairment disease is prevented or treated by overexpressing Wnt2b and Atoh1 to induce inner hair cell regeneration.
• the hearing impairment disease is a disease related to genetic defects, environmental damage or aging.
• it can be a related disease caused by a gene mutation.
• it can be a related disease caused by noise or drugs.
• Another example could be related diseases caused by aging.
• the hearing impairment disease can be a disease related to cell damage, etc. Specifically, it can be cochlear hair cell damage, supporting cell damage, etc., and more specifically, it can be cochlear hair cell damage caused by genetic mutation, supporting cell damage caused by genetic mutation, etc. Cell damage caused by noise, cell damage caused by drugs, or cell damage caused by aging.
• the fusion adeno-associated virus is used as a vector to deliver the target product.
• the present invention also provides a method for treating hearing impairment, which method includes administering an effective amount of the fusion adeno-associated virus of the present invention, the host cell, vector system or drug of the present invention to a subject in need. composition or conjugate.
• the actual dosage that is most appropriate for an individual patient can usually be determined by the physician and will vary based on the age, weight, and response of the particular individual.
• the fusion adeno-associated virus or host cell or vector system or pharmaceutical composition of the present invention can be administered to a patient.
• a patient Those skilled in the art will be able to determine appropriate administration methods and dosages.
• One or more therapeutic genes are delivered by the fusion adeno-associated virus of the present invention, which can be used alone or in combination with other therapeutic methods or therapeutic components.
• the fused adeno-associated viruses of the present invention are used to infect cells, thereby delivering genes and/or linked (eg, but not limited to, covalently linked) biologically active polypeptides to the cells. Accordingly, the present invention provides a method of delivering a transgene to a cell by subjecting one or more of the fusogenic adeno-associated disease genes of the invention to The virus or conjugate is injected into the cell to infect the cell, wherein the fused adeno-associated virus or conjugate contains one or more transgenes.
• the present invention also provides a method for producing a stable fusion adeno-associated virus vector production cell line, which includes:
• the AAV vector producing cells are mammalian cells.
• the mammalian cells are selected from HEK293 cells, CHO cells, Jurkat cells, K562 cells, PerC6 cells, HeLa cells or derivatives thereof.
• the mammalian host cell is, or is derived from, a HEK293 cell.
• the HEK293 cells are HEK293T cells.
• genomic sequences of the various serotypes of AAV as well as the sequences of the native ITR, Rep protein and capsid protein are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. The disclosures of which are incorporated herein by reference for AAV nucleic acid and amino acid sequences.
• the active compound when used in combination with other therapeutic agents, the active compound is co-administered with other therapeutic agents.
• Co-administered means simultaneous administration by the same or different routes in the same formulation or in two different formulations, or sequential administration by 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 fusion adeno-associated viruses and methods of the present invention can be used to prevent hearing damage, and can be administered as a preventive treatment before hearing damage or after a period of time after exposure to an environment prone to hearing damage. .
• vector refers to a macromolecule or combination of macromolecules that contains or is bound 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 vehicles.
• AAV is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or its derivatives.
• heterologous polynucleotide sequences usually for Genetically transform cells with sequences of interest.
• heterologous polynucleotides are flanked by at least one, typically two, AAV inverted terminal repeats (ITRs).
• AAV virus or "AAV virion” or “AAV vector particle” refers to a viral particle of an AAV vector containing at least one AAV capsid protein and one enveloped polynucleotide.
• packing refers to a series of intracellular processes that lead to the assembly and encapsulation of AAV particles.
• AAV rep and cap genes refer to polynucleotide sequences encoding the replication and packaging proteins of adeno-associated viruses.
• AAV rep and cap refer to AAV "packaging genes”.
• helper virus for AAV refers to a virus that enables AAV to be replicated and packaged in mammalian cells.
• a variety of such AAV helper viruses are known in the art, including adenovirus, herpesvirus, and poxvirus (eg, vaccinia).
• infectious virus or viral particle is one that contains cells that are tropic for the delivery of polynucleotide components into that viral species. The term need not imply that the virus has any ability to replicate.
• producer cell refers to a DNA genome with the AAV packaging genes (rep and cap genes), the required helper viral genes, and the recombinant AAV vector stably integrated into the host cell genome (e.g., consisting of two AAV inverted terminal repeats (ITR) cell lines flanked by the target transgene).
• AAV packaging genes rep and cap genes
• helper viral genes e.g., the required helper viral genes
• ITR inverted terminal repeats
• the term "individual” generally includes humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., who may benefit from treatment with the formulation, kit or combination. .
• terapéuticaally effective amount generally refers to an amount that, over an appropriate period of administration, is effective in treating the conditions listed above.
• treatment and prevention include alleviating the symptoms of a specific condition or preventing or reducing the risk of developing a specific condition.
• prevention is understood to mean reducing the severity of the onset of a specific condition. Treatment can also reduce the severity of existing conditions or the frequency of flare-ups.
• the subject or individual for therapeutic or preventive treatment 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).
• the subject is preferably a primate.
• the subject is most preferably a human.
• the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in this technical field. conventional technology. These techniques are well described in the existing literature.
• Example 1 Obtaining the new adeno-associated virus AAV-M9 (also known as AAV-WM01)
• the capsid sequences of 13 AAV serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13) found in humans and non-human primates were used as Parents undergo DNA family reshuffling.
• a total of 4 ⁇ g of the parental capsid sequences were mixed in an equimolar ratio for DNA family shuffling, and treated with 0.04U DNase I at 25°C for 30 seconds to randomly break the complete parental capsid sequences into fragments of different lengths.
• the obtained DNA family shuffling library plasmid and the adenovirus helper plasmid pHelper were co-transfected into HEK-293T cells in appropriate amounts.
• the pHelper plasmid (the complete sequence of the plasmid is the same as shown in SEQ ID NO: 12 of the AAV-ie patent document CN110437317A). Cells per 15 cm dish were transfected with only 20 ng of DNA family shuffled library plasmid.
• the culture medium was collected 72 hours after transfection, and the cells and culture medium were collected 48 hours later. Lyse the cells with 110mM citrate buffer (pH 4.2) to release AAV. After centrifugation, the virus-containing supernatant is neutralized with 1/5 volume of 2M HEPES, and then a final concentration of 8% polyethylene glycol 8000 and 500mM sodium chloride, overnight at 4°C to precipitate the virus. After centrifugation, resuspend the pellet in PBS containing 2mM Mg 2+ and add benzonase at a final concentration of 100U/mL to digest nucleic acids at 37°C for at least 1 hour.
• the virus suspension was purified by ultracentrifugation with iodixanol density gradient solution (15%, 25%, 40% and 60%), and then the AAV2Rep gene-specific primer (WPRE-F: GTCAGGCAACGTGGCGTGGTGTG (SEQ ID NO. 8); WPRE -R:GGCGATGAGTTCCGCCGTGGC (SEQ ID NO:9)), virus titer determined by qPCR.
• WPRE-F GTCAGGCAACGTGGCGTGGTGTG (SEQ ID NO. 8); WPRE -R:GGCGATGAGTTCCGCCGTGGC (SEQ ID NO:9)
• the present invention can recover the capsid gene of AAV infected cells from the total DNA of tissue cells.
• Total tissue DNA was extracted with Trizol, and the AAV capsid gene was recovered from the total DNA using PCR to complete an in vivo screening of the AAV library.
• the fragments obtained by PCR are cloned into the library vector to obtain the next round of library plasmids, which are packaged into viruses again.
• the in vivo screening process can be repeated.
• the recovered AAV capsid genes are subjected to third-generation sequencing. .
• Some mutant AAVs with high abundance can theoretically efficiently transduce cochlear cells.
• the AAV-M9 obtained as shown in SEQ ID NO:1 is one of the high-abundance mutants.
• AAV-M9 was a chimera of AAV1, 2, 6, and 7 and contained an S430I mutation.
• a 3D homology model of the AAV-M9 capsid was generated using residues from its VP3 region through amino acid sequence alignment with its parental serotype. Both the inner (B) and outer (C) surfaces of the capsid reveal the presence of capsid fragments from four parental AAVs and indicate that the triple-symmetric axon of AAV-M9 is composed of AAV1 and AAV6.
• AAV2, 6, and 7 assembled into a channel with a five-fold symmetry axis, and AAV6 formed a depression at the two-fold symmetry axis (Fig. 2).
• the sequence of the encoding gene of AAV-M9 is shown in SEQ ID No: 1, and the amino acid sequence is shown in SEQ ID No: 2.
• the computer simulated AAV-M9 structure diagram is shown in Figure 2.
• Example 2 In vivo verification of the new adeno-associated virus AAV-M9-CMV-EGFP in the hearing system
• the sequence of the AAV-M9 coding gene obtained from the above sequencing results was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. to obtain the Rep-Cap plasmid of AAV-M9, namely pAAV-M9.
• the obtained Rep-Cap plasmid pAAV-M9 of AAV-M9, the genomic plasmid pAAV-CMV-EGFP expressing a green fluorescent protein EGFP, and the pHelper plasmid were co-transfected into HEK-293T cells in appropriate amounts, using iodine dialkanol. Purify the AAV virus by gradient ultra-high speed centrifugation, measure the virus titer at 1E+12-1E+13GC/mL as the appropriate concentration, and place it at -80°C for later use.
• mice were sacrificed by cervical dislocation, the cochlea of the injected ear was removed, placed in 4% paraformaldehyde, and fixed at room temperature for 2 hours. Wash three times with PBS for 5 minutes each time. Then use 0.5mM EDTA to decalcify and soak at room temperature for 2 hours to soften the cochlea. Dissect the basement membrane with an ophthalmic scalpel under a stereomicroscope.
• Figure 3a is a tile of the cochlea at the hair cell level injected with AAV-ie and AAV-M9-CMV-EGFP viruses
• Figure 3b is a tile of the cochlea at the supporting cell level injected with AAV-ie and AAV-M9-CMV-EGFP viruses
• shown in purple is Myo7a
• a marker protein of hair cells is shown in green
• Figure 3c shows the effects of AAV-ie and AAV-M9-CMV-EGFP viruses on hair cells and Dieters Cells (- The statistical data of supporting cells) showed that AAV-M9-CMV-EGFP specifically infects supporting cells in the cochlea of young mice, but does not have the ability to infect hair cells. Dieters Cells are considered to be the supporting cells with the most regenerative potential, so AAV-M9 has great potential in gene therapy of supporting cells and hair cell regeneration.
• AAV-M9-CMV-EGFP was injected into the cochlea of C57BL/6J mice at 2-3 days of age through the round window. After the virus is fully expressed for 10-14 days, the mice are sacrificed by cervical dislocation, and the cochlea of the injected ear is removed. At the same time, the cochlea of a C57BL/6J mouse of the same age is taken as a control. The cochlea is placed in 4% paraformaldehyde and fixed at room temperature for 2 Hour. Wash three times with PBS for 5 minutes each time. Then use 0.5mM EDTA to decalcify and soak at room temperature for 2 hours to soften the cochlea.
• AAV-ie and AAV-M9-CMV-EGFP were injected into the cochlea of C57BL/6J mice 30 days after birth through the posterior semicircular canal. After the virus was fully infected for 10-14 days to express EGFP, the mice were sacrificed by cervical dislocation, and the injected ears were removed. The cochlea was placed in 4% paraformaldehyde and fixed at room temperature for 2 hours. Wash three times with PBS for 5 minutes each time. Then use 0.5mM EDTA to decalcify and soak at room temperature overnight to soften the cochlea. Dissect the basement membrane with an ophthalmic scalpel under a stereomicroscope.
• anti-Myo7a antibody was used to label hair cells to prepare cochlear samples.
• virus-infected cells express green fluorescent protein in their nuclei.
• the excitation light wavelengths are 488nm and 647nm respectively.
• the in vivo detection results are shown in Figure 5a.
• This picture shows the cochlear tiles injected with AAV-ie and AAV-M9-CMV-EGFP viruses. Shown in purple is Myo7a, a hair cell marker protein, and shown in green is the fluorescent protein expressed by virus-infected cells.
• AAV-M9 specifically infects inner hair cells in the cochlea of adult mice, but does not have the ability to infect outer hair cells.
• the statistical results show that the infection rate of AAV-M9 on the outer hair cells of adult mice is 0.33% ⁇ 0.333, and the infection rate of AAV-ie on the outer hair cells of adult mice is 56.33% ⁇ 1.202, as shown in Figure 5b;
• AAV The infection rate of -M9 on the inner hair cells of adult mice was 98.67% ⁇ 0.667, and the infection rate of AAV-ie on the inner hair cells of adult mice was 100% ⁇ 0, as shown in Figure 5c.
• AAV-M9 is highly efficient and specific in infecting inner hair cells in the cochlea of adult mice and can be used for gene therapy of inner hair cells.
• AAV-M9-CMV-EGFP was injected into the cochlea of 30-day-old C57BL/6J mice through the round window. After the virus was fully expressed for 10-14 days, the mice were sacrificed by cervical dislocation, and the cochlea of the injected ear was removed. At the same time, the cochlea of a C57BL/6J mouse of the same age was taken as a control. The cochlea was placed in 4% paraformaldehyde and solidified at room temperature. Set 2 hours. Wash three times with PBS for 5 minutes each time. Then use 0.5mM EDTA to decalcify and soak at room temperature for 2 hours to soften the cochlea.
• Example 3 In vivo verification of adeno-associated virus AAV-M9-CMV-EGFP in the visual system
• the needle Insert the needle at an angle of 50° between the posterior edge of the cornea and sclera and the iris plane, and slowly inject the adeno-associated virus AAV-M9-CMV-EGFP constructed in 2.1 in Example 2, and drop The concentration is 1.5E13gc/mL. After the injection, the glass needle stays for 30 seconds and then slowly pulled out. After smearing erythromycin ointment on the wound, the mice were placed in a mouse cage in a 41°C water bath to keep warm. After the mice regained consciousness, they were moved to the mouse room for breeding. After 10 days, remove the eye cup.
• mice were sacrificed by spinal dislocation, the eyeballs were removed and the cornea was punctured with a 1mL syringe to release aqueous humor.
• the eyeballs were placed in small petri dishes containing PBS solution and dissected under a microscope. Clamp the cornea with pointed forceps, poke the ophthalmic scissors through the wound in the cornea, cut off the cornea circumferentially, and use the forceps to remove the lens, leaving about 2mm of the optic nerve.
• the eyecups were fixed in 4% PFA for 12 hours at 4°C.
• the fixed eyecups were dehydrated in 30% sucrose for 12 hours at 4°C. After cryosectioning, sections with better results and complete tissue were selected for staining under a fluorescence microscope.
• Immunofluorescence results showed that AAV-M9 could infect the RPE layer.
• the in vivo detection results are shown in Figure 7.
• This picture shows a retinal section injected with AAV-M9-CMV-EGFP virus. Green shows the fluorescent protein expressed by virus infection, and blue shows the cell nucleus.
• the results show that AAV-M9-CMV-EGFP also has a high infection rate on the retinal RPE layer and can be used for ophthalmic gene therapy.
• the fusion adeno-associated virus AAV-M9 of the present invention and its application in the prevention and/or treatment of ear diseases or eye diseases effectively overcome various shortcomings in the prior art and have high industrial utilization value.

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