WO2024066780A1

WO2024066780A1 - Fusion type novel adeno-associated virus and use thereof

Info

Publication number: WO2024066780A1
Application number: PCT/CN2023/113302
Authority: WO
Inventors: 钟桂生; 储岑凤
Original assignee: 上海玮美基因科技有限责任公司
Priority date: 2022-09-30
Filing date: 2023-08-16
Publication date: 2024-04-04
Also published as: CN117801116A

Abstract

Provided are a fusion type adeno-associated virus and a gene delivery effect thereof in the lung and eyes. The fusion type adeno-associated virus comprises a fusion peptide fragment formed by fusion of peptide fragments of serotype AAV1 and AAV6 or a variant thereof, efficiently infecting the lung at a low dose. The adeno-associated virus can also efficiently infect a retinal pigment epithelium (RPE) layer for gene delivery to the eyes. The fusion type adeno-associated virus is used as a safe carrier, and has a wide application prospect in the treatment of a lung or eye disease.

Description

A new fusion adeno-associated virus and its application

Technical Field

The present invention relates to the field of biomedicine, and in particular to a fusion adeno-associated virus and its application, and in particular to the application of heterologous gene delivery in the lungs or eyes.

Background technique

At present, the number of people suffering from gene-related hereditary diseases worldwide is as high as 350 million, and about 80% of them are directly caused by gene mutations. Traditional drug treatment methods use large or small molecules to act on protein targets to exert their efficacy, and cannot effectively treat hereditary diseases such as gene mutations. Emerging gene therapy methods can introduce heterologous genes, small RNAs or gene editing tools into target cells to correct or compensate for diseases caused by gene defects and abnormalities, without the need for chemical drug intervention, radiotherapy or surgical treatment, to achieve the treatment goal.

Current strategies for gene therapy include gene replacement, gene correction, gene enhancement, gene suppression, and gene inactivation, all of which require gene transfer. Currently, there are three major methods of gene transfer: physical, chemical, and biological. Among them, biological transfer is the most widely used in human cells. Biological methods mainly refer to virus-mediated gene transfer, which uses viruses as vectors to recombinantly transfer heterologous genes with viruses through recombinant technology, infect receptor cells to express the target gene to treat diseases. In recent decades, viral vector-mediated gene therapy has been widely used in clinical trials to treat cardiovascular, muscle, metabolic, nervous system, blood, sensory, and infectious diseases and cancer.

Adeno-associated virus (AAV), belonging to the family of Parvoviridae, is a single-stranded DNA defective virus with the simplest structure found so far, and its replication process usually requires the participation of helper viruses. Adeno-associated virus, as a viral vector for the introduction of heterologous genes, has many significant advantages. First, AAV is a non-pathogenic microorganism with high biosafety; second, AAV can infect not only dividing cells but also non-dividing cells, and its applicable host range is very wide; in addition, the genome of AAV is only about 5kb, which is convenient for recombination and operation; in addition, the physical properties of AAV are stable and easy to store, and high temperature of 56°C and pH fluctuations in the range of 3-9 do not affect its infection activity. The above characteristics have led to the widespread use of AAV as a gene transfer vector, making it a hot spot in gene therapy research. In 2017, the US FDA announced that it had approved the application for approval of Spark Therapeutics' drug Luxturna for the treatment of inherited retinal dystrophy (IRD), making it the first approved gene therapy drug using AAV as a vector. This gene therapy can repair the missing PRE65 gene in IRD patients through AAV-mediated delivery, improve the patient's photosensitivity and visual field, and achieve the effect of improving functional vision. This study strongly demonstrates the bright prospects of AAVs in the field of gene therapy.

The lung is an important target organ for gene therapy, where the lung is prone to the most common genetic diseases, such as cystic fibrosis (CF) and α1-antitrypsin deficiency (α1AT). At the same time, lung cancer is also the most common cause of death in humans. To date, about 400 clinical programs for human gene therapy have been approved, of which about 10% are for lung diseases. The data obtained from these human trials have successfully demonstrated that it is possible to transfer genes to human lungs, and the strategy of overexpressing heterologous genes to treat or control lung diseases has potential prospects. So far, the delivery of heterologous proteins or nucleic acids to the lungs through viral vectors is limited by the low infection efficiency of the current vectors, and effective infection cannot be formed under low-dose conditions. Therefore, a higher dose of virus is required when treating the disease, and a high dose of virus has a greater safety risk. Therefore, it is necessary to continuously develop and optimize new, efficient and specific recombinant adeno-associated viruses to improve drug efficacy and reduce safety risks.

In addition, about five million people worldwide suffer from congenital retinal dystrophy, which usually leads to blindness in childhood. These diseases are often caused by specific gene mutations, and about 150 such gene mutations have been found. These mutations can cause loss of function of photoreceptor cells or cells that form the retinal pigment epithelium, leading to blindness. However, current 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 viruses, each injection can only target a small number of cells near the injection point. Therefore, it is necessary to develop better viral vectors suitable for gene therapy of eye diseases, which can more effectively target retinal photoreceptor cells through minimally invasive administration, so as to treat hereditary blindness more safely and efficiently.

Summary of the invention

In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a fusion adeno-associated virus, which is used to solve the problems of low lung infection efficiency under low-dose infection conditions of AAV virus, high safety risks under high-dose infection, and difficulty in virus delivery in tissues such as the retina, for gene therapy. The present invention infects the proximal and distal lungs of mice with the modified fusion-type new adeno-associated virus vector in vivo, which can mediate the specific expression of the target gene in lung cells under low-dose virus infection. The new adeno-associated virus has broad application value and market prospects in the structural and functional analysis of lung cells, the establishment of disease models, and gene therapy. At the same time, the virus was injected into the mouse eyeball through the vitreous body and found that the virus vector has better infection characteristics than the current existing vectors, and also has broad application value and market prospects in the structural and functional analysis of ophthalmic cells, the establishment of disease models, and gene therapy.

To achieve the above objectives and other related objectives, the present invention provides a fusion adeno-associated virus AAV-WM03 capsid protein, which comprises a fusion peptide segment of serotypes AAV1 and AAV6 or a variant thereof.

The present invention also provides a nucleic acid, which encodes the capsid protein of the fusion adeno-associated virus AAV-WM03.

The present invention also provides a construct, which contains the nucleic acid.

The present invention also provides a host cell, wherein the host cell comprises the construct or the heterologous nucleic acid is integrated into the genome, or the host cell comprises the fusion adeno-associated virus AAV-WM03.

The present invention also provides a fusion-type adeno-associated virus AAV-WM03, the capsid structure of the fusion-type adeno-associated virus comprising any one of the fusion-type adeno-associated virus AAV-WM03 capsid proteins.

In addition, the present invention also provides a fusion adeno-associated virus vector system, comprising a packaging plasmid and an auxiliary plasmid. The packaging plasmid comprises the nucleic acid, the nucleic acid fragment and the construct.

The present invention also provides a fusion-type adeno-associated virus, which is obtained by packaging the fusion-type adeno-associated virus vector system through viruses.

The present invention also provides a pharmaceutical composition, which comprises the fusion adeno-associated virus and pharmaceutically acceptable excipients.

Finally, the present invention also provides the use of the fusion adeno-associated virus AAV-WM03 capsid protein, nucleic acid, construct, fusion adeno-associated virus, host cell, fusion adeno-associated virus vector system, pharmaceutical composition or conjugate in the preparation of drugs for treating diseases; preferably, use in the preparation of drugs for gene therapy diseases; more preferably, use in the preparation of gene therapy drugs for lung diseases or eye diseases.

As described above, the fusion-type novel adeno-associated virus of the present invention has the following advantages and beneficial effects: (1) It provides a novel adeno-associated virus AAV-WM03 that can efficiently infect lung cells under low-dose conditions of AAV virus infection; (2) It provides a novel adeno-associated virus vector AAV-WM03 that can mediate the specific expression of the target gene in the RPE layer of the retina of adult mice; (3) The novel adeno-associated virus vector AAV-WM03 mediates the expression of the target gene more flexibly, with higher safety, more convenient application and lower cost than traditional transgenic methods; (4) The novel specific adeno-associated virus vector AAV-WM03 provides an application basis for the development of gene therapy for the precise treatment of lung and eye-related diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1a. Schematic diagram of AAV-WM03 sequence.

Figure 1b. Sequence alignment of AAV-WM03 and its parental vector.

Figure 1c. Computer-simulated structure diagram of AAV-WM03.

Figure 2. AAV-WM03 DNA sequence alignment.

Figure 3a. Immunofluorescence images of lungs infected with high doses of AAV-WM03, AAV6, AAV6.2ff, and AAV9.

Figure 3b. Statistical graph and average infection status of proximal lung infection with high-dose AAV-WM03, AAV6, AAV6.2ff, and AAV9.

Figure 3c. Statistical graph and average infection status of distal lung infection with high-dose AAV-WM03, AAV6, AAV6.2ff, and AAV9.

Figure 4a. Immunofluorescence images of lungs infected with low-dose AAV-WM03, AAV6, AAV6.2ff, and AAV9.

Figure 4b. Statistical graph and average infection status of proximal lung infection with low-dose AAV-WM03, AAV6, AAV6.2ff, and AAV9.

Figure 4c. Statistical graph and average infection status of distal lung infection with low-dose AAV-WM03, AAV6, AAV6.2ff, and AAV9.

Figure 5. Infection of the RPE layer of the eyeball by AAV-WM03-CMV-EGFP.

Detailed ways

The present invention provides a fusion adeno-associated virus AAV-WM03 capsid protein, which comprises a peptide segment (fusion peptide segment or chimeric peptide segment) formed by fusion of peptide segments of serotypes AAV1 and AAV6 or a variant thereof.

The fusion peptide segment or its variant comprises one or more segments of the peptide segments of AAV1 and 6. In a specific embodiment, the fusion peptide segment consists of two peptide segments, namely the first peptide segment and the second peptide segment. The first peptide segment comprises a peptide segment from AAV1, and the second peptide segment comprises a peptide segment from AAV6. The first peptide segment and the second peptide segment are connected in sequence. Preferably, the first peptide segment comprises the amino acid fragment shown in SEQ ID No.1, and the second peptide segment comprises the amino acid fragment shown in SEQ ID No.2.

The peptide segment of AAV1 contains one or more mutated amino acid sequence sites. In a specific embodiment, the peptide segment of AAV1 contains one mutation site. Preferably, the position and mutation type of the mutation site are S268R.

In the fusion-type adeno-associated virus AAV-WM03 capsid protein, the peptide segments are directly fused to each other.

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 capsomeres, in which the genomic nucleic acid is encapsidated. An icosahedral symmetrical 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 capsomeres 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 capsomeres rotate 180° around the 2-fold symmetry axis and reset twice to form two intersecting triangular faces; there is also a 5-fold symmetry axis passing through two opposite vertices, and the capsomeres 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, of which Every 2 triangles intersect to form an edge, with a total of 30 edges; every 5 triangles are connected to form 12 vertices.

In the fusion adeno-associated virus capsid protein, the peptide segments of the serotypes AAV1 and AAV6 are assembled into the three-fold symmetry axis protrusions of the fusion adeno-associated virus AAV-WM03 capsid protein; the peptide segments of the serotypes AAV1 and AAV6 are assembled into the five-fold symmetry axis channels of the fusion adeno-associated virus AAV-WM03 capsid protein, and the computer simulated structure schematic diagram is shown in Figure 1c.

In some preferred embodiments, the fusion adeno-associated virus AAV-WM03 capsid protein comprises:

a) the amino acid sequence shown in SEQ ID No.4;

b) A polypeptide fragment having a sequence identity of 90% or more as SEQ ID No. 4 and having the function of the amino acid sequence specified in a).

Furthermore, the polypeptide fragment in b) specifically refers to: a polypeptide fragment obtained by replacing, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids of the amino acid sequence as shown in SEQ ID No.4, or by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids at the N-terminus and/or C-terminus, and having the function of the polypeptide fragment as shown in the amino acid sequence as SEQ ID No.4, and the amino acid sequence in b) may have a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% with SEQ ID No.4.

The nucleotide sequence corresponding to the amino acid sequence of SEQ ID No. 4 includes one or more. In a specific embodiment, the nucleotide sequence is the nucleotide sequence shown in SEQ ID No. 3.

The present invention also provides a construct, which contains the above nucleotide sequence. The construct is obtained by inserting the above nucleotide sequence 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, wherein the host cell comprises the above-mentioned construct or the above-mentioned nucleotide sequence is integrated into the genome, or the host cell comprises the fusion-type adeno-associated virus AAV-WM03 as described in any one of the above items.

Further, the host cell is selected from any one of mammalian cells (such as CHO, COS and N2A), plant cells, human cells (human cervical cancer cells such as HELA and human embryonic kidney cells such as HEK293T), bacterial cells (such as Escherichia coli, Streptomyces, Salmonella typhimurium), fungal cells (such as yeast), and insect cells (such as Sf9). Preferably, the host cell is an animal cell, and more preferably a human cell. The host cell is a passage cell or a primary cell, i.e., a cell directly isolated from an organism (such as a human). The host cell is an adherent cell or a suspended cell, i.e., a cell growing in the form of a suspension.

The present invention also provides a fusion-type adeno-associated virus AAV-WM03, wherein the fusion-type adeno-associated virus contains the fusion-type adeno-associated virus AAV-WM03 capsid protein as described in any one of the above items.

Furthermore, the fusion adeno-associated virus AAV-WM03 also includes a heterologous nucleotide sequence encoding a target product. The heterologous nucleotide sequence encoding the target product can be wrapped and carried by various capsid proteins. The heterologous nucleotide sequence encoding the target product is a construct, and the construct is a nucleic acid containing 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 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.

Furthermore, the target product is encoded as a nucleic acid or a protein, and the nucleic acid is selected from a small guide RNA (sgRNA) and an interfering RNA (RNAi). The sgRNA or RNAi targets a target selected from the epidermal growth factor receptor EGFR or the programmed death receptor PD-1. The gene encoding the protein is selected from the D subtype surfactant protein SFTPD, the B subtype surfactant protein SFTPB, the A1 subtype surfactant protein SFTPA1, interferon γIFN-γ, the cystic fibrosis transmembrane regulator CFTR or the aspartic protease Napsin A.

The present invention also provides an engineered cell transformed with the fusion-type adeno-associated virus as described above. The engineered cell comprises the fusion-type adeno-associated virus as described above. The cell is a eukaryotic cell and/or a prokaryotic cell.

As representative examples of suitable cells, any one of mammalian cells (such as CHO, COS and N2A), plant cells, human cells (human cervical cancer cells such as HELA and human embryonic kidney cells such as HEK293FT), bacterial cells (such as Escherichia coli, Streptomyces, Salmonella typhimurium), fungal cells (such as yeast), and insect cells (such as Sf9) is selected. Preferably, the host cell is an animal cell, and more preferably a human cell. The host cell is a passage cell or a primary cell, i.e., a cell directly isolated from an organism (such as a human). The host cell is an adherent cell or a suspended cell, i.e., a cell grown in the form of a suspension.

The present invention also provides a fusion adeno-associated virus vector system, which comprises a packaging plasmid, an expression plasmid and a helper plasmid. The packaging plasmid comprises the nucleic acid or nucleic acid fragment as described above. The expression plasmid comprises a heterologous nucleotide sequence encoding a target product.

Furthermore, the packaging plasmid also contains a rep gene fragment of an adeno-associated virus, wherein the rep gene fragment contains an intron, and the intron contains a transcription termination sequence.

Furthermore, the target product is a nucleic acid or a protein, and the nucleic acid is selected from small guide RNA (sgRNA) and interfering RNA (RNAi). The sgRNA or RNAi targets a target selected from epidermal growth factor receptor (EGFR) and programmed death receptor 1 (PD-1). The protein encoding gene is selected from surfactant protein subtype D (SFTPD), surfactant protein subtype B (SFTPB), surfactant protein subtype A1 (SFTPA1), interferon γIFN-γ, cystic fibrosis transmembrane regulator (CFTR) and aspartic protease Napsin A.

The packaging plasmid, expression plasmid, and helper virus plasmid are transferred into host cells, and the nucleic acid sequences therein are all integrated into the host cells to produce the fusion adeno-associated virus. In some embodiments, the nucleic acid sequences encoding the 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.

The present invention also provides a fusion adeno-associated virus, which is obtained by virus packaging of the fusion adeno-associated virus vector system as described above.

The present invention also provides a pharmaceutical composition, comprising the fusion-type adeno-associated virus as described above and a pharmaceutically acceptable excipient. The pharmaceutical composition or conjugate is administered by a systemic route or a local route, selected from inner ear administration, ophthalmic administration, intravenous administration, intramuscular administration, subcutaneous administration, oral administration, local contact, intraperitoneal administration and intralesional administration. The pharmaceutical composition or conjugate dosage form is one or more of an injection, a tablet, a capsule, an aerosol, an eye drop or a nasal drop.

The adjuvant includes various excipients and diluents, which are not necessary active ingredients and have no excessive toxicity after application. The adjuvant includes sterile water or 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.) or adhesives. The adjuvant also includes other low molecular weight polypeptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol or sorbitol. The adjuvant is selected from saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannoses or sugar alcohol isotonic solution when used for the aqueous solution of injection. The aqueous solution of injection includes a solubilizing agent. The solubilizing agent is selected from alcohol (ethanol), polyol (propylene glycol or PEG) and/or nonionic surfactant (Tween 80 or HCO-50). In the pharmaceutical composition provided by the present invention, the fusion adeno-associated virus AAV-WM03 is a single active ingredient, and can also be combined with one or more other active components useful for treating lung diseases or eye diseases to form a combined preparation. The active components are various other drugs for treating lung diseases or eye diseases. The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the fusion adeno-associated virus AAV-WM03 and the active ingredient of the pharmaceutical composition depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the bifunctional compound as an active ingredient is 1-1000 mg/kg/day, 1-3 mg/kg/day, 3-5 mg/kg/day, 5-10 mg/kg/day, 10-20 mg/kg/day, 20-30 mg/kg/day, 30-40 mg/kg/day, 40-60 mg/kg/day, 60-80 mg/kg/day, 80-100 mg/kg/day, 100-200 mg/kg/day, 200-500 mg/kg/day, or greater than 500 mg/kg/day. The present invention also provides a conjugate, which includes the fusion adeno-associated virus AAV-WM03 as described above or a connected biologically active polypeptide.

The pulmonary disease is selected from one or more of pneumonia, pulmonary fibrosis, and pulmonary tuberculosis.

The ophthalmic disease is also selected from age-related macular degeneration (AMD), choroidal neovascularization (CNV), choroidal neovascularization (CNV), Angiogenic membrane (CNVM), cystoid macular edema (CME), epiretinal membrane (ERM) and macular hole, angioid streaks, retinal detachment, diabetic retinopathy, diabetic macular edema (DME), atrophic lesions of retinal pigment epithelial cells (RPE), hypertrophic lesions of retinal pigment epithelial cells (RPE), retinal vein occlusion, chorioretinal vein occlusion, macular edema, pterygium conjunctiva, subretinal edema and intraretinal edema, retinitis pigmentosa, Stargardt's disease, glaucoma, inflammatory diseases, cataract, refractory abnormal phenomenon, keratoconus, retinopathy of prematurity, anterior angiogenesis, keratitis, posterior corneal angiogenesis or corneal transplantation or corneal shaping one or more. In some preferred embodiments, the ophthalmic disease is RPE layer related diseases.

The inflammation is selected from skin inflammation, vascular inflammation, allergy, autoimmune disease, fibrosis, scleroderma or transplant rejection; the autoimmune disease is selected from one or more of rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, Sjögren's syndrome or polymyositis.

The cancer is selected from lymphoma, hematological tumor or solid tumor; specifically, the cancer is selected from adrenal cortical 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, brain low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell The invention relates to a method for treating a leukemia or a diabetic renal cell carcinoma, wherein the leukemia or the diabetic renal cell carcinoma is selected from the group consisting of: a) cancer, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, b) prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma, multiple myeloma, acute lymphoid leukemia, chronic lymphoid leukemia, chronic myeloid leukemia, T-cell lymphoma or B-cell lymphoma; preferably, the tumor is one or more of lung cancer, colorectal cancer and/or melanocytoma.

The metabolic disease is selected from diabetes, selected from type I and type II diabetes and diseases and conditions related to diabetes; the metabolic disease is selected from one or more of atherosclerosis, cardiovascular disease, nephropathy, neuropathy, retinopathy, β-cell dysfunction, dyslipidemia, hyperglycemia, insulin resistance or chronic obstructive pulmonary disease. In some more preferred embodiments, the gene therapy refers to the treatment of lung diseases. The fusion adeno-associated virus or pharmaceutical composition can deliver the target product to lung cells under low-dose conditions of AAV virus infection to achieve the treatment of lung diseases.

The fusion adeno-associated virus AAV-WM03, host cell, vector system, pharmaceutical composition or conjugate of the present invention is used to deliver the target product to lung cells under low-dose conditions of AAV virus infection to achieve the treatment of lung diseases. The delivery of the target product is for non-diagnostic treatment purposes, including delivery of the target product to lung cells in vitro.

The lung disease may be caused by pulmonary fibrosis caused by environmental factors. Therefore, the present invention also provides the use of the fusion adeno-associated virus in a drug for treating lung diseases caused by environmental factors in an individual.

Further, the lung disease is a lung epithelial cell-related disease. In some specific embodiments, the lung epithelial Cell diseases can be prevented or treated by overexpressing Napsin A to inhibit cell phenotypic transformation and proliferation ability.

Furthermore, the lung disease is a disease related to gene defects, environmental damage or aging, for example, a disease related to gene mutation, etc., another example, a disease related to air pollution or drugs, and another example, a disease related to aging.

Furthermore, lung diseases are diseases related to cell damage, etc., specifically lung epithelial cell damage and/or fibroblast damage, more specifically lung epithelial cell damage caused by gene mutation, fibroblast damage caused by gene mutation, etc., cell damage caused by noise, cell damage caused by drugs, or cell damage caused by aging.

Furthermore, the fusion adeno-associated virus is used as a vector for delivering the target product.

The present invention also provides a method for treating a lung disease, comprising administering an effective amount of the fusion-type adeno-associated virus of the present invention, the host cell of the present invention, the vector system or the pharmaceutical composition or conjugate to a subject in need thereof. A physician can determine the actual dosage that is most suitable for a single patient and it will vary according to the age, weight and response of a specific individual.

In the present invention, 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 person skilled in the art can determine the appropriate administration method and dosage.

The delivery of one or more therapeutic genes by the fusion adeno-associated virus of the present invention can be used alone or in combination with other treatment methods or therapeutic components.

The fusion adeno-associated virus of the present invention is used to infect cells, thereby delivering genes and/or linked (e.g., but not limited to, covalently linked) biologically active polypeptides to cells. Therefore, the present invention provides a method for delivering heterologous genes to cells, the method infecting cells by allowing one or more fusion adeno-associated viruses or conjugates of the present invention to the cells, wherein the fusion adeno-associated virus or conjugate comprises one or more heterologous genes.

The present invention also provides a method for producing a stable fusion adeno-associated virus vector production cell line, comprising:

(a) introducing a fusion adeno-associated virus vector as defined herein into a culture of mammalian host cells; and/or,

(b) selecting within the culture mammalian host cells that have the nucleic acid sequence encoded on the vector integrated into an endogenous chromosome of the mammalian host cell.

The AAV vector production cell is 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.

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. The text is for AAV nucleic acid and amino acid sequences.

In the compounds of the present invention and their use, when the fusion adeno-associated virus is used in combination with other therapeutic agents, the active compound is co-administered with the other therapeutic agents. "Co-administered" means that they are administered simultaneously in the same preparation or in two different preparations via the same or different routes, or 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.

In certain embodiments, the fusion adeno-associated viruses and methods of the invention can be used to prevent lung disease and can be administered as a preventive treatment prior to lung damage or after a period of time after exposure to an environment susceptible to lung damage.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one skilled in the art.

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 delivery of the polypeptide to a cell. Illustrative vectors include, for example, plasmids, viral vectors, liposomes or other gene delivery vectors.

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

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).

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 an encapsidated polynucleotide.

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

The terms AAV "rep" and "cap" genes refer to polynucleotide sequences encoding the replication and packaging proteins of the adeno-associated virus. In this article, AAV rep and cap refer to the AAV "packaging genes".

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).

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.

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.

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

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.

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.

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 to mean 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.

In the present invention, the subject or individual for therapeutic or preventive treatment is preferably a mammal, such as, but not limited to, a human, a primate, livestock (such as sheep, cattle, horses, donkeys, pigs), a pet (such as dogs, cats), a laboratory test animal (such as mice, rabbits, rats, guinea pigs, hamsters), or a captured wild animal (such as foxes, deer). The subject is preferably a primate. The subject is most preferably a human.

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 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 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.

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.

Example 1. Acquisition of a novel adeno-associated virus AAV-WM03

a. Construction of Rep-Cap plasmid of AAV-WM03

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 natural serotype was modified by fusing the amino acid sequence SEQ ID No.1 derived from AAV1 and the amino acid sequence SEQ ID No.2 derived from AAV6, wherein the sequence of AAV1 contains the mutation site S268R (Figure 1a, b), together forming a new AAV serotype, named AAV-WM03, whose nucleotide sequence is SEQ ID No.3 and amino acid sequence is SEQ ID No.4. Both the inner and outer surfaces of the capsid show the presence of capsid fragments from the two parents AAV1 and AAV6, and indicate that the three-fold symmetry axis protrusions and the five-fold symmetry axis channels of AAV-WM03 are also composed of AAV1 and 6 (Figure 1c).

There is a relatively high homology between wild-type AAVs, so the nucleic acid sequences of different serotypes of AAV are highly similar. Therefore, under the premise of the consistency of the amino acid sequence, the corresponding nucleic acid sequence sources or combinations include multiple ones, among which a DNA sequence of the AAV-WM03 capsid protein can be translated, as shown in Figure 2, which is derived from AAV1, AAV3 and AAV13, and contains three base mutations of AAV3, corresponding to the S268R amino acid mutation (Figure 2), that is, the nucleic acid sequence of the fusion adeno-associated virus AAV-WM03, which is fused from serotypes AAV1 and AAV6, can also be composed of serotypes AAV1, AAV3 and AAV13.

b. Preparation and purification of AAV-WM03 virus

The sequence of the coding gene of AAV-WM03 obtained in the above sequencing results was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. to obtain the Rep-Cap plasmid of AAV-WM03, namely pAAV-WM03. The obtained Rep-Cap plasmid pAAV-WM03 of AAV-WM03, the genomic plasmid pAAV-CMV-EGFP expressing a green fluorescent protein EGFP, the nucleotide sequence SEQ ID No.5, and the pHelper plasmid (the full sequence of the plasmid is the same as that of SEQ ID NO.12 of the AAV-ie patent document CN110437317A) were co-transfected into HEK-293T cells at a plasmid dosage of 80, 60, and 110 μg per plate of cells, and the AAV virus was purified by iodide dialkyl gradient ultracentrifugation. The virus titer was measured at 2E+13GC/mL as the appropriate concentration, and it was placed at -80°C for use.

SEQ ID No.1

SEQ ID No.2

SEQ ID No. 3

SEQ ID No. 4

SEQ ID No. 5

Example 2 In vivo verification of the novel adeno-associated virus AAV-WM03-mNeonGreen in the lungs

AAV6 and AAV6.2ff are two AAV vectors known to be effective against lung infections. The experiment set up four test viruses, AAV6, AAV9, AAV6.2ff, and AAV-WM03, which were packaged with green fluorescent protein mNeonGreen, and set up high-dose groups (5E+10GC/mL) and low-dose groups (2.5E+10GC/mL), for a total of 8 groups. All test groups used 4 mice aged 6-8 weeks for intratracheal injection into the lungs. Anesthetize the anesthetized mice and fix them on the animal workbench in a dorsal position, straighten their front paws to both sides of the body and fix them with tape. Shave the mouse hair in part of the neck area, and use iodine to disinfect the shaved area. Make a small incision of 5 to 7 mm on the mouse neck with surgical scissors. Use forceps to clamp and fix the mouse trachea, insert the syringe needle with AAV into the trachea with the bevel facing up at 45° to the trachea, confirm that the needle is correctly inserted, slowly inject 50μL of AAV into the trachea, wait for 5s and slowly remove the needle. Suture the neck wound and put the mouse back in the cage to recover until it is fully awake. Samples were taken one week after the injection, and the tissues were fixed by perfusion with 4% PFA. The lung tissues were dissected and the morphology was recorded. The samples were prepared by frozen sectioning and stained for imaging.

After analyzing the fluorescence images, it can be seen that the infection efficiency of AAV-WM03 in the proximal and distal parts of the lungs is high under high doses, which is not lower than that of the currently commonly used natural serotype AAV6 and its mutants (Figure 3a). Among them, the infection efficiency of AAV-WM03 in the proximal part of the lungs under high dose conditions is 59.67±1.202, and the infection efficiency of AAV6 in the proximal part of the lungs used as a comparison with AAV-WM03 is 39.00±1.528, AAV6.2ff is 77.67±1.453 and A AV9 was 83.00±2.887 (Figure 3b); the infection efficiency of AAV-WM03 in the distal lung was 54.33±0.667, while the infection efficiency of AAV6 in the distal lung, which was used as a comparison with AAV-WM03, was 27.67±1.453, AAV6.2ff was 64.33±1.202 and AAV9 was 72.67±5.783 (Figure 3c), **p<0.005, ***p<0.0005, ****p<0.00005. At low doses, AAV-WM03 had an advantage over natural serotype AAV6 and its mutant AAV6.2ff in infecting lung tissues in the proximal and distal parts of the lungs (Figure 4a). Under low dose conditions, the infection efficiency of AAV-WM03 in the proximal part of the lungs was 61.00±1.528, while the infection efficiency of AAV6, which was used as a comparison with AAV-WM03, in the proximal part of the lungs was 59.67±0.882, AAV6.2ff was 41.00±1.155, and AAV9 was 6. 8.67±1.764 (Figure 4b); the infection efficiency of AAV-WM03 in the distal lung was 58.33±0.8819, and the infection efficiency of AAV6 in the distal lung compared with AAV-WM03 was 21.33±2.028, AAV6.2ff was 41.67±2.186 and AAV9 was 13.00±2.082 (Figure 4c), *p<0.05, ***p<0.0005, ****p<0.00005.

Example 3 In vivo verification of the novel adeno-associated virus AAV-WM03 in the visual system

Place an anesthetized adult mouse under a microscope and drop 1-2 drops of tropicamide eye drops on the eyeball to dilate the pupil. 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 and hold a glass needle with 2μl of virus in the right hand. Insert the needle at 50° between the posterior edge of the corneosclera and the iris plane and slowly inject AAV-WM03 with a titer of 1.5E13gc/ml. After the injection, the glass needle stays for 30s and then slowly withdraws. After applying erythromycin 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 11 days, the eye cup is taken. The mouse is killed by spinal dislocation. After removing the eyeball, the cornea is punctured with a 1ml syringe to release the aqueous humor. The eyeball is placed in a small culture dish filled with PBS solution and dissected under a microscope. Clamp the cornea with pointed forceps, poke the ophthalmic scissors into the wound of the cornea, cut the cornea circumferentially, remove the lens with forceps, and retain about 2mm of the optic nerve. The eye cup was fixed in 4% PFA for 12h at 4°C. The fixed eye cup was dehydrated in 30% sucrose for 12h at 4°C. After frozen sectioning, the sections with good effect and intact tissue were selected for staining under a fluorescence microscope. The immunofluorescence results showed that AAV-WM03 could infect the RPE layer, as shown in Figure 5.

Claims

A fusion adeno-associated virus AAV-WM03 capsid protein, characterized in that the fusion adeno-associated virus AAV-WM03 capsid protein comprises a fusion peptide segment of the peptide segments of serotypes AAV1 and AAV6 or a variant thereof.
The fusion adeno-associated virus AAV-WM03 capsid protein as described in claim 1 is characterized in that the fusion peptide segment comprises a first peptide segment and a second peptide segment connected in sequence; the first peptide segment comprises a peptide segment from AAV1, and the second peptide segment comprises a peptide segment from AAV6.
The fusion adeno-associated virus AAV-WM03 capsid protein as described in claim 1 is characterized in that the first peptide segment contains the amino acid fragment shown in SEQ ID No.1, and the second peptide segment contains the amino acid fragment shown in SEQ ID No.2.
The fusion adeno-associated virus AAV-WM03 capsid protein according to claim 1, characterized in that the fusion adeno-associated virus AAV-WM03 capsid protein comprises:

a) the amino acid sequence shown in SEQ ID No.4;

b) A polypeptide fragment having a sequence identity of 90% or more as SEQ ID No. 4 and having the function of the amino acid sequence specified in a).
A nucleic acid, characterized in that the nucleic acid encodes the fusion adeno-associated virus AAV-WM03 capsid protein as described in any one of claims 1 to 4.
A construct, characterized in that it contains the nucleic acid according to claim 5.
A host cell, characterized in that the host cell comprises the construct as described in claim 6 or the nucleic acid as described in claim 5 is integrated heterologously in the genome, or the host cell comprises the fusion adeno-associated virus AAV-WM03 capsid protein as described in any one of claims 1-4.
A fusion adeno-associated virus AAV-WM03, characterized in that the capsid structure of the fusion adeno-associated virus AAV-WM03 contains the fusion adeno-associated virus AAV-WM03 capsid protein as claimed in any one of claims 1 to 4.
The fusion adeno-associated virus AAV-WM03 as described in claim 8 is characterized in that the fusion adeno-associated virus also includes a heterologous nucleotide sequence encoding a target product; preferably, the target product is a nucleic acid or a protein; further, the nucleic acid is selected from a small guide RNA and an interfering RNA.
A cell, characterized in that the cell is transformed using the fusion adeno-associated virus AAV-WM03 as described in claim 8 or 9.
A fusion adeno-associated virus vector system, characterized in that the fusion adeno-associated virus vector system comprises a packaging plasmid, and the packaging plasmid comprises the nucleic acid described in claim 5.
The fusion adeno-associated virus vector system according to claim 11, characterized in that the packaging plasmid also contains a rep gene fragment of an adeno-associated virus.
The fusion adeno-associated virus vector system according to claim 11 is characterized in that the adeno-associated virus vector system also includes an expression plasmid, which contains a heterologous nucleotide responsible for encoding a target product; preferably, the target product is a nucleic acid or a protein; further, the nucleic acid is selected from a small guide RNA and an interfering RNA.
The fusion adeno-associated virus vector system according to any one of claims 11 to 13 is characterized in that the adeno-associated virus vector system also includes a helper virus plasmid or a helper virus, and the fusion adeno-associated virus vector system also includes a host cell.
A pharmaceutical composition or conjugate, characterized in that the pharmaceutical composition comprises the fusion adeno-associated virus AAV-WM03 as described in any one of claims 8 or 9 and a pharmaceutically acceptable excipient; the conjugate comprises the fusion adeno-associated virus AAV-WM03 as described in any one of claims 8 or 9 and a biologically active polypeptide connected thereto.
The pharmaceutical composition or conjugate according to claim 15, characterized in that the pharmaceutical composition or conjugate dosage form is one or more of injection, tablet, capsule, aerosol, eye drops or nasal drops.
Use of the fusion adeno-associated virus AAV-WM03 capsid protein as described in any one of claims 1 to 4, or the nucleic acid as described in claim 5, or the construct as described in claim 6, the fusion adeno-associated virus AAV-WM03 as described in claim 8 or 9, or the host cell as described in claim 7 or the cell as described in claim 10, the fusion adeno-associated virus vector system as described in any one of claims 11 to 13, or the pharmaceutical composition or conjugate as described in claim 15 or 16 in the preparation of a medicament for preventing or treating a disease.
The use according to claim 17, characterized in that it comprises at least one of the following:

1) The disease is selected from one or more of pulmonary diseases, ophthalmic diseases, inflammation, tumors, metabolic diseases, pain, and neurodegenerative inflammatory diseases;

2) The pharmaceutical composition or conjugate is used to prepare gene therapy drugs.
The use according to claim 18, characterized in that it comprises at least one of the following:

1) The pulmonary disease or ophthalmic disease is a disease related to cell damage;

2) The lung disease or eye disease is a related disease caused by a gene defect;

3) The lung disease or eye disease is a related disease caused by environmental factors;

4) The lung disease or eye disease reported is a disease related to aging;

5) The lung disease or eye disease is a related disease caused by a primary tumor;

6) The pulmonary disease or ophthalmic disease is a disease related to cancer cell migration;

7) The ophthalmic disease is a disease related to the RPE layer.