WO2023143366A1 - Variant adeno-associated virus and application thereof in disease treatment - Google Patents

Variant adeno-associated virus and application thereof in disease treatment Download PDF

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WO2023143366A1
WO2023143366A1 PCT/CN2023/073135 CN2023073135W WO2023143366A1 WO 2023143366 A1 WO2023143366 A1 WO 2023143366A1 CN 2023073135 W CN2023073135 W CN 2023073135W WO 2023143366 A1 WO2023143366 A1 WO 2023143366A1
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associated virus
aav
adeno
cells
capsid protein
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Chinese (zh)
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钟桂生
储岑凤
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上海玮美基因科技有限责任公司
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Definitions

  • the invention relates to the field of biotechnology, and relates to a mutant adeno-associated virus and its application in disease treatment, in particular to AAV-ie-K558R and its application in mediating cochlear gene therapy and hair cell regeneration.
  • the cochlea is composed of multiple types of cells, including hair cells, supporting cells, and spiral ganglion neurons, responsible for converting mechanical energy into electrical signals for hearing.
  • the cells that make up the cochlea are critical to hearing. Genetic and environmental factors can lead to dysfunction of the cochlea and auditory system. Although sensorineural deafness can be caused by genetic mutations in cochlear hair cells (HCs) and supporting cells (SCs), nongenetic factors, such as noise, ototoxic drugs, or aging, can also induce deafness by damaging HCs. In either case, the damage was irreversible in mammals that do not have the capacity to regenerate cochlear cells. While current treatments, such as hearing aids and cochlear implants, can alleviate hearing loss in some patients, these methods do not support their sensitivity and perception of natural sounds in noisy environments.
  • HCs in the cochlea include two distinct types: outer hair cells (OHCs), which amplify sound, and inner hair cells (IHCs), which convert mechanical energy into electrical signals.
  • OOCs outer hair cells
  • IHCs inner hair cells
  • HCs anchor sensory epithelial cells to the basement membrane, which is critical for maintaining the environment in which HCs function properly.
  • SCs have the potential to transdifferentiate into HC-like cells
  • HCs regeneration has been considered as a potential approach for the treatment of acquired deafness caused by non-genetic factors.
  • Adeno-associated viruses have proven to be highly safe in both animal models and humans, and are widely used to deliver genetic material to cells for gene therapy in many different organs and diseases.
  • Anc80L65 is a promising vector for delivering Harmonin for the treatment of Deafness caused by HCs dysfunction.
  • its efficiency in transducing HCs needs to be improved.
  • the present invention has developed a synthetic AAV, AAV-ie, which can target SCs and HCs; and transdifferentiate SCs into HC-like cells by delivering the transcription factor Atoh1 to regenerate HC-like cells.
  • AAV AAV-ie
  • its targeting efficiency to SCs or HCs needs to be further improved, especially in the basal region of the cochlea.
  • the purpose of the present invention is to provide a mutant adeno-associated virus and its application in mediating cochlear gene therapy and hair cell regeneration, which is used to solve the problem of AAV in the prior art.
  • the efficiency of gene therapy and supporting cell transduction is not high, and it is impossible to efficiently use gene therapy or induce hair cell regeneration for the treatment of hearing impairment diseases.
  • the present invention provides a mutant adeno-associated virus and its application in mediating cochlear gene therapy and hair cell regeneration.
  • One of the objectives of the present invention is to provide a mutant adeno-associated virus capsid protein.
  • the capsid protein includes a mutation of the 558th amino acid K.
  • the mutation of amino acid K at position 558 is preferably K558R mutation.
  • Another object of the present invention is to provide a nucleic acid encoding the nucleotide sequence of the mutant adeno-associated virus capsid protein as described above.
  • Another object of the present invention is to provide a construct comprising a nucleic acid as described above.
  • Another object of the present invention is to provide a host cell comprising the above-mentioned construct or exogenous nucleic acid as above integrated in the genome.
  • Another object of the present invention is to provide a mutant adeno-associated virus, the capsid structure of the mutant adeno-associated virus contains the above-mentioned mutant adeno-associated virus capsid protein.
  • Another object of the present invention is to provide host cells transformed with the mutant adeno-associated virus as described above.
  • Another object of the present invention is to provide a mutant adeno-associated virus vector system, which includes a packaging plasmid, and the packaging plasmid contains the above-mentioned nucleic acid fragment.
  • Another object of the present invention is to provide a mutant adeno-associated virus, which is obtained from the above-mentioned mutant adeno-associated virus vector system through virus packaging.
  • Another object of the present invention is to provide a pharmaceutical composition, which comprises the above-mentioned mutant adeno-associated virus and a pharmaceutically acceptable carrier.
  • Another object of the present invention is to provide a use of the above-mentioned mutant adeno-associated virus, host cell, vector system, and pharmaceutical composition in the preparation of medicines for treating diseases.
  • the beneficial effects of the present invention include:
  • the mutant adeno-associated virus provided by the present invention has reduced ubiquitination or phosphorylation, can efficiently transduce hair cells and supporting cells in the cochlea (such as transducing newborn mice), and can make Prestin knockout mice Partial recovery of the hearing loss in the human body can send Atoh1 into the cochlear supporting cells to generate HC-like cells.
  • the mutated adeno-associated virus of the present invention is a safe carrier, and the gene therapy for hearing loss-related diseases using the carrier is safe and effective in humans, and has no negative effects on the auditory and vestibular systems, for example, on HCs It has no significant effect on morphology, will not cause loss of hair cells, will not affect hearing threshold, will not make individual gait or vestibular function abnormal, etc., and has clinical potential in the treatment of hearing loss caused by hair cell death.
  • Capsid protein amino acid mutations enhance AAV transduction.
  • AAV-ie-K558R is a safe vector.
  • FIG. 3 AAV-ie-K558R-Prestin restored auditory function in Prestin KO mice.
  • Thresholds are determined by the presence of peak 1 and are represented by colored traces. Scales apply to all families.
  • the contralateral ear of an uninjected Prestin knockout mouse was used as a negative control (blue), and an uninjected WT mouse was used as a positive control (purple).
  • Data are shown as mean ⁇ SEM. Significance (*P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001) was calculated by multiple t-test between the AAV-injected group and the contralateral non-injected group.
  • FIG. 4 AAV-ie-K558R-Atoh1 can regenerate HC-like cells in neonatal mice.
  • FIG. 1 Transduction efficiency of AAV-ie variants. Immunofluorescent images of cochlea, hair cell layer transduced with AAV-ie S/K mutant vector. All cochleae were harvested at P14 after microinjection with 1.5 ⁇ l of AAV stock solution at P3, stained with anti-Myo7a antibody (magenta) and imaged for NLS-mNeonGreen fluorescence (green).
  • K39R K61R, K137R, K142R, K143R, K161R, K258R, K332R, K492R, K546R, K551R, K558R, K676R, K699R, K703R, K717R, S225A , S269A, S314A, S392A, S393A, S425A, S431A, S491A, S505A, S539A, S675A, S679A.
  • AAV-ie-K558R broadly transduces mouse cochlear and vestibular sensory epithelial cells.
  • FIG. 1 The figure shows that AAV-ie-K558R efficiently transduces cochlear hair cells and various types of supporting cells.
  • Scale bar 20 ⁇ m
  • FIG. 7 AAV-ie-K558R does not affect vestibular function.
  • FIG. 8 Generation and validation of Prestin knockout mice.
  • Prestin knockout mice were constructed by CRISPR base replacement. Two stop codons were simultaneously introduced in the coding sequence of exon 4 and exon 11 of Prestin to cause early transcription termination.
  • (b) Perform PCR amplification around the mutation site with the primers described in Methods to verify the genotype of Prestin knockout mice and perform sequencing detection.
  • AAV-ie-K558R-Prestin enables the expression of Prestin in both OHCs and IHCs.
  • AAV-ie-K558R was used to package a single-stranded (ss) AAV genome expressing Prestin through the constitutive CAG promoter.
  • Phalloidin green was used to label the morphology of F-actin and HCs.
  • Dapi blue color was used to label the nuclei.
  • Prestin knockout mice do not express Prestin.
  • Scale bar 10 ⁇ m
  • AAV-ie-K558R-Prestin enables the expression of Prestin in HCs and other cell types.
  • AAV-ie-Prestin can induce the expression of Prestin in HCs and other cell types, but to a lesser extent. (Scale bar: 10 ⁇ m).
  • AAV-ie-Atoh1 induces HC-like cells in neonatal mice. Scanning images of the apical, middle and basal regions of the cochlea injected with AAV-ie-Atoh1 (1 ⁇ 1010GCs) at P14. (Scale bar: 10 ⁇ m)
  • AAV-ie-K558R-Atoh1 induces HC-like cells in the GER region.
  • Scale bar 5 ⁇ m
  • FIG. 12 Flow chart of construction of AAV-ie-K558R vector in Example 1.
  • Adeno-associated virus is a single-stranded DNA virus containing two open reading frames (rep and cap).
  • the rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for genome replication, and the cap gene expresses three structural proteins (VP1-3) that assemble to form the viral capsid.
  • the present invention is based on the wild-type adeno-associated virus AAV-DJ, inserts the amino acid fragment shown in SEQ ID NO:1 between N589 and R590 of the capsid protein VP1 shown in SEQ ID NO:3, and obtains mutant adenocarcinoma Related virus AAV-ie (seeing patent literature CN110437317 A), then produced a series of mutations on the amino acid sequence of AAV-ie capsid protein VP1, to manipulate the phosphorylation or ubiquitination of AAV-ie in cells, the construction obtained Various mutants.
  • the present invention obtains a mutant adeno-associated virus AAV-ie-K558R containing a specific capsid protein amino acid mutant, which can efficiently transduce HCs and SCs, and is suitable for correcting dysfunctional gene mutations and HC-like cell regeneration, etc.
  • the present invention provides a mutant adeno-associated virus capsid protein, the capsid protein compared with the wild type Adeno-associated virus AAV-DJ capsid protein VP1, including the mutation of amino acid K at position 558.
  • the mutation of amino acid K at position 558 is K558R mutation.
  • the mutation of the 558th amino acid K includes amino acid deletion or substitution.
  • the substitution refers to replacing the 558th amino acid residue K with other non-K amino acids or their derivatives, such as G, A, V, L, I, P, F, W, M, Y, S, T , C, N, Q, D, E, R, H amino acid residues.
  • the amino acid fragment shown in SEQ ID NO.1 is inserted between N589 and R590 of the wild-type adeno-associated virus AAV-DJ capsid protein VP1 to obtain the adeno-associated virus AAV-ie capsid protein VP1, and then The 558th amino acid K is mutated to R, and the adeno-associated virus AAV-ie-K558R capsid protein VP1 as shown in SEQ ID NO.5 is obtained.
  • amino acid sequence of the wild-type AAV-DJ capsid protein VP1 is shown in SEQ ID NO.3.
  • amino acid sequence of the capsid protein VP1 of the adeno-associated virus AAV-ie is shown in SEQ ID NO.4.
  • amino acid sequence of the adeno-associated virus AAV-ie-K558R capsid protein VP1 is shown in SEQ ID NO.5.
  • the present invention also provides a nucleic acid encoding the mutant adeno-associated virus capsid protein as described above.
  • the present invention also provides a construct comprising the above-mentioned nucleic acid.
  • the construct can usually be constructed by inserting the above nucleic acid into an appropriate expression vector, and those skilled in the art can select an appropriate expression vector.
  • the present invention also provides a host cell, which contains the above-mentioned construct or the above-mentioned exogenous nucleic acid integrated in the genome.
  • mammalian cells such as CHO or COS
  • plant cells such as CHO or COS
  • human cells human embryonic kidney cells such as HEK293FT
  • bacterial cells such as Escherichia coli, Streptomyces sp., Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Sf9
  • a person skilled in the art can select a suitable host based on the teachings herein.
  • said 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 such as a human.
  • the host cells may be adherent cells or suspended cells, ie cells grown in suspension.
  • the present invention also provides a mutant adeno-associated virus, which contains the above-mentioned mutant adeno-associated virus capsid protein VP1.
  • the mutant adeno-associated virus is AAV-ie-K558R.
  • the mutant adeno-associated virus AAV-ie-K558R contains the amino acid fragment shown in SEQ ID NO.1 between N589 and R590 of the capsid protein VP1 and the 558th position Amino acid mutations.
  • the mutated adeno-associated virus can be produced by replacing the nucleotide encoding capsid protein VP1 with the nucleotide encoding the mutated adeno-associated virus AAV-ie-K558R capsid in the vector system for producing wild-type adeno-associated virus AAV-DJ. Obtained by post-nucleotide packaging of the coat protein VP1.
  • the adeno-associated virus AAV-ie-K558R compared with the adeno-associated virus AAV-ie (obtained with reference to patent document CN110437317 A), contains a mutation of the 558th amino acid.
  • the mutated adeno-associated virus can be produced by replacing the nucleotide encoding capsid protein VP1 with the nucleotide encoding the mutated adeno-associated virus AAV-ie-K558R capsid protein in the vector system for producing adeno-associated virus AAV-ie It is obtained by post-nucleotide packaging of VP1; or obtained by mutating the adeno-associated virus AAV-ie.
  • the Rep-Cap plasmid sequence used is shown in SEQ ID NO.2 (same as SEQ ID NO.6 in the AAV-ie patent document CN 110437317A).
  • the mutant adeno-associated virus also includes a heterologous nucleotide sequence encoding the target product, and the heterologous nucleotide sequence encoding the target product can be carried by various capsid proteins.
  • the above-mentioned heterologous nucleotide sequence encoding the product of interest may generally be a construct, and the construct may 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 an appropriate expression vector, and those skilled in the art can select an appropriate expression vector.
  • the above-mentioned expression vector can include but not limited to pAAV-CAG, pAAV- TRE, pAAV-EF1a, pAAV-GFAP promoter, pAAV-Lgr5 promoter, pAAV-Sox2 promoter expression vector, etc.
  • the mutated adeno-associated virus when the mutated adeno-associated virus encodes a heterologous nucleotide sequence of the target product, the mutated adeno-associated virus contains a capsid, the viral vector carries a transgene encoding the gene product, and the transgene base Because it is regulated by the regulatory sequence directing its expression in the host cell; in some preferred embodiments, the amino acid sequence of the capsid protein is shown in SEQ ID NO:5.
  • the target product can be nucleic acid or protein
  • the nucleic acid can be small guide RNA (sgRNA), interfering RNA (RNAi), etc.
  • the protein-coding gene can be Prestin, Atoh1.
  • the adeno-associated virus AAV-ie-K558R can be used as a carrier material to introduce exogenous genes into the cells of the subject. Compared with the parental wild-type AAV-DJ and the adeno-associated virus AAV-ie, the AAV-ie - K558R significantly increases the transduction efficiency of hair cells and supporting cells.
  • the present invention also provides an engineered host cell obtained by transforming the mutant adeno-associated virus as described above.
  • the engineered host cell contains the aforementioned mutant adeno-associated virus.
  • the host cells may be eukaryotic cells and/or prokaryotic cells.
  • mammalian cells such as CHO or COS
  • plant cells such as CHO or COS
  • human cells human embryonic kidney cells such as HEK293FT
  • bacterial cells such as Escherichia coli, Streptomyces sp., Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Sf9
  • a person skilled in the art can select a suitable host based on the teachings herein.
  • said 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 such as a human.
  • the host cells may be adherent cells or suspended cells, ie cells grown in suspension.
  • the present invention also provides a mutant adeno-associated virus vector system, the vector system comprises a packaging plasmid, and the packaging plasmid contains the above-mentioned nucleic acid fragment.
  • the packaging plasmid also contains the rep gene fragment of the adeno-associated virus.
  • the rep gene includes an intron
  • the intron includes a transcription termination sequence.
  • the adeno-associated virus vector system also includes an expression plasmid, and the expression plasmid contains heterologous nucleotides responsible for encoding the target product.
  • the adeno-associated virus vector system also includes a helper virus plasmid.
  • the adeno-associated virus vector system also includes host cells.
  • the packaging plasmid, expression plasmid and helper virus plasmid are transferred into the host cell, and the nucleic acid sequences thereof are all integrated in the host cell to produce the mutant adeno-associated virus.
  • the nucleic acid sequences are all integrated together at a single locus within the genome of the host cell.
  • the nucleic acid sequences encoding the various genes are present as separate expression cassettes, which prevent Risk of any 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 mutant adeno-associated virus, which is obtained from the above-mentioned mutant adeno-associated virus vector system through virus packaging.
  • the present invention also provides a pharmaceutical composition, which comprises the above-mentioned adeno-associated virus and a pharmaceutically acceptable carrier.
  • the acceptable carrier is such as sterile water or physiological saline, stabilizer, excipient, antioxidant (ascorbic acid, etc.), buffer (phosphoric acid, citric acid, other organic acids, etc.), preservative, surfactant (PEG, Tween, etc.), chelating agents (EDTA, etc.), adhesives, etc.
  • it may also contain other low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulin; amino acids such as glycine, glutamine, asparagine, arginine and lysine; carbohydrates such as polysaccharides and monosaccharides or Carbohydrates; sugar alcohols such as mannitol or sorbitol.
  • aqueous solution for injection such as physiological saline
  • isotonic solution containing glucose or other auxiliary drugs such as D-sorbitol, D-mannose, D-mannitol, sodium chloride
  • appropriate Solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), nonionic surfactants (Tween 80, HCO-50) and the like.
  • the AAV-ie-K558R can be a single active ingredient, or can be combined with one or more other active ingredients useful for hearing loss to form a joint preparation.
  • the other active components can be other various drugs that can be used for the treatment of hearing impairment.
  • the content of the active ingredient in the composition is generally a safe and effective amount, and the safe and effective amount should be adjustable for those skilled in the art, for example, the administration of the active ingredient of the AAV-ie-K558R and the pharmaceutical composition
  • the dosage usually depends on the body weight of the patient, the type of application, the condition and severity of the disease, for example, the dosage of the bifunctional compound as the active ingredient can usually be 1-1000 mg/kg/day, 20-200 mg/kg/day day, 1 ⁇ 3mg/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 ⁇ 150mg/kg/day, 150 ⁇ 200mg/kg/day, 200 ⁇ 300mg/kg/day, 300 ⁇ 500mg/kg/day, or 500 ⁇
  • the mutated adeno-associated virus provided by the present invention can be adapted to a suitable administration method, which 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 administration mode.
  • the present invention also provides the use of the above-mentioned mutant adeno-associated virus, host cell, vector system or pharmaceutical composition in the preparation of medicines for preventing and/or treating diseases;
  • diseases include, but are not limited to, one or more of hearing impairment, inflammation, tumor, metabolic disease, pain, neurodegenerative inflammatory disease, and the like.
  • the hearing impairment disease is selected from hearing loss, deafness and tinnitus.
  • the inflammation is selected from skin inflammation, vascular inflammation, allergy, autoimmune disease, fibrogenesis, 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, hematological tumor or solid tumor; specifically selected from adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid Tumors, diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, chromophobe renal cell carcinoma, clear cell renal cell carcinoma, renal papillary cell carcinoma, acute myeloid carcinoma Leukemia, low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, Malignant sarcoma, melanom
  • the metabolic disease is selected from diabetes, including type I and type II diabetes and diabetes-related diseases and disorders; the metabolic disease includes, but is not limited to, atherosclerosis, cardiovascular disease, nephropathy, neuropathy, retinopathy, beta - One or more of cellular dysfunction, dyslipidemia, hyperglycemia, insulin resistance, chronic obstructive pulmonary disease, etc.
  • the gene therapy refers to the treatment of hearing impairment diseases.
  • the mutated adeno-associated virus or the pharmaceutical composition can be used to treat hearing impairment diseases by delivering the target product to individual hair cells and/or supporting cells.
  • the target product delivery may be a non-diagnostic treatment
  • the purpose for example, can be in vitro, delivery of the product of interest to isolated hair cells and/or supporting cells.
  • the hair cells typically include outer hair cells and/or inner hair cells.
  • the target product is nucleic acid or protein
  • the nucleic acid can be small guide RNA (sgRNA), interfering RNA (RNAi) and the like.
  • the hearing impairment disease may be caused by cochlear damage caused by environmental factors. Therefore, the present invention also provides the use of the above-mentioned mutant adeno-associated virus in the medicine for treating hearing impairment caused by environmental factors in individuals.
  • the hearing impairment disease is a disease related to hair cells and/or supporting cells and/or spiral neuron cells.
  • the hearing impairment disease is a disease related to gene defect, environmental damage or aging, for example, it may be a related disease caused by gene mutation, and another example may be a related disease caused by noise or drugs, Another example may be related diseases caused by aging.
  • the hearing impairment disease can be related diseases such as cell damage, specifically cochlear hair cell damage, supporting cell damage, etc., more specifically cochlear hair cell damage caused by gene mutation, supporting cell damage caused by gene mutation, etc. Cell damage caused by noise, cell damage caused by drugs, or cell damage caused by aging.
  • mutant adeno-associated virus is used as a carrier for delivering the target product.
  • the present invention also provides a method for treating hearing impairment, the method comprising administering an effective amount of the mutant adeno-associated virus of the present invention, the host cell of the present invention, the vector system or the drug to a subject in need thereof combination.
  • the actual dosage which will be most suitable for an individual patient can be determined by a physician and will vary according to the age, weight and response of a particular individual.
  • mutant adeno-associated virus or host cell or vector system or pharmaceutical composition of the present invention can be administered to patients.
  • Those skilled in the art can determine the appropriate mode of administration and dosage.
  • the delivery of one or more therapeutic genes by the mutant adeno-associated virus of the present invention can be used alone or in combination with other therapeutic methods or therapeutic components.
  • the present invention also provides a conjugate, which includes the above-mentioned mutant adeno-associated virus or linked biologically active polypeptide.
  • the variant adeno-associated virus of the invention is 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 invention provides a method of delivering a transgene to a cell by infecting the cell with one or more variant adeno-associated viruses or conjugates of the invention, wherein the variant adeno-associated virus The virus or conjugate contains One or more transgenes.
  • the present invention also provides a method for producing a stable mutant adeno-associated virus vector production cell line, comprising:
  • 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.
  • the active compound when used in combination with other therapeutic agents, the active compound is co-administered with other therapeutic agents.
  • Co-administration means simultaneous administration via the same or different routes in the same formulation or in two different formulations, or sequential administration via the same or different routes.
  • Sequential administration means having a time difference in seconds, minutes, hours or days between the administration of two or more different compounds.
  • the mutant adeno-associated virus of the present invention and methods thereof can be used to prevent hearing loss, and can be administered as a prophylactic treatment before hearing loss or after a period of time after exposure to an environment prone to hearing loss .
  • a "carrier” refers to a macromolecule or combination of macromolecules that contains or binds to a polypeptide and can be used to mediate delivery of the polypeptide to cells.
  • 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.
  • references herein to a "recombinant AAV vector” refers to an AAV vector containing a heterologous polynucleotide sequence, typically a sequence of interest for genetically transforming cells.
  • the heterologous polynucleotide is flanked by at least one, usually two AAV terminal inverted repeats (ITRs).
  • AAV virus or "AAV virus particle” or “AAV vector particle” refers to a virus particle of an AAV vector comprising at least one AAV capsid protein and one enveloped polynucleotide. If the particle contains a heterologous polynucleotide (ie, a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is generally referred to as an "AAV vector particle", or "AAV vector”.
  • AAV vector particle ie, a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell
  • references herein to "packaging” refer to a series of intracellular processes leading to the assembly and encapsulation of AAV particles.
  • references herein to the AAV "rep” and “cap” genes refer to the polynucleotide sequences encoding the replication and capsid proteins of the adeno-associated virus.
  • AAV rep and cap refer to AAV "packaging genes" in the text.
  • helper virus for AAV refers to a virus that enables AAV to be replicated and packaged by mammalian cells.
  • a variety of such AAV helper viruses are known in the art, including adenoviruses, herpes viruses, and pox viruses (eg, vaccinia).
  • an "infectious" virus or virus particle is a cell containing a tropism capable of delivering a polynucleotide component into the virus species.
  • the term need not imply that the virus has any ability to replicate.
  • producer cell refers to a DNA genome having 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., composed of two AAV inverted terminal repeats (ITR) flanking target transgene) cell line.
  • AAV packaging genes rep and cap genes
  • helper viral genes e.g., the required helper viral genes
  • ITR inverted terminal repeats
  • the "individual” mentioned herein generally includes humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., which may be affected by treatment with the preparation, kit or combination preparation. benefit.
  • the “therapeutically effective dose” mentioned herein generally refers to an amount that can achieve the effect of treating the diseases listed above after an appropriate administration period.
  • treatment and prevention include alleviating the symptoms of a particular condition or preventing Stop or reduce the risk of a specific condition.
  • prevention is understood as reducing the severity of the onset of a particular condition. Treatment may also reduce the severity of pre-existing conditions or the frequency of exacerbations.
  • 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, cows, 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.
  • transfection transformation
  • transformation transformation
  • transfection transformation
  • transduction insertion of viral vectors into a target cell. Insertion of vectors is commonly referred to as transformation of bacterial cells and transfection of eukaryotic cells, although insertion of viral vectors may also be referred to as transduction.
  • non-viral transfection methods including but not limited to the use of physical methods (e.g., electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, magnetic transfection, gene gun or particle bombardment), chemical reagents (eg, calcium phosphate, hyperbranched organic compounds, or cationic polymers), or cationic lipids (eg, lipofection).
  • physical methods e.g., electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, magnetic transfection, gene gun or particle bombardment
  • chemical reagents eg, calcium phosphate, hyperbranched organic compounds, or cationic polymers
  • cationic lipids eg, lipofection
  • 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 the technical field conventional technology.
  • myosin 7A Myo7a, #25-6790 Proteus Biosciences, 1:1000
  • Sox2 Sox-2, #sc-17320, Santa Cruz Biotechnology, 1:1000
  • Flag Flag, #F3165, Sigma Aldrich, 1:1000
  • NeuN NeuN, #12943S, Cell Signaling Technology, 1:500.
  • Secondary antibodies Secondary antibodies of three different marker colors (TRIC, FITC, Cy5) for the secondary antibody markers anti-rat, mouse, rabbit, and goat, from Invitrogen.
  • DMEM Hyclone Company
  • fetal bovine serum Lensa Company
  • supplement N2 ThermoFisher Company
  • ampicillin ThermoFisher Company
  • double antibody ThermoFisher Company
  • Cell and tissue culture consumables Common consumables including various culture dishes, centrifuge tubes, pipettes, and disposable filters were purchased from Corning.
  • Example 1 The mutation of amino acids on the surface of the capsid protein improves the cochlear transduction efficiency of AAV-ie in vivo (1.1) Protein ubiquitination is related to AAV transduction efficiency, and blocking ubiquitination can improve the transduction efficiency of AAV in vitro and in vivo conduction efficiency.
  • Previous studies in the present invention have shown that AAV-ie can transduce rodent and human cochlear cells. However, the transduction efficiency of AAV-ie in the basal region of the cochlea is very low.
  • the present invention believes that mutating amino acids of the capsid protein on the exposed side of AAV-ie can improve its transduction efficiency (Fig. 1a). In order to verify this hypothesis, the present invention constructed 28 AAV-ie variants with different capsid protein sequences (Table 1).
  • AAV-ie-K558R The construction of AAV-ie-K558R is used as an example below, and the construction steps of other AAV-ie variants are similar.
  • the packaging of AAV requires three plasmids: the genome plasmid containing the gene of interest, and the Capsid plasmid (Rep-Cap plasmid) and Helper plasmid.
  • the sequence of the Cap protein in the Capsid plasmid determines the different serotypes of AAV, which in turn affects the preference of AAV-infected cells. Therefore, modification of Cap protein can lead to new AAV serotypes.
  • AAV-ie-K558R is generated by mutating a single amino acid K to R at position 558 on the Cap protein sequence of the parental AAV-ie.
  • design primers for the fragment from 558 amino acids to the Sma1 restriction site design a forward primer at sequence 558 of the Cap protein, introduce the K558R mutation, and add a 15-20nt homology arm that binds to the recombinant fragment 1 at the 5' end of the primer (Primer 3).
  • a reverse primer was designed at the Sma1 restriction site, and a 15-20nt homology arm (Primer 4) combined with the linearized AAV-ie capsid vector was added to the 5' end of the primer.
  • Recombinant fragment 2 was generated by polymerase chain reaction (PCR).
  • Recombinant Fragment 2 (Axygen: AP-GX-250G) was recovered. The recovered fragments were detected by Nanodrop2000.
  • the primers are designed as follows, gray indicates the mutated base of K558R introduced in the primers:
  • Forward primer Primer 1 5'-TTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGAC-3' (SEQ ID NO.6);
  • Reverse primer Primer 4 5'-CGCCCGCTGTTTAAACGCCCGGGCTGTAGTTAATGATTAACCCG-3' (SEQ ID NO.9).
  • the linearized AAV-ie capsid vector and the recombinant fragments 1 and 2 obtained by PCR reaction were recombined to produce AAV-ie-K558R.
  • Recombination system linearized AAV-iecapsid vector, 50ng; recombinant fragment 1, 30 ng; recombinant fragment 2, 30 ng; recombinant ligase, 1 ⁇ L; recombinant ligation buffer, 5 ⁇ L; , resulting in recombinant products.
  • Transformation steps are as follows: Thaw 100 ⁇ L of competent cells (TransGen: CD201) on ice; mix 10 ⁇ L of recombinant product with competent cells, and place on ice for 20 minutes; heat shock at 42°C for 60 seconds; place on ice for 2 minutes, add 400 ⁇ L Recover LB medium (MDBio: L001-1kg), shake for 30 minutes; take 70 ⁇ L ampicillin-coated plate (50 ⁇ g/ml, incubator at 37°C, and incubate for 14 hours.
  • the steps are as follows: centrifuge the bacterial solution at 4000 rpm for 10 minutes, discard the supernatant medium; add 350 ⁇ L of buffer S1, blow off the bacteria, and transfer to a 2ml centrifuge tube; add 250 ⁇ L of buffer S2, turn it upside down 8 times ; Add 250 ⁇ L of buffer S3, invert and mix 6 times to produce a precipitate; centrifuge at 12,000 rpm for 10 minutes, take the supernatant to pass through the column; centrifuge for 1 minute, discard the waste liquid, add 500 ⁇ L of W1, centrifuge for 1 minute, and discard Waste liquid; add 750 ⁇ L of W2, centrifuge, discard the supernatant; add 500 ⁇ L of W2, centrifuge, discard the supernatant; idle for 1 minute; add 50 ⁇ L of eluent, let stand for 2 minutes, and centrifuge. After concentration detection of the obtained plasmid, 10 ⁇ L was taken for sequencing, and the positive plasmid was stored at -20°
  • the resulting Rep-Cap plasmid expresses a genomic plasmid pAAV-CAG-mNeonGreen (full sequence of the plasmid as shown in SEQ ID NO.12) of a green fluorescent protein mNeonGreen (GenBank: LC279210.1) (same as AAV-ie patent document CN 110437317A SEQ ID NO.11)), pHelper plasmid (full sequence of the plasmid is shown in SEQ ID NO.13 (same as SEQ ID NO.12 of AAV-ie patent document CN 110437317A) shown) in HEK-293T with an appropriate amount
  • the AAV virus was purified by ultrahigh-speed centrifugation using an iodideanol gradient, and the virus titer was measured at a suitable concentration of 1E+12-1E+13GC/mL, and placed at -80°C for later use.
  • capsid protein variants and AAV viruses produced by their parental capsid proteins (AAV-ie and AAV-DJ, respectively) in HEK 293T cells cultured with DMEM+10% fetal bovine serum.
  • the MOI value of virus addition was 1000.
  • the expression of green fluorescent protein mNeonGreen was observed by fluorescence microscope.
  • the present invention uses an equal amount (4.5 ⁇ 10 9 ) of AAV-ie-
  • AAV-ie variants in (1.1) were screened in vivo by K558R particles injected into the cochlea of neonatal mouse P3 through the round window membrane (RWM) (Fig. 1b).
  • the cochlea at P14 stage was taken for immunostaining: the hair cells were labeled with Myo7a antibody, and the nuclear localization signal (NLS)-mNeonGreen signal showed the nuclear localization of the transduced cells in the cochlea.
  • the transduction efficiency of these AAV-ie variants was analyzed in the present invention and compared with AAV-ie under the same conditions.
  • the present invention also evaluated the targeting efficiency of AAV-ie-K558R in the vestibular system responsible for detecting linear motion and sensing gravity. It was observed that NLS-mNeonGreen was expressed in the whole mouse sensory epithelium, and AAV-ie-K558R was transfected in HCs and SCs. Conductivity is close to 100% (Figure 6).
  • AAV-ie-K558R can efficiently target different types of cells in the cochlea, revealing that it can serve as a suitable vector to mediate gene correction of auditory diseases or to regenerate HC-like cells in the cochlea or vestibular system. cell potential.
  • AAV-ie was a safe carrier and did not show any toxic effects on HCs and auditory function.
  • the present invention detects whether AAV-ie-K558R has security characteristics similar to AAV-ie.
  • AAV-ie-K558R injection group (3 mice in each group): AAV-ie-K558R-NLS-mNeonGreen (1 ⁇ 10 10 GCs) was injected into each ear of the mice through the round window membrane (RWM);
  • WT control group (Control, 3 mice in each group): AAV-ie-NLS-mNeonGreen (1 ⁇ 10 10 GCs) was injected into each ear of the mice through the round window membrane (RWM).
  • the auditory brainstem responses (ABRs) reflecting the auditory function of living animals and the distortion product otoacoustic emissions (DPOAE) reflecting the integrity of outer hair cells were measured to verify whether AAV-ie-K558R injection affects hearing. Also, the threshold showed no significant difference between the injected and control groups (Fig. 2e, 2f).
  • AAV-ie-K558R had similar side effects such as circling behavior or other gait abnormalities on the behavior of animals with severe vestibular impairment.
  • step 2.1 After four weeks of treatment in step 2.1, no AAV-ie-K558R note was observed in the open path-following test Indicate any abnormalities in gait or circulatory behavior.
  • AAV-ie-K558R has no negative effects on the auditory and vestibular systems.
  • Prestin knockout mice Using the Crispr-Cas system to simultaneously introduce two stop codons into the coding sequences of Prestin exon 4 and exon 11, resulting in the premature stop of prestin protein translation, achieving knockout of prestin in Expression of outer hairs in the cochlea to construct Prestin knockout mice.
  • the primers described in the method (SEQ ID NO.42-45: forward primer 1: 5'-CCACCACGTTTAGTAGCATC-3', reverse primer 1: 5'-ACTGTGATGAACATGAGCCA-3', forward primer 2: 5'-AGAGCACACCTGCGCTCTTC-3', reverse primer 2: 5'-AGTGTGGATGTCAGGCAGAGTA-3') Perform PCR amplification around the mutation site to verify the genotype of the Prestin knockout mice, and perform sequencing detection.
  • Example 4 AAV-ie-K558R-Prestin can partially restore the deafness of Prestin knockout mice
  • Prestin knockout mice in which the Prestin protein expression gene is deleted in OHCs. Prestin is a key molecule localized only in OHCs and plays a key role in mediating the electromotive force of OHCs. In Prestin knockout mice, the auditory threshold was significantly higher than in wild type (WT) (Fig. 3a-b). Prestin knockout mice did not exhibit circling behavior or other gait abnormalities (data not shown).
  • the AAV-ie-K558R-Prestin virus was injected into P1-2 ("P1-2" means 1 day or 2 days after birth) Prestin knockout mice to study its role in the treatment of OHCs Effects of genetic hearing disorders triggered by defective gene function in the medium.
  • P1-2 means 1 day or 2 days after birth
  • Prestin knockout mice to study its role in the treatment of OHCs Effects of genetic hearing disorders triggered by defective gene function in the medium.
  • the present invention conducted ABR experiments on different groups of mice.
  • step 4.2 One month after step 4.2 was processed, the ABR threshold of each group of mice was measured, and a partial reduction of the ABR threshold of the AAV-ie-K558R-Prestin experimental group was observed (Fig. 3b, 3c); DPOAE results showed that AAV-ie- The K558R-Prestin has 10dB relief at 16kHz.
  • AAV-ie-K558R can be used as a potential carrier to deliver genes to OHCs and be used for gene therapy in a deaf mouse model with hair cell deficiency.
  • Atoh1 is packaged into the AAV-ie-K558R vector
  • Atoh1 is packaged into the AAV-ie vector
  • HC regeneration may help restore hearing loss caused by aging, noise, or ototoxic drugs.
  • the transcription factor Atoh1 was shown to enable the transdifferentiation of SCs into HCs.
  • Earlier studies of the present invention showed that AAV-ie can deliver Atoh1 to SCs and make them transdifferentiate into HC-like cells.
  • AAV-ie-K558R-Atoh1 To assess the potential of AAV-ie-K558R vectors for HC regeneration, we used AAV-ie-K558R-Atoh1 to deliver mouse Atoh1 into neonatal mouse cochlea in vivo (Fig. 4a).
  • AAV-ie-Atoh1 injection group AAV-ie-Atoh1 (1 ⁇ 10 10 GCs per ear) was injected into the cochlea by RWM at P3.
  • AAV-ie-K558R-Atoh1 injection group AAV-ie-K558R-Atoh1 virus (1 ⁇ 10 10 GCs per ear) was injected into the cochlea by RWM at P3.
  • the cochlea was harvested at P14.
  • some HC-like cells expressing Myo7a appeared in the sensory area (Fig. 4b).
  • the AAV-ie-K558R-Atoh1 injected group a large number of new Myo7a-expressing HCs in the epithelial ridge (GER) region was observed.
  • some new HC-like cells in the AAV-ie-Atoh1-injected group and AAV-ie-K558R-Atoh1-injected group had sustained expression of Sox2, implying that these newly regenerated HCs might be in different developmental stages stage (Fig. 4b).
  • Atoh1 overexpression methods such as the genetic method used in previous studies, there was no significant difference in the number of regenerated HCs in the AAV-ie-K558R-Atoh1 injection group in this example, which indicates that AAV-ie-K558R is a highly efficient HCs regenerate viral vectors.
  • this example quantifies the morphology of newly regenerated HCs cells by SEM.
  • AAV-ie-K558R-Atoh1 induced the regeneration of many HC-like cells, and hair bundles grew out of the HC-like cells below the IHC region (Fig. 4c).
  • AAV-ie-Atoh1 was also able to induce regeneration of HC-like cells (Fig. 10).
  • SEM proved that AAV-ie-K558R-Atoh1 could induce some HC-like cells in the GER region ( Figure 11), while AAV-ie-Atoh1 could not produce similar effects.
  • the present invention reduces ubiquitination or phosphorylation by mutating the amino acids of the AAV-ie exposed side capsid protein Optimized for AAV-ie.
  • the findings of the present invention show that the newly generated AAV variant AAV-ie-K558 is safe and beneficial for both hair cell regeneration and gene therapy. Instead of high targeting efficiency, most AAV-ie variants showed lower targeting efficiency, suggesting that reduced ubiquitination or phosphorylation does not necessarily lead to higher transduction efficiency. Therefore, the exact mechanism of transduction efficiency awaits further investigation. These results also reveal the importance of performing screening experiments to determine the optimal AAV for different tissues. Nonetheless, the results of the present invention demonstrate that peptide insertion and amino acid mutations are feasible for engineering suitable AAVs to efficiently transduce cells in the cochlea and other tissues.
  • AAV-ie-K558R can partially restore the auditory function of Prestin knockout mice, because it has always been a major challenge to effectively deliver genes to OHCs, so this is a major progress in the field of gene therapy for auditory diseases.
  • the present invention achieved only partial restoration of auditory function with AAV-ie-K558R.
  • AAV-ie-K558R can also transduce IHCs, and the overexpression of Prestin in IHCs may lead to IHCs dysfunction, thereby preventing the full recovery of auditory function.
  • the expression level of Prestin in OHCs may have to be precisely controlled.
  • CAG gene regulatory promoter
  • AAV-ie-K558R-Atoh1 induces many new HCs in sensory regions with comparable efficiency to previously genetic mice. After measuring the regeneration efficiency of adenovirus and strong immune response, AAV-ie-K558R has more advantages in HC regeneration.
  • AAV-ie-K558R is the first AAV vector that can be used for gene therapy in a mouse model of deafness and can induce the regeneration of HC-like cells in neonatal mice.
  • the further development and improvement of AAV-ie-K558R will play a crucial role in gene therapy of different auditory diseases or better efficacy of HC cell regeneration.
  • AV-ie-K558R-mediated gene therapy not only restores hearing function in a deaf mouse model caused by genetic dysfunction of HCs or SCs, but also relieves environmental and age-induced deafness, which is especially important for hearing impairment The treatment has great application potential.

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Abstract

A variant adeno-associated virus and the use thereof in gene treatment and cell regeneration of a mediated cochlea. The variant adeno-associated virus can efficiently transduce hair cells and/or support cells in the cochlea, promotes regeneration of HC-like cells, and helps restoring hearing. The variant adeno-associated virus is used as a safe vector, and has great clinical potential in treating a hearing impairment disease caused by cochlear injury, and especially in treating hearing loss caused by hair cell death.

Description

变异型腺相关病毒及其在疾病治疗中的应用Mutant adeno-associated virus and its application in disease treatment 技术领域technical field
本发明涉及生物技术领域，涉及一种变异型腺相关病毒及其在疾病治疗中的应用，特别涉及AAV-ie-K558R及其在介导耳蜗基因治疗和毛细胞再生中的应用。The invention relates to the field of biotechnology, and relates to a mutant adeno-associated virus and its application in disease treatment, in particular to AAV-ie-K558R and its application in mediating cochlear gene therapy and hair cell regeneration.
背景技术Background technique
耳蜗由多种类型的细胞组成，包括毛细胞、支持细胞和螺旋神经节神经元，负责将机械能转换为电信号，以实现听觉。组成耳蜗的细胞，对听力至关重要。遗传和环境因素可导致耳蜗和听觉系统的功能障碍。虽然感音神经性耳聋可由耳蜗毛细胞(HCs)和支持细胞(SCs)的基因突变导致，但非基因的因素，如噪音、耳毒性药物或老化，也可通过损伤HCs诱发耳聋。在任何一种情况下，这些损伤对于不具备再生耳蜗细胞能力的哺乳动物来说都是不可逆的。虽然目前的治疗方法，如助听器和人工耳蜗，可以缓解一些患者的听力损失，但这些方法并不能支持他们在嘈杂环境中对自然声音的敏感性和感知能力。The cochlea is composed of multiple types of cells, including hair cells, supporting cells, and spiral ganglion neurons, responsible for converting mechanical energy into electrical signals for hearing. The cells that make up the cochlea are critical to hearing. Genetic and environmental factors can lead to dysfunction of the cochlea and auditory system. Although sensorineural deafness can be caused by genetic mutations in cochlear hair cells (HCs) and supporting cells (SCs), nongenetic factors, such as noise, ototoxic drugs, or aging, can also induce deafness by damaging HCs. In either case, the damage was irreversible in mammals that do not have the capacity to regenerate cochlear cells. While current treatments, such as hearing aids and cochlear implants, can alleviate hearing loss in some patients, these methods do not support their sensitivity and perception of natural sounds in noisy environments.
耳蜗中的HCs包括两种不同的类型：负责放大声音的外毛细胞(OHCs)和将机械能转化为电信号的内毛细胞(IHCs)。HCs将感觉上皮细胞固定在基底膜上，这对维持HCs正常功能的环境至关重要。值得注意的是，由于SCs有可能转分化为类HC(HC-like)细胞，因此HCs再生被认为是一种用于治疗由非遗传性因素引发的后天获得性耳聋的潜在方法。HCs in the cochlea include two distinct types: outer hair cells (OHCs), which amplify sound, and inner hair cells (IHCs), which convert mechanical energy into electrical signals. HCs anchor sensory epithelial cells to the basement membrane, which is critical for maintaining the environment in which HCs function properly. Notably, since SCs have the potential to transdifferentiate into HC-like cells, HCs regeneration has been considered as a potential approach for the treatment of acquired deafness caused by non-genetic factors.
基因治疗已经成为治疗遗传性疾病的重要手段。事实上，目前的研究进展也证明了它在治疗听力损失方面的潜力。当一些基因，如tmc1，clrn和otof，被传递到耳蜗时，可以恢复动物模型的听力功能。之前的研究表明，成年耳蜗的HCs再生可以恢复耳聋豚鼠模型的听觉功能，但还需要进一步研究，将这一发现扩展到其他动物模型。Gene therapy has become an important means of treating genetic diseases. In fact, current research advances also demonstrate its potential in the treatment of hearing loss. When some genes, such as tmc1, clrn and otof, were delivered to the cochlea, hearing function could be restored in animal models. Previous studies have shown that regeneration of HCs in the adult cochlea can restore auditory function in a guinea pig model of deafness, but further research is needed to extend this finding to other animal models.
近年来，基因治疗已经成为耳聋的一种有前景的治疗方法。基因治疗耳聋的一个主要挑战是如何有效地将基因传递给耳蜗的特定细胞。腺相关病毒(AAVs)已被证明在动物模型和人类中都具有很高的安全性，并被广泛用于向细胞传递遗传物质，用于许多不同器官和疾病的基因治疗。在听力领域，早期的研究发现，Anc80L65是一个很有前景的载体，可以传递Harmonin来治疗由 HCs功能障碍引发的耳聋。然而，其转导HCs的效率还有待提高。本发明已经开发的一种合成的AAV，AAV-ie，可以靶向SCs和HCs；并通过传递转录因子Atoh1使SCs转分化为类HC细胞以再生类HC细胞。然而，它对SCs或HCs的靶向效率还有待进一步提高，特别是在耳蜗的基底区。In recent years, gene therapy has emerged as a promising treatment for deafness. A major challenge in gene therapy for deafness is how to efficiently deliver genes to specific cells of the cochlea. Adeno-associated viruses (AAVs) have proven to be highly safe in both animal models and humans, and are widely used to deliver genetic material to cells for gene therapy in many different organs and diseases. In the field of hearing, early studies have found that Anc80L65 is a promising vector for delivering Harmonin for the treatment of Deafness caused by HCs dysfunction. However, its efficiency in transducing HCs needs to be improved. The present invention has developed a synthetic AAV, AAV-ie, which can target SCs and HCs; and transdifferentiate SCs into HC-like cells by delivering the transcription factor Atoh1 to regenerate HC-like cells. However, its targeting efficiency to SCs or HCs needs to be further improved, especially in the basal region of the cochlea.
发明内容Contents of the invention
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种变异型腺相关病毒及其在介导耳蜗基因治疗和毛细胞再生中的应用，用于解决现有技术中AAV对毛细胞和支持细胞转导效率不高，无法高效率利用基因治疗或者诱导毛细胞再生以用于治疗听力障碍疾病等问题。In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a mutant adeno-associated virus and its application in mediating cochlear gene therapy and hair cell regeneration, which is used to solve the problem of AAV in the prior art. The efficiency of gene therapy and supporting cell transduction is not high, and it is impossible to efficiently use gene therapy or induce hair cell regeneration for the treatment of hearing impairment diseases.
为实现上述目的及其他相关目的，本发明提供一种变异型腺相关病毒及其在介导耳蜗基因治疗和毛细胞再生中的应用。To achieve the above and other related purposes, the present invention provides a mutant adeno-associated virus and its application in mediating cochlear gene therapy and hair cell regeneration.
本发明的目的之一是提供一种变异型腺相关病毒衣壳蛋白，所述衣壳蛋白相比野生型腺相关病毒AAV-DJ衣壳蛋白VP1，包括第558位氨基酸K的突变。所述第558位氨基酸K的突变优选为K558R突变。One of the objectives of the present invention is to provide a mutant adeno-associated virus capsid protein. Compared with the wild-type adeno-associated virus AAV-DJ capsid protein VP1, the capsid protein includes a mutation of the 558th amino acid K. The mutation of amino acid K at position 558 is preferably K558R mutation.
本发明的另一目的是提供一种核酸，所述核酸编码如上所述的变异型腺相关病毒衣壳蛋白的核苷酸序列。Another object of the present invention is to provide a nucleic acid encoding the nucleotide sequence of the mutant adeno-associated virus capsid protein as described above.
本发明的另一目的是提供一种构建体，所述构建体含有如上所述的核酸。Another object of the present invention is to provide a construct comprising a nucleic acid as described above.
本发明的另一目的是提供一种宿主细胞，所述宿主细胞包含如上所述的构建体或基因组中整合有外源的如上所述的核酸。Another object of the present invention is to provide a host cell comprising the above-mentioned construct or exogenous nucleic acid as above integrated in the genome.
本发明的另一目的是提供一种变异型腺相关病毒，所述变异型腺相关病毒的衣壳结构含有如上所述的变异型腺相关病毒衣壳蛋白。Another object of the present invention is to provide a mutant adeno-associated virus, the capsid structure of the mutant adeno-associated virus contains the above-mentioned mutant adeno-associated virus capsid protein.
本发明的另一目的是提供使用如上所述的变异型腺相关病毒转化的宿主细胞。Another object of the present invention is to provide host cells transformed with the mutant adeno-associated virus as described above.
本发明的另一目的是提供一种变异型腺相关病毒载体系统，包含包装质粒，所述包装质粒中包含如上所述的核酸片段。Another object of the present invention is to provide a mutant adeno-associated virus vector system, which includes a packaging plasmid, and the packaging plasmid contains the above-mentioned nucleic acid fragment.
本发明的另一目的是提供一种变异型腺相关病毒，由如上所述的变异型腺相关病毒载体系统经病毒包装获得。Another object of the present invention is to provide a mutant adeno-associated virus, which is obtained from the above-mentioned mutant adeno-associated virus vector system through virus packaging.
本发明的另一目的是提供一种药物组合物，所述药物组合物包含如上所述的变异型腺相关病毒以及药学上可接受的载体。 Another object of the present invention is to provide a pharmaceutical composition, which comprises the above-mentioned mutant adeno-associated virus and a pharmaceutically acceptable carrier.
本发明的另一目的是提供一种如上所述的变异型腺相关病毒、宿主细胞、载体系统、药物组合物在制备治疗疾病的药物中的用途。Another object of the present invention is to provide a use of the above-mentioned mutant adeno-associated virus, host cell, vector system, and pharmaceutical composition in the preparation of medicines for treating diseases.
相对于现有技术，本发明的有益效果包括：Compared with the prior art, the beneficial effects of the present invention include:
本发明提供的所述变异型腺相关病毒具有减少的泛素化或磷酸化，能高效地转导耳蜗(如转导新生小鼠)中的毛细胞和支持细胞，可以使Prestin敲除小鼠的听力损失部分恢复，可以将Atoh1送入耳蜗支持细胞，产生类HC(HC-like)细胞。本发明所述的变异型腺相关病毒是一个安全的载体，使用该载体的听力损伤相关疾病的基因治疗在人中是安全和有效的，对听觉和前庭系统没有负面作用，例如，对HCs的形态没有显著影响，不会造成毛细胞的损失，也不会影响听力阈值，不会使个体步态或者前庭功能异常等，在治疗由毛细胞死亡引起的听力损失方面具有临床潜力。The mutant adeno-associated virus provided by the present invention has reduced ubiquitination or phosphorylation, can efficiently transduce hair cells and supporting cells in the cochlea (such as transducing newborn mice), and can make Prestin knockout mice Partial recovery of the hearing loss in the human body can send Atoh1 into the cochlear supporting cells to generate HC-like cells. The mutated adeno-associated virus of the present invention is a safe carrier, and the gene therapy for hearing loss-related diseases using the carrier is safe and effective in humans, and has no negative effects on the auditory and vestibular systems, for example, on HCs It has no significant effect on morphology, will not cause loss of hair cells, will not affect hearing threshold, will not make individual gait or vestibular function abnormal, etc., and has clinical potential in the treatment of hearing loss caused by hair cell death.
附图说明Description of drawings
图1.衣壳蛋白氨基酸突变增强了AAV的转导。(a)氨基酸突变策略。磷酸化部位的丝氨酸(S)被丙氨酸(A)取代，泛素化部位的赖氨酸(K)被精氨酸(R)取代。选择结构最相似的氨基酸是为了在防止磷酸化或泛素化修饰的同时，避免对衣壳蛋白产生任何重大的属性变化。(b)AAV突变体载体的筛选示意图。P3小鼠通过圆窗注射AAV-ie突变体，剂量为每耳4.5×109个含基因组(颗粒)(GCs)，病毒注射后11天将耳蜗解剖成虚线所示的三个部分，分别代表低频(顶端)、中频(中部)和高频(基底)。然后固定耳蜗并进行免疫组化标记，接着进行共聚焦成像。(c)AAV-ie和AAV-ie-S/K突变体载体在同等剂量下(4.5×109GCs)对外毛细胞和内毛细胞的转导效率。AAV-ie-K558R显示出与AAV-ie相当的转导效率。(d,e)比较AAV-ie和AAV-ie-K558R的转导效率。对病毒表达的NLS-mNeonGreen(绿色)进行成像，对Myo7a(品红色)和SOX2(红色)进行染色。鉴于虽然AAV-ie和AAV-ie-K558R都能有效地转导整个耳蜗的毛细胞和支持细胞，但AAV-ie-K558R在部分耳蜗显示出相同甚至更高的转导效率。(Scale bar:50μm)(f)AAV-ie和AAV-ie-K558R转导效率的统计分析。这里显示的数据是平均值±SEM。AAV-ie和AAV-ie-K558R之间进行了显著性检验，P值通过t检验计算。*p<0.05，**p<0.01，和***p<0.001。Figure 1. Capsid protein amino acid mutations enhance AAV transduction. (a) Amino acid mutation strategy. Serine (S) at the phosphorylation site is replaced by alanine (A), and lysine (K) at the ubiquitination site is replaced by arginine (R). The most structurally similar amino acids were chosen to avoid any major property changes to the capsid protein while preventing phosphorylation or ubiquitination modifications. (b) Schematic diagram of screening of AAV mutant vectors. P3 mice were injected with AAV-ie mutants through the round window at a dose of 4.5× 109 genome-containing (particles) (GCs) per ear, and the cochlea was dissected into three parts indicated by dotted lines 11 days after virus injection, representing Low frequency (top), mid frequency (middle) and high frequency (base). Cochleae were then fixed and immunohistochemically labeled, followed by confocal imaging. (c) Transduction efficiency of AAV-ie and AAV-ie-S/K mutant vectors at the same dose (4.5×10 9 GCs) to outer hair cells and inner hair cells. AAV-ie-K558R showed comparable transduction efficiency to AAV-ie. (d,e) Comparison of the transduction efficiency of AAV-ie and AAV-ie-K558R. Virus-expressed NLS-mNeonGreen (green) was imaged, stained for Myo7a (magenta) and SOX2 (red). Whereas, although both AAV-ie and AAV-ie-K558R could efficiently transduce hair cells and supporting cells of the whole cochlea, AAV-ie-K558R showed the same or even higher transduction efficiency in partial cochlea. (Scale bar: 50 μm) (f) Statistical analysis of transduction efficiency of AAV-ie and AAV-ie-K558R. Data shown here are mean ± SEM. Significance tests were performed between AAV-ie and AAV-ie-K558R, and P values were calculated by t-test. *p<0.05, **p<0.01, and ***p<0.001.
图2.AAV-ie-K558R是一个安全的载体。(a)AAV-ie-K558R研究的实验装置示意 图，研究载体的安全性。(b)WT对照组和AAV-ie-K558R-NLS-mNeonGreen注射的耳蜗的顶端、中部和基底区域的代表性SEM图像。(Scale bar:10μm)(c)未注射的WT小鼠和AAV-ie-K558R-NLS-mNeonGreen注射的耳蜗顶端、中间和基底区域每100μm的OHCs和IHCs的数量。(d)P14 WT对照组和AAV-ie K558R-NLS-mNeonGreen注射的耳蜗的外毛细胞(OHCs)和内毛细胞(IHCs)的放大SEM图像，在顶端、中部和基底区域。(Scale bar:1μm)(e,f)注射AAV-ie-K558R-NLS-mNeonGreen的耳朵在P3注射病毒后第27天和未注射的对侧耳朵的ABR和DPOAE阈值。病毒注射和AAV载体对小鼠听力没有影响。Figure 2. AAV-ie-K558R is a safe vector. (a) Schematic of the experimental setup for AAV-ie-K558R research Figure, research vector safety. (b) Representative SEM images of the apical, middle and basal regions of the WT control group and AAV-ie-K558R-NLS-mNeonGreen injected cochlea. (Scale bar: 10 μm) (c) The number of OHCs and IHCs per 100 μm in the apical, middle and basal regions of the cochlea from uninjected WT mice and AAV-ie-K558R-NLS-mNeonGreen injected. (d) Magnified SEM images of outer hair cells (OHCs) and inner hair cells (IHCs) of P14 WT control and AAV-ie K558R-NLS-mNeonGreen-injected cochlea, in the apical, middle, and basal regions. (Scale bar: 1 μm) (e,f) ABR and DPOAE thresholds of AAV-ie-K558R-NLS-mNeonGreen-injected ears at P3 on day 27 after virus injection and non-injected contralateral ears. Viral injections and AAV vectors had no effect on hearing in mice.
图3.AAV-ie-K558R-Prestin恢复了Prestin KO小鼠的听觉功能。(a)Prestin敲除小鼠听力恢复实验示意图。AAV-ie和AAV-ie-K558R载体被用来包装一个单链基因组，表达由CAG启动子驱动的Prestin。(b)未注射的WT小鼠、未经处理的Prestin敲除小鼠和注射了AAV-ie-Prestin或AAV-ie-K558R-Prestin的Prestin敲除小鼠在P28记录的ABR波形家族。ABRs是用以16kHz的声音变化的，声压水平以10dB为单位递增。阈值由峰1的存在决定，并由彩色痕迹表示。比例尺适用于所有家族。(c,d)注射AAV-ie-Prestin(黑色)或AAV-ie-K558R-Prestin(红色)的Prestin敲除小鼠的ABR和DPOAE阈值是刺激频率的函数。未注射的Prestin敲除小鼠的对侧耳朵被用作阴性对照(蓝色)，未注射的WT小鼠被用作阳性对照(紫色)。数据显示为平均值±SEM。显著性(*P<0.05，**P<0.01，***P<0.001)用AAV注射组和对侧未注射组的多重t检验计算。Figure 3. AAV-ie-K558R-Prestin restored auditory function in Prestin KO mice. (a) Schematic diagram of hearing recovery experiment in Prestin knockout mice. AAV-ie and AAV-ie-K558R vectors were used to package a single-stranded genome expressing Prestin driven by the CAG promoter. (b) Family of ABR waveforms recorded at P28 from uninjected WT mice, untreated Prestin knockout mice, and Prestin knockout mice injected with AAV-ie-Prestin or AAV-ie-K558R-Prestin. ABRs are used to vary the sound at 16kHz, and the sound pressure level is incremented in 10dB increments. Thresholds are determined by the presence of peak 1 and are represented by colored traces. Scales apply to all families. (c,d) ABR and DPOAE thresholds of Prestin knockout mice injected with AAV-ie-Prestin (black) or AAV-ie-K558R-Prestin (red) as a function of stimulation frequency. The contralateral ear of an uninjected Prestin knockout mouse was used as a negative control (blue), and an uninjected WT mouse was used as a positive control (purple). Data are shown as mean ± SEM. Significance (*P<0.05, **P<0.01, ***P<0.001) was calculated by multiple t-test between the AAV-injected group and the contralateral non-injected group.
图4.AAV-ie-K558R-Atoh1能使新生小鼠的HC样细胞再生。(a)AAV诱导的毛细胞再生在WT C57BL/6新生小鼠的AAV诱导毛细胞再生示意图。(b)用AAV-ie-Atoh1和AAV-ie-K558R-Atoh1转导的耳蜗的免疫荧光成像，剂量为1×1010GCs。(Scale bar:20μm)(c)P14时注射了AAV-ie-K558R-Atoh1的耳蜗的顶端、中部和基底区域的SEM图像。顶行，三行OHCs的编号。底行，方框内显示由AAV-ie-K558R-Atoh1再生的未成熟(白色)和成熟(黄色)HC样细胞。(Scale bar:5μm)。(d)中所示的未成熟和成熟的再生HC样细胞的放大的SEM图像，并进行了编号。在这些再生细胞中观察到立体细胞，而静纤毛被人为地染成了红色。不成熟的再生细胞没有明显的极性，而那些长出动纤毛(黄色箭头)并有一定极性的再生细胞被认为是比较成熟的。(Scale bar:1μm) Figure 4. AAV-ie-K558R-Atoh1 can regenerate HC-like cells in neonatal mice. (a) Schematic diagram of AAV-induced hair cell regeneration in WT C57BL/6 neonatal mice. (b) Immunofluorescence imaging of cochlea transduced with AAV-ie-Atoh1 and AAV-ie-K558R-Atoh1 at a dose of 1 × 10 10 GCs. (Scale bar: 20 μm) (c) SEM images of the apical, middle and basal regions of the cochlea injected with AAV-ie-K558R-Atoh1 at P14. Top row, numbering of the three OHCs. Bottom row, boxes show immature (white) and mature (yellow) HC-like cells regenerated by AAV-ie-K558R-Atoh1. (Scale bar: 5μm). Magnified SEM images of immature and mature regenerating HC-like cells shown in (d) and numbered. Stereocytic cells were observed in these regenerative cells, while stereocilia were artificially stained red. Immature regenerative cells have no apparent polarity, while those that develop kinetocilia (yellow arrows) and have some polarity are considered more mature. (Scale bar: 1μm)
图5.AAV-ie变体的转导效率。AAV-ie S/K突变体载体转导耳蜗、毛细胞层的免疫荧光图像。所有耳蜗在P3时用1.5微升AAV原液显微注射后，在P14时收获，用抗Myo7a抗体(品红色)染色，并对NLS-mNeonGreen荧光(绿色)进行成像。(Scale bar:50μm)从左至右、从上至下依次为K39R、K61R、K137R、K142R、K143R、K161R、K258R、K332R、K492R、K546R、K551R、K558R、K676R、K699R、K703R、K717R、S225A、S269A、S314A、S392A、S393A、S425A、S431A、S491A、S505A、S539A、S675A、S679A。Figure 5. Transduction efficiency of AAV-ie variants. Immunofluorescent images of cochlea, hair cell layer transduced with AAV-ie S/K mutant vector. All cochleae were harvested at P14 after microinjection with 1.5 μl of AAV stock solution at P3, stained with anti-Myo7a antibody (magenta) and imaged for NLS-mNeonGreen fluorescence (green). (Scale bar: 50μm) From left to right, top to bottom are K39R, K61R, K137R, K142R, K143R, K161R, K258R, K332R, K492R, K546R, K551R, K558R, K676R, K699R, K703R, K717R, S225A , S269A, S314A, S392A, S393A, S425A, S431A, S491A, S505A, S539A, S675A, S679A.
图6.AAV-ie-K558R广泛转导小鼠耳蜗、前庭感觉上皮细胞。(a)AAV-ie-K558R在其他细胞类型中转导的实验设置示意图。P3 WT小鼠注射AAV-ie-K558R-NLS-mNeonGreen，剂量为1×1010GCs。注射后11天收获了组织。(b)注射AAV-ie-K558R-NLS-mNeonGreen的耳蜗冷冻切片，用抗SOX2(红色)和Myo7a(品红色)的抗体染色，并对NLS-mNeonGreen的荧光(绿色)进行成像。图中显示AAV-ie-K558R有效地转导了耳蜗毛细胞和各种类型的支持细胞。(Scale bar:20μm)(c)左图，染色的小鼠支持细胞的代表性共聚焦图像。右图，左图中方框内区域的放大图。(d)与c中一样，为椭圆形毛细胞层。比例尺(c和d)：左，100μm，右：10μm。Figure 6. AAV-ie-K558R broadly transduces mouse cochlear and vestibular sensory epithelial cells. (a) Schematic of the experimental setup for AAV-ie-K558R transduction in other cell types. P3 WT mice were injected with AAV-ie-K558R-NLS-mNeonGreen at a dose of 1×10 10 GCs. Tissues were harvested 11 days after injection. (b) Cryosections of cochlea injected with AAV-ie-K558R-NLS-mNeonGreen, stained with antibodies against SOX2 (red) and Myo7a (magenta), and imaged for fluorescence of NLS-mNeonGreen (green). The figure shows that AAV-ie-K558R efficiently transduces cochlear hair cells and various types of supporting cells. (Scale bar: 20 μm) (c) Left panel, representative confocal images of stained mouse Sertoli cells. Right panel, enlarged view of the boxed area in the left panel. (d) As in c, the oval hair cell layer. Scale bars (c and d): left, 100 μm, right: 10 μm.
图7.AAV-ie-K558R不影响前庭功能。(a)在P2注射了AAV-ie-K558R的P28小鼠的窄梁行走试验路径轨迹(120秒)。(b)与a相同，但没有注射。(c)每分钟旋转次数。(d)每秒速度。(e)注射AAV-ie-K558R的小鼠和对照组小鼠穿越80厘米窄梁。Figure 7. AAV-ie-K558R does not affect vestibular function. (a) Narrow beam walking test path trajectory (120 sec) of P28 mice injected with AAV-ie-K558R at P2. (b) Same as a, but without injection. (c) Rotations per minute. (d) Speed per second. (e) AAV-ie-K558R-injected mice and control mice crossed an 80 cm narrow beam.
图8.Prestin敲除小鼠的产生和验证。(a)Prestin敲除小鼠是CRISPR的碱基替换构建的。在Prestin第4外显子和第11外显子的编码序列中同时引入两个终止密码子，以导致早期转录终止。(b)通过方法中描述的引物在突变位点周围进行PCR扩增，验证Prestin敲除小鼠的基因型，并进行测序检测。(c)通过对WT小鼠和Prestin敲除小鼠的Prestin特异性抗体(绿色)进行免疫组织化学检测，清楚地显示Prestin敲除小鼠的OHCs中没有Prestin表达。(Scale bar:20μm)Figure 8. Generation and validation of Prestin knockout mice. (a) Prestin knockout mice were constructed by CRISPR base replacement. Two stop codons were simultaneously introduced in the coding sequence of exon 4 and exon 11 of Prestin to cause early transcription termination. (b) Perform PCR amplification around the mutation site with the primers described in Methods to verify the genotype of Prestin knockout mice and perform sequencing detection. (c) Immunohistochemical detection of Prestin-specific antibodies (green) from WT and Prestin-knockout mice clearly shows no Prestin expression in OHCs of Prestin-knockout mice. (Scale bar:20μm)
图9.AAV-ie-K558R-Prestin使Prestin在OHCs和IHCs中都能表达。(a)AAV-ie-K558R被用来包装一个单链(ss)AAV基因组，该基因组通过组成型CAG启动子表达Prestin。(b)Phalloidin(绿色)被用来标记F-actin和HCs的形态。Dapi(蓝 色)被用来标记细胞核。Prestin敲除小鼠不表达Prestin。(Scale bar:10μm)(c)AAV-ie-K558R-Prestin使Prestin在HCs和其他类型的细胞中表达。(Scale bar:10μm)(d)AAV-ie-Prestin能诱导Prestin在HCs和其他类型细胞中的表达，但程度较低。(Scale bar:10μm)。Figure 9. AAV-ie-K558R-Prestin enables the expression of Prestin in both OHCs and IHCs. (a) AAV-ie-K558R was used to package a single-stranded (ss) AAV genome expressing Prestin through the constitutive CAG promoter. (b) Phalloidin (green) was used to label the morphology of F-actin and HCs. Dapi (blue color) was used to label the nuclei. Prestin knockout mice do not express Prestin. (Scale bar: 10 μm) (c) AAV-ie-K558R-Prestin enables the expression of Prestin in HCs and other cell types. (Scale bar: 10 μm) (d) AAV-ie-Prestin can induce the expression of Prestin in HCs and other cell types, but to a lesser extent. (Scale bar: 10μm).
图10.AAV-ie-Atoh1诱导新生小鼠的HC样细胞。注射了AAV-ie-Atoh1(1×1010GCs)的耳蜗在P14的顶端、中部和基底区域的扫描图像。(Scale bar:10μm)Figure 10. AAV-ie-Atoh1 induces HC-like cells in neonatal mice. Scanning images of the apical, middle and basal regions of the cochlea injected with AAV-ie-Atoh1 (1×1010GCs) at P14. (Scale bar: 10μm)
图11.AAV-ie-K558R-Atoh1诱导GER区域的HC样细胞。(a)用AAV-ie-Atoh1和AAV-ie-K558R-Atoh1以1×1010GCs的剂量转导的耳蜗GER的免疫荧光成像。AAV-ie-Atoh1和AAV-ie-K558R-Atoh1都能在GER区域产生Myo7a阳性细胞。(Scale bar:5μm)(b)耳蜗注射AAV-ie-Atoh1和AAV-ie-K558R-Atoh1后，在GER区域的P14的扫描图像。SEM图像验证了载体诱导的类似HC的细胞再生，形成了纤毛束。(Scale bar:left,20μm,right,1μm.)Figure 11. AAV-ie-K558R-Atoh1 induces HC-like cells in the GER region. (a) Immunofluorescent imaging of cochlear GER transduced with AAV-ie-Atoh1 and AAV-ie-K558R-Atoh1 at a dose of 1×10 10 GCs. Both AAV-ie-Atoh1 and AAV-ie-K558R-Atoh1 can generate Myo7a-positive cells in the GER region. (Scale bar: 5 μm) (b) Scanning images of P14 in the GER region after cochlear injection of AAV-ie-Atoh1 and AAV-ie-K558R-Atoh1. SEM images verified vehicle-induced regeneration of HC-like cells, forming cilia bundles. (Scale bar: left, 20μm, right, 1μm.)
图12.实施例1中AAV-ie-K558R载体的构建流程图。Fig. 12. Flow chart of construction of AAV-ie-K558R vector in Example 1.
具体实施方式Detailed ways
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.
腺相关病毒(AAV)是一种含有两个开放读框(rep和cap)的单链DNA病毒。rep基因编码基因组复制所需的4种蛋白质(Rep78、Rep68、Rep52和Rep40)，cap基因表达装配形成病毒衣壳的3种结构蛋白(VP1-3)。本发明基于野生型腺伴随病毒AAV-DJ，在其氨基酸序列如SEQ ID NO:3所示的衣壳蛋白VP1的N589与R590之间插入SEQ ID NO:1所示的氨基酸片段，获得变异性腺相关病毒AAV-ie(参见专利文献CN110437317 A)，然后在AAV-ie衣壳蛋白VP1的氨基酸序列上产生了一系列突变，以操纵AAV-ie在细胞中的磷酸化或泛素化，构建获得多种突变体。本发明经过多次研发筛选，获得一个包含特定的衣壳蛋白氨基酸突变体的变异型腺相关病毒AAV-ie-K558R，它可以高效地转导HCs和SCs，适用于纠正功能障碍的基因突变和类HC细胞再生等。Adeno-associated virus (AAV) is a single-stranded DNA virus containing two open reading frames (rep and cap). The rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for genome replication, and the cap gene expresses three structural proteins (VP1-3) that assemble to form the viral capsid. The present invention is based on the wild-type adeno-associated virus AAV-DJ, inserts the amino acid fragment shown in SEQ ID NO:1 between N589 and R590 of the capsid protein VP1 shown in SEQ ID NO:3, and obtains mutant adenocarcinoma Related virus AAV-ie (seeing patent literature CN110437317 A), then produced a series of mutations on the amino acid sequence of AAV-ie capsid protein VP1, to manipulate the phosphorylation or ubiquitination of AAV-ie in cells, the construction obtained Various mutants. After many times of research and development and screening, the present invention obtains a mutant adeno-associated virus AAV-ie-K558R containing a specific capsid protein amino acid mutant, which can efficiently transduce HCs and SCs, and is suitable for correcting dysfunctional gene mutations and HC-like cell regeneration, etc.
本发明提供了一种变异型腺相关病毒衣壳蛋白，所述衣壳蛋白相比野生型 腺相关病毒AAV-DJ衣壳蛋白VP1，包括第558位氨基酸K的突变。The present invention provides a mutant adeno-associated virus capsid protein, the capsid protein compared with the wild type Adeno-associated virus AAV-DJ capsid protein VP1, including the mutation of amino acid K at position 558.
作为优选，所述第558位氨基酸K的突变为K558R突变。所述第558位氨基酸K的突变包括氨基酸的缺失或替代。所述替代是指将第558位氨基酸残基K替换为其他非K氨基酸或其衍生物，如替换为G、A、V、L、I、P、F、W、M、Y、S、T、C、N、Q、D、E、R、H氨基酸残基。Preferably, the mutation of amino acid K at position 558 is K558R mutation. The mutation of the 558th amino acid K includes amino acid deletion or substitution. The substitution refers to replacing the 558th amino acid residue K with other non-K amino acids or their derivatives, such as G, A, V, L, I, P, F, W, M, Y, S, T , C, N, Q, D, E, R, H amino acid residues.
作为优选，将野生型腺相关病毒AAV-DJ衣壳蛋白VP1的N589与R590之间，插入如SEQ ID NO.1所示的氨基酸片段，获得腺相关病毒AAV-ie衣壳蛋白VP1，然后将第558位氨基酸K突变为R，获得如SEQ ID NO.5所示的腺相关病毒AAV-ie-K558R衣壳蛋白VP1。As preferably, the amino acid fragment shown in SEQ ID NO.1 is inserted between N589 and R590 of the wild-type adeno-associated virus AAV-DJ capsid protein VP1 to obtain the adeno-associated virus AAV-ie capsid protein VP1, and then The 558th amino acid K is mutated to R, and the adeno-associated virus AAV-ie-K558R capsid protein VP1 as shown in SEQ ID NO.5 is obtained.
DGTLAVPFK(SEQ ID NO.1)。DGTLAVPFK (SEQ ID NO. 1).
在本发明一些实施方式中，所述野生型AAV-DJ衣壳蛋白VP1的氨基酸序列如SEQ ID NO.3所示。In some embodiments of the present invention, the amino acid sequence of the wild-type AAV-DJ capsid protein VP1 is shown in SEQ ID NO.3.
在本发明一些实施方式中，所述腺相关病毒AAV-ie衣壳蛋白VP1的氨基酸序列如SEQ ID NO.4所示。In some embodiments of the present invention, the amino acid sequence of the capsid protein VP1 of the adeno-associated virus AAV-ie is shown in SEQ ID NO.4.


所述腺相关病毒AAV-ie-K558R衣壳蛋白VP1的氨基酸序列如SEQ ID NO.5所示。The amino acid sequence of the adeno-associated virus AAV-ie-K558R capsid protein VP1 is shown in SEQ ID NO.5.
本发明还提供了一种核酸，所述核酸编码如上所述的变异型腺相关病毒衣壳蛋白。 The present invention also provides a nucleic acid encoding the mutant adeno-associated virus capsid protein as described above.
本发明还提供了一种构建体，所述构建体含有上述的核酸。所述构建体通常可以通过将上述核酸插入合适的表达载体中构建获得，本领域技术人员可选择合适的表达载体。The present invention also provides a construct comprising the above-mentioned nucleic acid. The construct can usually be constructed by inserting the above nucleic acid into an appropriate expression vector, and those skilled in the art can select an appropriate expression vector.
本发明还提供了一种宿主细胞，所述宿主细胞包含上述的构建体或基因组中整合有外源的上述核酸。The present invention also provides a host cell, which contains the above-mentioned construct or the above-mentioned exogenous nucleic acid integrated in the genome.
作为适当宿主细胞的代表性示例，可举例哺乳动物细胞(如CHO或COS)、植物细胞、人细胞(人胚胎肾细胞如HEK293FT)、细菌细胞(如大肠杆菌、链霉菌属、鼠伤寒沙门氏菌)、真菌细胞(如酵母)、昆虫细胞(如Sf9)等。本领域技术人员根据本文教导能够选择合适的宿主。优选地，所述宿主细胞是动物细胞，且更优选是人细胞。宿主细胞可以是培养的细胞或原代细胞，即直接从生物体(如人)中分离。该宿主细胞可以是粘附性细胞或悬浮的细胞，即悬液形式生长的细胞。As representative examples of suitable host cells, 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 sp., Salmonella typhimurium) can be exemplified , fungal cells (such as yeast), insect cells (such as Sf9), etc. A person skilled in the art can select a suitable host based on the teachings herein. Preferably, said 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 such as a human. The host cells may be adherent cells or suspended cells, ie cells grown in suspension.
本发明还提供了一种变异型腺相关病毒，所述变异型腺相关病毒含有如上述变异型腺相关病毒衣壳蛋白VP1。在一些优选实施方式中，所述变异型腺相关病毒为AAV-ie-K558R。The present invention also provides a mutant adeno-associated virus, which contains the above-mentioned mutant adeno-associated virus capsid protein VP1. In some preferred embodiments, the mutant adeno-associated virus is AAV-ie-K558R.
所述变异型腺相关病毒AAV-ie-K558R相比野生型腺相关病毒AAV-DJ，在衣壳蛋白VP1的N589与R590之间包含如SEQ ID NO.1所示的氨基酸片段以及第558位氨基酸的突变。所述变异型腺相关病毒可以通过将生产野生型腺相关病毒AAV-DJ的载体系统中，将编码衣壳蛋白VP1的核苷酸替换为编码所述变异型腺相关病毒AAV-ie-K558R衣壳蛋白VP1的核苷酸后包装获得。Compared with the wild-type adeno-associated virus AAV-DJ, the mutant adeno-associated virus AAV-ie-K558R contains the amino acid fragment shown in SEQ ID NO.1 between N589 and R590 of the capsid protein VP1 and the 558th position Amino acid mutations. The mutated adeno-associated virus can be produced by replacing the nucleotide encoding capsid protein VP1 with the nucleotide encoding the mutated adeno-associated virus AAV-ie-K558R capsid in the vector system for producing wild-type adeno-associated virus AAV-DJ. Obtained by post-nucleotide packaging of the coat protein VP1.
所述腺相关病毒AAV-ie-K558R相比腺相关病毒AAV-ie(可参考专利文献CN110437317 A获得)，包含第558位氨基酸的突变。所述变异型腺相关病毒可以通过将生产腺相关病毒AAV-ie的载体系统中，将编码衣壳蛋白VP1的核苷酸替换为编码所述变异型腺相关病毒AAV-ie-K558R衣壳蛋白VP1的核苷酸后包装获得；或通过将腺相关病毒AAV-ie进行突变获得。The adeno-associated virus AAV-ie-K558R, compared with the adeno-associated virus AAV-ie (obtained with reference to patent document CN110437317 A), contains a mutation of the 558th amino acid. The mutated adeno-associated virus can be produced by replacing the nucleotide encoding capsid protein VP1 with the nucleotide encoding the mutated adeno-associated virus AAV-ie-K558R capsid protein in the vector system for producing adeno-associated virus AAV-ie It is obtained by post-nucleotide packaging of VP1; or obtained by mutating the adeno-associated virus AAV-ie.
在一些实施方式中，所述AAV-ie的包装过程中，采用的Rep-Cap质粒序列如SEQ ID NO.2所示(同AAV-ie专利文献CN 110437317A的SEQ ID NO.6)。In some embodiments, during the packaging process of the AAV-ie, the Rep-Cap plasmid sequence used is shown in SEQ ID NO.2 (same as SEQ ID NO.6 in the AAV-ie patent document CN 110437317A).
SEQ ID NO.2SEQ ID NO.2








进一步地，所述变异型腺相关病毒还包括编码目的产物的异源核苷酸序列，所述编码目的产物的异源核苷酸序可以是各种衣壳蛋白能包裹携带的。上述编码目的产物的异源核苷酸序列通常可以是构建体，所述构建体通常可以含有编码目的产物的核酸。所述构建体通常可以通过将编码目的产物的核酸插入合适的表达载体中构建获得，本领域技术人员可选择合适的表达载体，例如，上述表达载体可以是包括但不限于pAAV-CAG、pAAV-TRE、pAAV-EF1a、pAAV-GFAP promoter、pAAV-Lgr5 promoter、pAAV-Sox2 promoter表达载体等。本发明中，当所述变异型腺相关病毒编码目的产物的异源核苷酸序列时，所述变异型腺相关病毒含有衣壳，所述病毒载体携有编码基因产物的转基因，所述转基 因受指导其在宿主细胞内表达的调控序列的调控；在一些优选实施方式中，所述衣壳蛋白的氨基酸序列如SEQ ID NO:5所示。Further, the mutant adeno-associated virus also includes a heterologous nucleotide sequence encoding the target product, and the heterologous nucleotide sequence encoding the target product can be carried by various capsid proteins. The above-mentioned heterologous nucleotide sequence encoding the product of interest may generally be a construct, and the construct may 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 an appropriate expression vector, and those skilled in the art can select an appropriate expression vector. For example, the above-mentioned expression vector can include but not limited to pAAV-CAG, pAAV- TRE, pAAV-EF1a, pAAV-GFAP promoter, pAAV-Lgr5 promoter, pAAV-Sox2 promoter expression vector, etc. In the present invention, when the mutated adeno-associated virus encodes a heterologous nucleotide sequence of the target product, the mutated adeno-associated virus contains a capsid, the viral vector carries a transgene encoding the gene product, and the transgene base Because it is regulated by the regulatory sequence directing its expression in the host cell; in some preferred embodiments, the amino acid sequence of the capsid protein is shown in SEQ ID NO:5.
进一步地，所述目的产物可以为核酸或蛋白质，所述核酸可以是小向导RNA(sgRNA)、干扰RNA(RNAi)等，所述蛋白质编码基因可以是Prestin、Atoh1。Further, the target product can be nucleic acid or protein, the nucleic acid can be small guide RNA (sgRNA), interfering RNA (RNAi), etc., and the protein-coding gene can be Prestin, Atoh1.
所述腺相关病毒AAV-ie-K558R可以作为载体材料，将外源的基因导入受试个体的细胞中，相比于亲本野生型AAV-DJ和腺相关病毒AAV-ie，所述AAV-ie-K558R对毛细胞和支持细胞的转导效率显著提高。The adeno-associated virus AAV-ie-K558R can be used as a carrier material to introduce exogenous genes into the cells of the subject. Compared with the parental wild-type AAV-DJ and the adeno-associated virus AAV-ie, the AAV-ie - K558R significantly increases the transduction efficiency of hair cells and supporting cells.
本发明还提供了一种使用如上所述的变异型腺相关病毒转化获得的工程化宿主细胞。所述工程化宿主细胞包含上述变异型腺相关病毒。所述宿主细胞可以是真核细胞和/或原核细胞。The present invention also provides an engineered host cell obtained by transforming the mutant adeno-associated virus as described above. The engineered host cell contains the aforementioned mutant adeno-associated virus. The host cells may be eukaryotic cells and/or prokaryotic cells.
作为适当宿主细胞的代表性示例，可举例哺乳动物细胞(如CHO或COS)、植物细胞、人细胞(人胚胎肾细胞如HEK293FT)、细菌细胞(如大肠杆菌、链霉菌属、鼠伤寒沙门氏菌)、真菌细胞(如酵母)、昆虫细胞(如Sf9)等。本领域技术人员根据本文教导能够选择合适的宿主。优选地，所述宿主细胞是动物细胞，且更优选是人细胞。宿主细胞可以是培养的细胞或原代细胞，即直接从生物体(如人)中分离。该宿主细胞可以是粘附性细胞或悬浮的细胞，即悬液形式生长的细胞。As representative examples of suitable host cells, 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 sp., Salmonella typhimurium) can be exemplified , fungal cells (such as yeast), insect cells (such as Sf9), etc. A person skilled in the art can select a suitable host based on the teachings herein. Preferably, said 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 such as a human. The host cells may be adherent cells or suspended cells, ie cells grown in suspension.
本发明还提供了一种变异型腺相关病毒载体系统，所述载体系统包含包装质粒，所述包装质粒中包含如上所述的核酸片段。The present invention also provides a mutant adeno-associated virus vector system, the vector system comprises a packaging plasmid, and the packaging plasmid contains the above-mentioned nucleic acid fragment.
进一步地，所述包装质粒中还包含腺相关病毒的rep基因片段。其中所述rep基因包含内含子，所述内含子包含转录终止序列。Further, the packaging plasmid also contains the rep gene fragment of the adeno-associated virus. Wherein the rep gene includes an intron, and the intron includes a transcription termination sequence.
进一步地，所述腺相关病毒载体系统还包括表达质粒，所述表达质粒包含负责编码目的产物的异源核苷酸。Further, the adeno-associated virus vector system also includes an expression plasmid, and the expression plasmid contains heterologous nucleotides responsible for encoding the target product.
进一步地，所述腺相关病毒载体系统还包括辅助病毒质粒。Further, the adeno-associated virus vector system also includes a helper virus plasmid.
进一步地，所述腺相关病毒载体系统还包括宿主细胞。Further, the adeno-associated virus vector system also includes host cells.
将所述的包装质粒、表达质粒、辅助病毒质粒转入宿主细胞，其中的核酸序列全部整合于宿主细胞中，以生产所述变异型腺相关病毒。在一些实施方式中，所述核酸序列全部一起整合在所述宿主细胞细胞基因组内的单一基因座处。在一些实施方式中，编码各种基因的核酸序列作为单独的表达盒存在，其防止 任何重组以形成能够复制的病毒的风险；编码rep和cap基因的核酸序列存在于同一表达盒中。The packaging plasmid, expression plasmid and helper virus plasmid are transferred into the host cell, and the nucleic acid sequences thereof are all integrated in the host cell to produce the mutant adeno-associated virus. In some embodiments, the nucleic acid sequences are all integrated together at a single locus within the genome of the host cell. In some embodiments, the nucleic acid sequences encoding the various genes are present as separate expression cassettes, which prevent Risk of any 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.
本发明还提供了一种变异型腺相关病毒，由如上所述的变异型腺相关病毒载体系统经病毒包装获得。The present invention also provides a mutant adeno-associated virus, which is obtained from the above-mentioned mutant adeno-associated virus vector system through virus packaging.
本发明还提供了一种药物组合物，所述药物组合物包含如上所述的腺相关病毒以及药学上可接受的载体。The present invention also provides a pharmaceutical composition, which comprises the above-mentioned adeno-associated virus and a pharmaceutically acceptable carrier.
所述可接受的载体例如无菌水或生理盐水、稳定剂、赋形剂、抗氧化剂(抗坏血酸等)、缓冲剂(磷酸、枸橼酸、其它的有机酸等)、防腐剂、表面活性剂(PEG、Tween等)、螯合剂(EDTA等)、粘合剂等。而且，也可含有其它低分子量的多肽；血清白蛋白、明胶或免疫球蛋白等蛋白质；甘氨酸、谷酰胺、天冬酰胺、精氨酸和赖氨酸等氨基酸；多糖和单糖等糖类或碳水化物；甘露糖醇或山梨糖醇等糖醇。当制备用于注射的水溶液时，例如生理盐水、含有葡萄糖或其它的辅助药物的等渗溶液，如D-山梨糖醇、D-甘露糖、D-甘露糖醇、氯化钠，可并用适当的增溶剂例如醇(乙醇等)、多元醇(丙二醇，PEG等)、非离子表面活性剂(吐温80，HCO-50)等。The acceptable carrier is such as sterile water or physiological saline, stabilizer, excipient, antioxidant (ascorbic acid, etc.), buffer (phosphoric acid, citric acid, other organic acids, etc.), preservative, surfactant (PEG, Tween, etc.), chelating agents (EDTA, etc.), adhesives, etc. Moreover, it may also contain other low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulin; amino acids such as glycine, glutamine, asparagine, arginine and lysine; carbohydrates such as polysaccharides and monosaccharides or Carbohydrates; sugar alcohols such as mannitol or sorbitol. When preparing an aqueous solution for injection, such as physiological saline, isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, appropriate Solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), nonionic surfactants (Tween 80, HCO-50) and the like.
本发明所提供的药物组合物中，所述AAV-ie-K558R可以是单一有效成分，也可以与其他一种或多种对于听力损伤有用的活性组分进行组合，构成联合制剂。所述其他活性组分可以是其他各种可以用于治疗听力损伤的药物。组合物中活性组分的含量通常为安全有效量，所述安全有效量对于本领域技术人员来说应该是可以调整的，例如，所述AAV-ie-K558R和药物组合物的活性成分的施用量通常依赖于患者的体重、应用的类型、疾病的病情和严重程度，例如，作为活性成分的所述双功能化合物的施用量通常可以为1～1000mg/kg/day、20～200mg/kg/day、1～3mg/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、100～150mg/kg/day、150～200mg/kg/day、200～300mg/kg/day、300～500mg/kg/day、或500～1000mg/kg/day。In the pharmaceutical composition provided by the present invention, the AAV-ie-K558R can be a single active ingredient, or can be combined with one or more other active ingredients useful for hearing loss to form a joint preparation. The other active components can be other various drugs that can be used for the treatment of hearing impairment. The content of the active ingredient in the composition is generally a safe and effective amount, and the safe and effective amount should be adjustable for those skilled in the art, for example, the administration of the active ingredient of the AAV-ie-K558R and the pharmaceutical composition The dosage usually depends on the body weight of the patient, the type of application, the condition and severity of the disease, for example, the dosage of the bifunctional compound as the active ingredient can usually be 1-1000 mg/kg/day, 20-200 mg/kg/day day, 1～3mg/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, 100～150mg/kg/day, 150～200mg/kg/day, 200～300mg/kg/day, 300～ 500mg/kg/day, or 500~1000mg/kg/day.
本发明所提供的变异型腺相关病毒可以适应于合适的给药方式，可以是注射到耳蜗、眼睛、肌肉、神经系统或者血液循环系统。本领域技术人员可根据给药方式，选择合适的剂量。 The mutated adeno-associated virus provided by the present invention can be adapted to a suitable administration method, which 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 administration mode.
本发明还提供了上述变异型腺相关病毒、宿主细胞、载体系统或药物组合物在制备预防和/或治疗疾病药物中的用途；优选地，在用于制备预防和/或治疗基因治疗疾病的药物中的用途；所述疾病包括但不限于是听力障碍疾病、炎症、肿瘤、代谢疾病、疼痛、神经退行性炎症疾病等中的一种或几种。The present invention also provides the use of the above-mentioned mutant adeno-associated virus, host cell, vector system or pharmaceutical composition in the preparation of medicines for preventing and/or treating diseases; Use in medicine; the diseases include, but are not limited to, one or more of hearing impairment, inflammation, tumor, metabolic disease, pain, neurodegenerative inflammatory disease, and the like.
所述听力障碍疾病选自听力下降、耳聋、耳鸣。The hearing impairment disease is selected from hearing loss, deafness and tinnitus.
所述炎症选自皮肤炎症、脉管炎症、变态反应、自身免疫性疾病、纤维组织形成、硬皮病或移植物排斥；所述自身免疫性疾病选自风湿关节炎、系统性硬化症、系统性红斑狼疮、类口眼干燥综合征、多发性肌炎等中的一种或多种。The inflammation is selected from skin inflammation, vascular inflammation, allergy, autoimmune disease, fibrogenesis, 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.
所述癌症选自淋巴瘤、血液瘤或实体瘤；具体选自肾上腺皮质癌、膀胱尿路上皮癌、乳腺癌、宫颈鳞状细胞癌、宫颈内腺癌、胆管癌、结肠腺癌、淋巴样肿瘤、弥散性大B细胞淋巴瘤、食管癌、多形性成胶质细胞瘤、头颈部鳞状细胞癌、肾嫌色细胞癌、肾透明细胞癌、肾乳头状细胞癌、急性髓性白血病、脑低度胶质瘤、肝细胞癌、肺腺癌、肺鳞状细胞癌、间皮细胞癌、卵巢癌、胰腺癌、嗜铬细胞瘤和副神经节瘤、前列腺癌、直肠癌、恶性肉瘤、黑色素瘤、胃癌、睾丸生殖细胞肿瘤、甲状腺癌、胸腺癌、子宫内膜癌、子宫肉瘤、葡萄膜黑色素瘤、多发性骨髓瘤、急性淋系白血病、慢性淋系白血病、慢性髓性白血病、T细胞淋巴瘤、B细胞淋巴瘤肿瘤细胞中的一种或多种；优选地，所述肿瘤为结直肠癌和/或黑色素细胞瘤等中的一种或几种。The cancer is selected from lymphoma, hematological tumor or solid tumor; specifically selected from adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid Tumors, diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, chromophobe renal cell carcinoma, clear cell renal cell carcinoma, renal papillary cell carcinoma, acute myeloid carcinoma Leukemia, low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, Malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymus cancer, endometrial cancer, uterine sarcoma, uveal melanoma, multiple myeloma, acute lymphoid leukemia, chronic lymphoid leukemia, chronic myeloid One or more of leukemia, T-cell lymphoma, B-cell lymphoma tumor cells; preferably, the tumor is one or more of colorectal cancer and/or melanoma, etc.
所述代谢疾病选自糖尿病，包括I型和II型糖尿病以及与糖尿病相关的疾病和病症；所述代谢性疾病包括但不限于动脉粥样硬化，心血管疾病，肾病，神经病，视网膜病，β-细胞机能障碍，血脂异常，高血糖，胰岛素抵抗、慢性阻塞性肺病等中的一种或几种。The metabolic disease is selected from diabetes, including type I and type II diabetes and diabetes-related diseases and disorders; the metabolic disease includes, but is not limited to, atherosclerosis, cardiovascular disease, nephropathy, neuropathy, retinopathy, beta - One or more of cellular dysfunction, dyslipidemia, hyperglycemia, insulin resistance, chronic obstructive pulmonary disease, etc.
在一些更优选实施方式中，所述基因治疗是指听力障碍疾病的治疗。所述变异型腺相关病毒或药物组合物可通过将目的产物递送至个体的毛细胞和/或支持细胞以实现听力障碍疾病的治疗。In some more preferred embodiments, the gene therapy refers to the treatment of hearing impairment diseases. The mutated adeno-associated virus or the pharmaceutical composition can be used to treat hearing impairment diseases by delivering the target product to individual hair cells and/or supporting cells.
本发明所述变异型腺相关病毒、宿主细胞、载体系统或药物组合物在用于将目的产物递送至个体的毛细胞和/或支持细胞中的用途中，所述目的产物递送可以是非诊断治疗目的的，例如，可以是体外的，对离体的毛细胞和/或支持细胞进行目的产物递送。所述毛细胞通常包括外毛细胞和/或内毛细胞。 In the use of the mutant adeno-associated virus, host cell, carrier system or pharmaceutical composition of the present invention for delivering the target product to the hair cells and/or supporting cells of an individual, the target product delivery may be a non-diagnostic treatment The purpose, for example, can be in vitro, delivery of the product of interest to isolated hair cells and/or supporting cells. The hair cells typically include outer hair cells and/or inner hair cells.
进一步地，所述目的产物为核酸或蛋白质，所述核酸可以是小向导RNA(sgRNA)、干扰RNA(RNAi)等。Further, the target product is nucleic acid or protein, and the nucleic acid can be small guide RNA (sgRNA), interfering RNA (RNAi) and the like.
本发明中，所述听力障碍疾病可以是由环境因素引起的耳蜗损伤导致的。因此，本发明还提供了上述变异型腺相关病毒在治疗个体中环境因素导致的听力障碍疾病的药物中的用途。In the present invention, the hearing impairment disease may be caused by cochlear damage caused by environmental factors. Therefore, the present invention also provides the use of the above-mentioned mutant adeno-associated virus in the medicine for treating hearing impairment caused by environmental factors in individuals.
进一步地，所述听力障碍疾病为毛细胞和/或支持细胞和/或螺旋神经元细胞相关疾病。Further, the hearing impairment disease is a disease related to hair cells and/or supporting cells and/or spiral neuron cells.
进一步地，所述听力障碍疾病为基因缺陷、环境损伤或衰老的相关疾病，例如，可以是基因突变等所导致的相关疾病，再例如，可以是噪声所致的、药物所导致的相关疾病，再例如，可以是衰老所导致的相关疾病。Further, the hearing impairment disease is a disease related to gene defect, environmental damage or aging, for example, it may be a related disease caused by gene mutation, and another example may be a related disease caused by noise or drugs, Another example may be related diseases caused by aging.
进一步地，听力障碍疾病可以为细胞损伤等的相关疾病，具体可以是耳蜗毛细胞损伤、支持细胞损伤等，更具体可以是基因突变导致的耳蜗毛细胞损伤、基因突变导致的支持细胞损伤等、噪声所致的细胞损伤、药物所导致的细胞损伤或者衰老所导致的细胞损伤。Further, the hearing impairment disease can be related diseases such as cell damage, specifically cochlear hair cell damage, supporting cell damage, etc., more specifically cochlear hair cell damage caused by gene mutation, supporting cell damage caused by gene mutation, etc. Cell damage caused by noise, cell damage caused by drugs, or cell damage caused by aging.
进一步地，所述变异型腺相关病毒作为递送目的产物的载体。Further, the mutant adeno-associated virus is used as a carrier for delivering the target product.
本发明还提供了一种治疗听力障碍疾病的方法，所述方法包括向有此需要的对象给予有效量的本发明所述的变异型腺相关病毒、本发明所述宿主细胞、载体系统或药物组合物。通常，医师都可确定对单个患者而言最适用的实际剂量且其会根据特定个体的年龄、体重和反应而变化。The present invention also provides a method for treating hearing impairment, the method comprising administering an effective amount of the mutant adeno-associated virus of the present invention, the host cell of the present invention, the vector system or the drug to a subject in need thereof combination. In general, the actual dosage which will be most suitable for an individual patient can be determined by a physician and will vary according to the age, weight and response of a particular individual.
在本发明中，可向患者给予本发明的变异型腺相关病毒或宿主细胞或载体系统或药物组合物。本领域技术人员能够确定适当的给药方式和剂量。In the present invention, the mutant adeno-associated virus or host cell or vector system or pharmaceutical composition of the present invention can be administered to patients. Those skilled in the art can determine the appropriate mode of administration and dosage.
通过本发明所述变异型腺相关病毒递送一种或多种治疗基因，可单独使用或与其他治疗方法或治疗组分联用。The delivery of one or more therapeutic genes by the mutant adeno-associated virus of the present invention can be used alone or in combination with other therapeutic methods or therapeutic components.
本发明还提供了一种偶联物，所述偶联物包括如上所述的变异型腺相关病毒或连接的生物活性多肽。The present invention also provides a conjugate, which includes the above-mentioned mutant adeno-associated virus or linked biologically active polypeptide.
本发明的变异型腺相关病毒用于感染细胞，从而将基因和/或连接的(例如但不限于，共价连接的)生物活性多肽递送至细胞。因此，本发明提供了一种将转基因递送至细胞的方法，所述方法通过使一个或多个本发明的变异型腺相关病毒或偶联物至细胞来感染细胞，其中所述变异型腺相关病毒或偶联物包含 一个或多个转基因。The variant adeno-associated virus of the invention is 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 invention provides a method of delivering a transgene to a cell by infecting the cell with one or more variant adeno-associated viruses or conjugates of the invention, wherein the variant adeno-associated virus The virus or conjugate contains One or more transgenes.
本发明还提供了产生稳定的变异型腺相关病毒载体的生产细胞系的方法，其包括：The present invention also provides a method for producing a stable mutant adeno-associated virus vector production cell line, comprising:
(a)将如本文所定义的变异型腺相关病毒载体引入哺乳动物宿主细胞的培养物中；和(a) introducing a variant adeno-associated viral vector as defined herein into a culture of mammalian host cells; and
(b)在所述培养物内选择具有在整合至所述哺乳动物宿主细胞的内源染色体中的载体上编码的核酸序列的哺乳动物宿主细胞。(b) selecting within said culture a mammalian host cell having a nucleic acid sequence encoded on a vector integrated into an endogenous chromosome of said mammalian host cell.
所述AAV载体生产细胞是哺乳动物细胞。在一些实施方式中，所述哺乳动物细胞选自HEK293细胞、CHO细胞、Jurkat细胞、K562细胞、PerC6细胞、HeLa细胞或其衍生物。在一些实施方式中，所述哺乳动物宿主细胞是HEK293细胞，或源自HEK293细胞。在一些实施方式中，所述HEK293细胞是HEK293T细胞。The AAV vector producing cells are mammalian cells. In some embodiments, the mammalian cells are 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, or is derived from, a HEK293 cell. In some embodiments, the HEK293 cells are HEK293T cells.
AAV的各种血清型的基因组序列以及天然ITR、Rep蛋白和衣壳亚基的序列是本领域中已知的。此类序列可见于文献中或公共数据库诸如GenBank中。其公开内容通过引用并入本文用于教导AAV核酸和氨基酸序列。The genome sequences of the various serotypes of AAV as well as the sequences of the native ITRs, Rep proteins and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. The disclosure thereof is incorporated herein by reference for the teaching of AAV nucleic acid and amino acid sequences.
本发明化合物及其应用中，当所述变异型腺相关病毒与其他治疗剂联用时，所述活性化合物与其他治疗剂共同给予。“共同给予”表示在同一制剂中或在两种不同制剂中经由相同或不同途径同时给予，或通过相同或不同途径顺次给予。“顺次”给予表示在两种或多种不同化合物的给药之间具有以秒、分钟、小时或天计的时间差异。In the compounds of the present invention and their applications, when the mutant adeno-associated virus is used in combination with other therapeutic agents, the active compound is co-administered with other therapeutic agents. "Co-administration" means simultaneous administration via the same or different routes in the same formulation or in two different formulations, or sequential administration via the same or different routes. "Sequential" administration means having a time difference in seconds, minutes, hours or days between the administration of two or more different compounds.
在某些实施方式中，本发明的变异型腺相关病毒及其方法可用于预防听力损伤，可作为预防性治疗方法在听力损伤之前施用或在接触易造成听力损伤的环境后的一段时间后施用。In certain embodiments, the mutant adeno-associated virus of the present invention and methods thereof can be used to prevent hearing loss, and can be administered as a prophylactic treatment before hearing loss or after a period of time after exposure to an environment prone to hearing loss .
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.
本文述及“载体”指含有多肽或与多肽结合并可用于介导多肽递送至细胞的大分子或大分子的联合体。说明性的载体包括，例如质粒、病毒载体、脂质体或其它基因递送载体。 As used herein, a "carrier" refers to a macromolecule or combination of macromolecules that contains or binds to a polypeptide and can be used to mediate delivery of the polypeptide to cells. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, or other gene delivery vehicles.
本文述及“AAV”是腺相关病毒的缩写，可用于指病毒自身或其衍生物。As used herein, "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or its derivatives.
本文述及“重组AAV载体”指含有异源性多核苷酸序列的AAV载体，通常是用于遗传性转化细胞的感兴趣的序列。总体上，异源多核苷酸的两侧连接有至少一条，一般是两条AAV末端反向重复序列(ITR)。Reference herein to a "recombinant AAV vector" refers to an AAV vector containing a heterologous polynucleotide sequence, typically a sequence of interest for genetically transforming cells. Generally, the heterologous polynucleotide is flanked by at least one, usually two AAV terminal inverted repeats (ITRs).
本文述及“AAV病毒”或“AAV病毒颗粒”或“AAV载体颗粒”指至少含有一种AAV衣壳蛋白和一种包裹的多核苷酸的AAV载体的病毒颗粒。如果该颗粒含有异源性多核苷酸(即，除野生型AAV基因组以外的多核苷酸，例如要递送至哺乳动物细胞的转基因)，一般称为“AAV载体颗粒”、或“AAV载体”。Reference herein to "AAV virus" or "AAV virus particle" or "AAV vector particle" refers to a virus particle of an AAV vector comprising at least one AAV capsid protein and one enveloped polynucleotide. If the particle contains a heterologous polynucleotide (ie, a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is generally referred to as an "AAV vector particle", or "AAV vector".
本文述及“包装”指导致AAV颗粒装配和包裹的一系列胞内过程。References herein to "packaging" refer to a series of intracellular processes leading to the assembly and encapsulation of AAV particles.
本文述及AAV“rep”和“cap”基因指编码腺相关病毒的复制和包裹蛋白的多核苷酸序列。文中AAV rep和cap指AAV“包装基因”。References herein to the AAV "rep" and "cap" genes refer to the polynucleotide sequences encoding the replication and capsid proteins of the adeno-associated virus. AAV rep and cap refer to AAV "packaging genes" in the text.
本文述及AAV的“辅助病毒”指能使AAV被哺乳动物细胞复制和包装的病毒。各种这样的AAV辅助病毒是本领域已知的，包括腺病毒、疱疹病毒和痘病毒(例如牛痘)。Reference herein to "helper virus" for AAV refers to a virus that enables AAV to be replicated and packaged by mammalian cells. A variety of such AAV helper viruses are known in the art, including adenoviruses, herpes viruses, and pox viruses (eg, vaccinia).
本文述及“感染性”病毒或病毒颗粒是含有能将多核苷酸成分递送进该病毒种类具有嗜性的细胞。该术语无需暗示病毒有任何复制能力。As referred to herein, an "infectious" virus or virus particle is a cell containing a tropism capable of delivering a polynucleotide component into the virus species. The term need not imply that the virus has any ability to replicate.
术语“生产细胞”是指具有稳定整合至宿主细胞基因组中的AAV包装基因(rep和cap基因)、所需的辅助病毒基因和重组AAV载体的DNA基因组(例如，由两个AAV反向末端重复(ITR)侧接的目标转基因)的细胞系。The term "producer cell" refers to a DNA genome having 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., composed of two AAV inverted terminal repeats (ITR) flanking target transgene) cell line.
本文述及“包含”、“含有”等应理解为是包括性的意思，而没有排他性或穷尽的意思；即“包括但不限于”的意思。The words "comprising", "containing", etc. mentioned in this article should be understood as inclusive rather than exclusive or exhaustive; that is, "including but not limited to".
本文述及“个体”通常包括人类、非人类的灵长类，如哺乳动物、狗、猫、马、羊、猪、牛等，其可因利用所述制剂、试剂盒或联合制剂进行治疗而获益。The "individual" mentioned herein generally includes humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., which may be affected by treatment with the preparation, kit or combination preparation. benefit.
本文述及“治疗有效量”通常指一用量在经过适当的给药期间后，能够达到治疗如上所列出的疾病的效果。The "therapeutically effective dose" mentioned herein generally refers to an amount that can achieve the effect of treating the diseases listed above after an appropriate administration period.
本文述及“治疗性”和“预防性”应理解为其最宽的意义。术语“治疗性”不一定暗示哺乳动物接受治疗直至完全恢复。类似地，“预防性”不一定表示对象最终不会感染疾病病症。因此，治疗和预防包括缓解具体病症的症状或防 止或降低具体病症产生的风险。术语“预防”可理解为降低具体病症发作的严重程度。治疗也可降低已有病症的严重程度或急性发作的频率。References herein to "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 the disease condition. Thus, treatment and prevention include alleviating the symptoms of a particular condition or preventing Stop or reduce the risk of a specific condition. The term "prevention" is understood as reducing the severity of the onset of a particular condition. Treatment may also reduce the severity of pre-existing conditions or the frequency of exacerbations.
本发明中，进行治疗性或预防性治疗的对象或个体优选哺乳动物，例如但不限于人、灵长类、牲畜(如绵羊、牛、马、驴、猪)、宠物(如狗、猫)、实验室试验动物(如小鼠、家兔、大鼠、豚鼠、仓鼠)或被捕获的野生动物(如狐狸、鹿)。所述对象优选灵长类。所述对象最优选人。In the present invention, 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, cows, 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.
本文述及“转染(transfection)”、“转化(transformation)”和“转导(transduction)”可用于描述将非哺乳动物或病毒的载体插入靶细胞。插入载体通常称为细菌细胞的转化和真核细胞的转染，尽管插入病毒载体也可称为转导。技术人员将意识到通常使用的不同的非病毒转染方法，其包括但不限于使用物理方法(例如，电穿孔、细胞挤压(cell squeezing)、声致穿孔、光学转染、原生质体融合、穿刺转染(impalefection)、磁转染、基因枪或颗粒轰击)、化学试剂(例如，磷酸钙、高度支化的有机化合物或阳离子聚合物)或阳离子脂质(例如，脂质转染)。许多转染方法需要使载体DNA溶液与细胞接触，然后使其生长并针对标记基因表达进行选择。References herein to "transfection", "transformation" and "transduction" may be used to describe the insertion of a non-mammalian or viral vector into a target cell. Insertion of vectors is commonly referred to as transformation of bacterial cells and transfection of eukaryotic cells, although insertion of viral vectors may also be referred to as transduction. The skilled artisan will be aware of the different non-viral transfection methods commonly used, including but not limited to the use of physical methods (e.g., electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, magnetic transfection, gene gun or particle bombardment), chemical reagents (eg, calcium phosphate, hyperbranched organic compounds, or cationic polymers), or cationic lipids (eg, lipofection). Many transfection methods require contacting a solution of carrier DNA with cells, which are then grown and selected for marker gene expression.
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention; in the description and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural forms.
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the examples, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the examples of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。 Unless otherwise stated, 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 the technical field conventional technology.
材料与方法：Materials and Methods:
材料与来源：Materials and sources:
幼鼠：上海灵畅生物科技有限公司Pups: Shanghai Lingchang Biotechnology Co., Ltd.
各种AAV：南京金斯瑞公司合成构建Various AAVs: synthetically constructed by Nanjing GenScript
驴血清：上海翊圣生物科技有限公司Donkey serum: Shanghai Yisheng Biotechnology Co., Ltd.
各个抗体与染色时的稀释比：The dilution ratio of each antibody and staining:
一抗：myosin 7A(Myo7a,#25-6790 Proteus Biosciences,1:1000),Sox2(Sox-2,#sc-17320,Santa Cruz Biotechnology,1:1000),Flag(Flag,#F3165,Sigma Aldrich,1:1000),NeuN(NeuN,#12943S,Cell Signaling Technology,1:500)。Primary antibody: myosin 7A (Myo7a, #25-6790 Proteus Biosciences, 1:1000), Sox2 (Sox-2, #sc-17320, Santa Cruz Biotechnology, 1:1000), Flag (Flag, #F3165, Sigma Aldrich, 1:1000), NeuN (NeuN, #12943S, Cell Signaling Technology, 1:500).
二抗：二抗标记物anti-rat、mouse、rabbit及goat的各三种不同标记颜色(TRIC,FITC,Cy5)的二抗，来源于invitrogen公司。Secondary antibodies: Secondary antibodies of three different marker colors (TRIC, FITC, Cy5) for the secondary antibody markers anti-rat, mouse, rabbit, and goat, from Invitrogen.
细胞和组织培养试剂：DMEM(Hyclone公司)，胎牛血清(Lensa公司)，添加物N2(ThermoFisher公司)，氨苄西林(ThermoFisher公司)，双抗(ThermoFisher公司)。Cell and tissue culture reagents: DMEM (Hyclone Company), fetal bovine serum (Lensa Company), supplement N2 (ThermoFisher Company), ampicillin (ThermoFisher Company), double antibody (ThermoFisher Company).
细胞与组织培养耗材：包括各类培养皿、离心管、移液管，一次性滤器等常用耗材购于Corning公司。Cell and tissue culture consumables: Common consumables including various culture dishes, centrifuge tubes, pipettes, and disposable filters were purchased from Corning.
实施例1衣壳蛋白表面氨基酸的突变提高了AAV-ie在体内的耳蜗转导效率(1.1)蛋白泛素化与AAV转导效率有关，阻断泛素化可以提高AAV在体外和体内的转导效率。本发明以前的研究表明，AAV-ie可以转导啮齿动物和人类的耳蜗细胞。然而，AAV-ie在耳蜗基底区的转导效率很低。本发明认为，在AAV-ie暴露侧突变衣壳蛋白氨基酸可以提高其转导效率(图1a)。为了验证这一假设，本发明构建了28个衣壳蛋白序列不同的AAV-ie变体(表1)。Example 1 The mutation of amino acids on the surface of the capsid protein improves the cochlear transduction efficiency of AAV-ie in vivo (1.1) Protein ubiquitination is related to AAV transduction efficiency, and blocking ubiquitination can improve the transduction efficiency of AAV in vitro and in vivo conduction efficiency. Previous studies in the present invention have shown that AAV-ie can transduce rodent and human cochlear cells. However, the transduction efficiency of AAV-ie in the basal region of the cochlea is very low. The present invention believes that mutating amino acids of the capsid protein on the exposed side of AAV-ie can improve its transduction efficiency (Fig. 1a). In order to verify this hypothesis, the present invention constructed 28 AAV-ie variants with different capsid protein sequences (Table 1).
(1.2)AAV-ie变体的构建(1.2) Construction of AAV-ie variants
以下以AAV-ie-K558R的构建作为示例性说明，其他AAV-ie变体的构建步骤相似。The construction of AAV-ie-K558R is used as an example below, and the construction steps of other AAV-ie variants are similar.
AAV-ie-K558R的构建：Construction of AAV-ie-K558R:
AAV的包装需要三个质粒：含有目的基因的基因组质粒、Capsid质粒 (Rep-Cap质粒)和Helper质粒。The packaging of AAV requires three plasmids: the genome plasmid containing the gene of interest, and the Capsid plasmid (Rep-Cap plasmid) and Helper plasmid.
其中，Capsid质粒中的Cap蛋白的序列决定了AAV的不同血清型，进而影响了AAV感染细胞的偏好性。因此，改造Cap蛋白能够得到新的AAV血清型。Among them, the sequence of the Cap protein in the Capsid plasmid determines the different serotypes of AAV, which in turn affects the preference of AAV-infected cells. Therefore, modification of Cap protein can lead to new AAV serotypes.
AAV-ie-K558R是在亲本AAV-ie的Cap蛋白序列上通过558位的单个氨基酸K突变成R而产生的。AAV-ie-K558R is generated by mutating a single amino acid K to R at position 558 on the Cap protein sequence of the parental AAV-ie.
(a)AAV-ie capsid载体线性化(a) AAV-ie capsid vector linearization
首先对于AAV-ie capsid的质粒进行线性化切割，通过Sma1和Xho1两个酶切位点，将其切开，产生AAV-ie capsid载体，回收线性化AAV-ie capsid片段(Axygen：AP-GX-250G)。回收后的片段经过Nanodrop2000检测浓度。First, linearize the plasmid of AAV-ie capsid, cut it through the two restriction sites of Sma1 and Xho1, generate the AAV-ie capsid vector, and recover the linearized AAV-ie capsid fragment (Axygen: AP-GX -250G). The recovered fragments were detected by Nanodrop2000.
(b)突变片段的构建(b) Construction of mutant fragments
首先，针对Xho1酶切位点到558氨基酸的片段设计引物，在Xho1酶切位点处设计正向引物，并在引物5’端加入15-20 nt与线性化AAV-ie capsid载体结合的同源臂(Primer 1)。再在558氨基酸处设计反向引物，并在5’端加入15-20nt与重组片段2结合的同源臂(Primer 2)，Primer2中引入K558R突变。通过聚合酶链式反应(PCR)产生重组片段1。回收重组片段1(Axygen：AP-GX-250G)。回收后的片段经过Nanodrop2000检测浓度。First, design primers for the fragment from the Xho1 restriction site to 558 amino acids, design a forward primer at the Xho1 restriction site, and add 15-20 nt synchronous peptides combined with the linearized AAV-ie capsid vector at the 5' end of the primers. Source arm (Primer 1). A reverse primer was designed at 558 amino acids, and a 15-20nt homology arm (Primer 2) combined with the recombinant fragment 2 was added at the 5' end, and the K558R mutation was introduced into Primer2. Recombinant fragment 1 was generated by polymerase chain reaction (PCR). Recombinant Fragment 1 (Axygen: AP-GX-250G) was recovered. The recovered fragments were detected by Nanodrop2000.
再针对558氨基酸到Sma1酶切位点片段设计引物，在Cap蛋白的序列558处设计正向引物，引入K558R突变，并在引物5’端中加入15-20nt与重组片段1结合的同源臂(Primer 3)。再在Sma1酶切位点处设计反向引物，并在引物5’端加入15-20nt与线性化AAV-ie capsid载体结合的同源臂(Primer 4)。通过聚合酶链式反应(PCR)产生重组片段2。回收重组片段2(Axygen：AP-GX-250G)。回收后的片段经过Nanodrop2000检测浓度。Then design primers for the fragment from 558 amino acids to the Sma1 restriction site, design a forward primer at sequence 558 of the Cap protein, introduce the K558R mutation, and add a 15-20nt homology arm that binds to the recombinant fragment 1 at the 5' end of the primer (Primer 3). A reverse primer was designed at the Sma1 restriction site, and a 15-20nt homology arm (Primer 4) combined with the linearized AAV-ie capsid vector was added to the 5' end of the primer. Recombinant fragment 2 was generated by polymerase chain reaction (PCR). Recombinant Fragment 2 (Axygen: AP-GX-250G) was recovered. The recovered fragments were detected by Nanodrop2000.
引物设计如下，灰色表明引物中引入K558R的突变碱基：The primers are designed as follows, gray indicates the mutated base of K558R introduced in the primers:
正向引物Primer 1：5’-TTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGAC-3’(SEQ ID NO.6)；Forward primer Primer 1: 5'-TTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGAC-3' (SEQ ID NO.6);
反向引物Primer 2： Reverse primer Primer 2:
正向引物Primer 3： Forward primer Primer 3:
反向引物Primer 4：5’-CGCCCGCTGTTTAAACGCCCGGGCTGTAGTTAATGATTAACCCG-3’(SEQ ID NO.9)。Reverse primer Primer 4: 5'-CGCCCGCTGTTTAAACGCCCGGGCTGTAGTTAATGATTAACCCG-3' (SEQ ID NO.9).
(c)AAV-ie-K558R载体的构建和纯化(c) Construction and purification of AAV-ie-K558R vector
利用多片段同源重组的方法，将线性化AAV-ie capsid载体和PCR反应获得的重组片段1，2进行重组，产生AAV-ie-K558R。重组体系为：线性化AAV-iecapsid载体，50ng；重组片段1，30 ng；重组片段2，30 ng；重组连接酶，1μL；重组连接buffer，5μL；ddH2O，补齐20μL；50℃反应20分钟，产生重组产物。Using the multi-fragment homologous recombination method, the linearized AAV-ie capsid vector and the recombinant fragments 1 and 2 obtained by PCR reaction were recombined to produce AAV-ie-K558R. Recombination system: linearized AAV-iecapsid vector, 50ng; recombinant fragment 1, 30 ng; recombinant fragment 2, 30 ng; recombinant ligase, 1 μL; recombinant ligation buffer, 5 μL; , resulting in recombinant products.
流程图如图12所示。The flowchart is shown in Figure 12.
(d)载体的转化与质粒提取(d) Vector transformation and plasmid extraction
转化步骤如下：取100μL感受态细胞(TransGen:CD201)在冰上解冻；10μL的重组产物与感受态细胞混合，冰上放置20分钟；42℃热激60秒；冰上放置2分钟，加入400μL复苏LB培养基(MDBio:L001-1kg)，摇床30分钟；取70μL涂氨苄平板(50μg/ml，37℃培养箱，培养14个小时。Transformation steps are as follows: Thaw 100 μL of competent cells (TransGen: CD201) on ice; mix 10 μL of recombinant product with competent cells, and place on ice for 20 minutes; heat shock at 42°C for 60 seconds; place on ice for 2 minutes, add 400 μL Recover LB medium (MDBio: L001-1kg), shake for 30 minutes; take 70 μL ampicillin-coated plate (50 μg/ml, incubator at 37°C, and incubate for 14 hours.
挑选单克隆菌，在4ml液体LB培养基中扩大培养，14小时后提取质粒(Axygene:AP-MN-P-250G)。Select monoclonal bacteria, expand culture in 4ml liquid LB medium, and extract plasmid (Axygene: AP-MN-P-250G) after 14 hours.
步骤如下：菌液经过4000转/分钟离心10分钟，倒掉上清培养基；加入350μL的buffer S1，将菌体吹散，转移到2ml离心管中；加入250μL的buffer S2，上下颠倒8次；加入250μL的buffer S3，颠倒混匀6次，产生沉淀；12000转/分钟离心10分钟，取上清过柱；离心1分钟，倒掉废液，加入500μL的W1，离心一分钟，倒掉废液；加入750μL的W2，离心，倒掉上清；加入500μL的W2，离心，倒掉上清；空转1分钟；加入50μL的洗脱液，静置2分钟，离心。获得质粒经过浓度检测，取10μL送测序，阳性质粒保存在-20℃。测序结果表明，获得质粒能够编码变异型衣壳蛋白VP1。The steps are as follows: centrifuge the bacterial solution at 4000 rpm for 10 minutes, discard the supernatant medium; add 350 μL of buffer S1, blow off the bacteria, and transfer to a 2ml centrifuge tube; add 250 μL of buffer S2, turn it upside down 8 times ; Add 250 μL of buffer S3, invert and mix 6 times to produce a precipitate; centrifuge at 12,000 rpm for 10 minutes, take the supernatant to pass through the column; centrifuge for 1 minute, discard the waste liquid, add 500 μL of W1, centrifuge for 1 minute, and discard Waste liquid; add 750 μL of W2, centrifuge, discard the supernatant; add 500 μL of W2, centrifuge, discard the supernatant; idle for 1 minute; add 50 μL of eluent, let stand for 2 minutes, and centrifuge. After concentration detection of the obtained plasmid, 10 μL was taken for sequencing, and the positive plasmid was stored at -20°C. Sequencing results showed that the obtained plasmid could encode the variant capsid protein VP1.
进一步的实验结果表明，制备获得的质粒能够表达变异型衣壳蛋白VP1，其中，AAV-ie-K558R capsid VP1的多核苷酸编码序列如SEQ ID NO.10所示，构建获得的Rep-Cap质粒的全序列如SEQ ID NO.11所示。Further experimental results show that the prepared plasmid can express mutant capsid protein VP1, wherein the polynucleotide coding sequence of AAV-ie-K558R capsid VP1 is shown in SEQ ID NO.10, and the obtained Rep-Cap plasmid is constructed The full sequence is shown in SEQ ID NO.11.
(e)AAV变异体(命名为AAV-ie-K558R)病毒的包装与纯化 (e) Packaging and purification of AAV variant (named AAV-ie-K558R) virus
将得到的Rep-Cap质粒，表达一个绿色荧光蛋白mNeonGreen(GenBank:LC279210.1)的基因组质粒pAAV-CAG-mNeonGreen(质粒全序列如SEQ ID NO.12所示(同AAV-ie专利文献CN 110437317A的SEQ ID NO.11))，pHelper质粒(质粒全序列如SEQ ID NO.13(同AAV-ie专利文献CN 110437317A的SEQ ID NO.12)所示)以合适的量共转于HEK-293T细胞中，采用碘二烷醇梯度超高速离心纯化AAV病毒，测量病毒滴度在1E+12-1E+13GC/mL为合适浓度，放置-80℃备用。The resulting Rep-Cap plasmid expresses a genomic plasmid pAAV-CAG-mNeonGreen (full sequence of the plasmid as shown in SEQ ID NO.12) of a green fluorescent protein mNeonGreen (GenBank: LC279210.1) (same as AAV-ie patent document CN 110437317A SEQ ID NO.11)), pHelper plasmid (full sequence of the plasmid is shown in SEQ ID NO.13 (same as SEQ ID NO.12 of AAV-ie patent document CN 110437317A) shown) in HEK-293T with an appropriate amount In the cells, the AAV virus was purified by ultrahigh-speed centrifugation using an iodideanol gradient, and the virus titer was measured at a suitable concentration of 1E+12-1E+13GC/mL, and placed at -80°C for later use.
在用DMEM+10％胎牛血清培养的HEK 293T细胞中加入衣壳蛋白变异体和其亲本衣壳蛋白产生的AAV病毒(分别为AAV-ie和AAV-DJ)。病毒加入的MOI值为1000。48小时后，用荧光显微镜观察了绿色荧光蛋白mNeonGreen的表达。Add capsid protein variants and AAV viruses produced by their parental capsid proteins (AAV-ie and AAV-DJ, respectively) in HEK 293T cells cultured with DMEM+10% fetal bovine serum. The MOI value of virus addition was 1000. After 48 hours, the expression of green fluorescent protein mNeonGreen was observed by fluorescence microscope.
结果显示：AAV变异体与其亲本AAV感染HEK 293T细胞的比例相似。The results showed that the proportion of AAV variants infected HEK 293T cells was similar to that of its parental AAV.
SEQ ID NO.10(AAV-ie-K558R capsid VP1):

SEQ ID NO.10(AAV-ie-K558R capsid VP1):

SEQ ID NO.11(AAV-ie-K558R Rep-Cap质粒):
SEQ ID NO.11 (AAV-ie-K558R Rep-Cap plasmid):
SEQ ID NO.12SEQ ID NO.12




SEQ ID NO.13 SEQ ID NO.13










(1.3)对AAV-ie变体进行筛选(1.3) Screening for AAV-ie variants
为了提高筛选的准确性，本发明将等量(4.5×109)的含基因组的AAV-ie- K558R颗粒通过圆窗膜(RWM)注射到新生小鼠P3的耳蜗中，来对(1.1)中的这些AAV-ie变体进行体内筛选(图1b)。取P14时期的耳蜗进行免疫染色：用Myo7a抗体标记毛细胞，核定位信号(NLS)-mNeonGreen信号显示转导的细胞在耳蜗中的细胞核定位。本发明对这些AAV-ie变体的转导效率进行了分析，并与相同条件下的AAV-ie进行了比较。如图1c所示，相对于AAV-ie，虽然大多数AAV-ie变体在耳蜗基底区的转导效率没有增加(图5)，有些甚至反而下降，但变体AAV-ie-K558R在耳蜗基底区的OHCs和IHCs都有显著提高的转导效率。本发明进一步比较AAV-ie-K558R和AAV-ie的转导情况，发现AAV-ie-K558R在耳蜗中间区域的OHCs和基底区域的SCs的转导效率都有显著提高(图1d-f)。本发明也评估了AAV-ie-K558R在负责检测直线运动和感知重力的前庭系统的靶向效率，观察到NLS-mNeonGreen在整个小鼠感觉上皮表达，AAV-ie-K558R在HCs和SCs的转导效率接近100％(图6)。In order to improve the accuracy of screening, the present invention uses an equal amount (4.5×10 9 ) of AAV-ie- These AAV-ie variants in (1.1) were screened in vivo by K558R particles injected into the cochlea of neonatal mouse P3 through the round window membrane (RWM) (Fig. 1b). The cochlea at P14 stage was taken for immunostaining: the hair cells were labeled with Myo7a antibody, and the nuclear localization signal (NLS)-mNeonGreen signal showed the nuclear localization of the transduced cells in the cochlea. The transduction efficiency of these AAV-ie variants was analyzed in the present invention and compared with AAV-ie under the same conditions. As shown in Figure 1c, compared with AAV-ie, although most AAV-ie variants did not increase the transduction efficiency in the cochlear basal region (Figure 5), some even decreased, but the variant AAV-ie-K558R Both OHCs and IHCs in the basal region had significantly improved transduction efficiencies. The present invention further compared the transduction of AAV-ie-K558R and AAV-ie, and found that the transduction efficiency of AAV-ie-K558R in OHCs in the middle region of the cochlea and in SCs in the basal region was significantly improved (Fig. 1d-f). The present invention also evaluated the targeting efficiency of AAV-ie-K558R in the vestibular system responsible for detecting linear motion and sensing gravity. It was observed that NLS-mNeonGreen was expressed in the whole mouse sensory epithelium, and AAV-ie-K558R was transfected in HCs and SCs. Conductivity is close to 100% (Figure 6).
这些实验结果共同证明了AAV-ie-K558R可以有效地靶向耳蜗中的不同类型的细胞，揭示了它可以作为合适的载体来介导听觉疾病的基因纠正或在耳蜗或前庭系统中再生类HC细胞的潜力。These experimental results collectively demonstrate that AAV-ie-K558R can efficiently target different types of cells in the cochlea, revealing that it can serve as a suitable vector to mediate gene correction of auditory diseases or to regenerate HC-like cells in the cochlea or vestibular system. cell potential.
表1.AAV-ie衣壳蛋白氨基酸突变的序列(大写字母代表编码氨基酸的密码子，红色字母代表变异后的碱基)。Table 1. Sequences of amino acid mutations in AAV-ie capsid protein (capital letters represent codons encoding amino acids, red letters represent mutated bases).




实施例2 AAV-ie-K558R的安全性验证Example 2 Security Verification of AAV-ie-K558R
本课题组早期的研究表明，AAV-ie是一个安全的载体，并对HCs和听觉功能没有表现出任何毒性的作用。本发明检测了AAV-ie-K558R是否具有与AAV-ie类似的安全特性。Early studies of our research group showed that AAV-ie was a safe carrier and did not show any toxic effects on HCs and auditory function. The present invention detects whether AAV-ie-K558R has security characteristics similar to AAV-ie.
2.1实验分组和处理2.1 Experimental grouping and processing
AAV-ie-K558R注射组(每组3只)：通过圆窗膜(RWM)给小鼠的每只耳朵注射AAV-ie-K558R-NLS-mNeonGreen(1×1010GCs)；AAV-ie-K558R injection group (3 mice in each group): AAV-ie-K558R-NLS-mNeonGreen (1×10 10 GCs) was injected into each ear of the mice through the round window membrane (RWM);
WT对照组(Control，每组3只)：通过圆窗膜(RWM)给小鼠的每只耳朵注射AAV-ie-NLS-mNeonGreen(1×1010GCs)。WT control group (Control, 3 mice in each group): AAV-ie-NLS-mNeonGreen (1×10 10 GCs) was injected into each ear of the mice through the round window membrane (RWM).
2.2对HCs的形态和数量的影响2.2 Effects on the morphology and quantity of HCs
经步骤2.1处理两个星期后，对耳蜗进行扫描电子显微镜(SEM)分析。SEM的结果表明，HCs在注射后没有或几乎没有形态上的变化，毛束方向正确(图2b，2d)。本实施例同时计算了视野中的HCs数量，观察到AAV注射组和对照组之间没有差异，表明没有出现明显的毛细胞损失(图2c)。Two weeks after the treatment in step 2.1, the cochlea was analyzed by scanning electron microscopy (SEM). The results of SEM showed that HCs showed little or no morphological changes after injection, and the orientation of hair bundles was correct (Fig. 2b, 2d). In this example, the number of HCs in the visual field was also counted, and no difference was observed between the AAV-injected group and the control group, indicating that no significant loss of hair cells occurred (Fig. 2c).
2.3测定AAV-ie-K558R对听力的影响2.3 Determination of the effect of AAV-ie-K558R on hearing
本实施例通过测定反映活体动物听觉功能的听觉脑干反应(ABRs)和反映外毛细胞完整性的畸变产物耳声发射(DPOAE)来验证AAV-ie-K558R注射是否影响听力。同样，在注射组和对照组之间，阈值没有表现出显著差异(图2e，2f)。In this example, the auditory brainstem responses (ABRs) reflecting the auditory function of living animals and the distortion product otoacoustic emissions (DPOAE) reflecting the integrity of outer hair cells were measured to verify whether AAV-ie-K558R injection affects hearing. Also, the threshold showed no significant difference between the injected and control groups (Fig. 2e, 2f).
2.4测定AAV-ie-K558R对听觉和前庭系统的影响2.4 Determination of the effects of AAV-ie-K558R on the auditory and vestibular systems
本实施例接下来检测了AAV-ie-K558R对表现严重前庭功能损伤的动物行为是否存在像转圈行为或其他步态异常等类似的副作用。Next, in this example, it was tested whether AAV-ie-K558R had similar side effects such as circling behavior or other gait abnormalities on the behavior of animals with severe vestibular impairment.
在步骤2.1处理四周后，在开放路径跟踪试验中未观察到AAV-ie-K558R注 射组任何步态或循环行为的异常。After four weeks of treatment in step 2.1, no AAV-ie-K558R note was observed in the open path-following test Indicate any abnormalities in gait or circulatory behavior.
在进一步的窄梁行走试验中，注射病毒的AAV-ie-K558R注射组小鼠和未注射病毒的对照组小鼠之间没有观察到明显的差异(图7)。In a further narrow-beam walking test, no significant difference was observed between virus-injected AAV-ie-K558R-injected mice and non-virus-injected control mice (Fig. 7).
根据这些结果，本发明得出结论，AAV-ie-K558R对听觉和前庭系统没有负面作用。From these results, the present inventors conclude that AAV-ie-K558R has no negative effects on the auditory and vestibular systems.
实施例3 Prestin敲除小鼠的产生和验证。Example 3 Production and verification of Prestin knockout mice.
构建Prestin敲除小鼠：利用Crispr-Cas体系进行在Prestin第4外显子和第11外显子的编码序列中同时引入两个终止密码子，导致prestin蛋白翻译提前停止，达到敲除prestin在耳蜗中外毛的表达，以构建Prestin敲除小鼠。Construction of Prestin knockout mice: Using the Crispr-Cas system to simultaneously introduce two stop codons into the coding sequences of Prestin exon 4 and exon 11, resulting in the premature stop of prestin protein translation, achieving knockout of prestin in Expression of outer hairs in the cochlea to construct Prestin knockout mice.
如图8b所示，通过方法中描述的引物(SEQ ID NO.42-45：正向引物1:5’-CCACCACGTTTAGTAGCATC-3’、反向引物1：5’-ACTGTGATGAACATGAGCCA-3’,正向引物2：5’-AGAGCACACCTGCGCTCTTC-3’，反向引物2：5’-AGTGTGGATGTCAGGCAGAGTA-3’)在突变位点周围进行PCR扩增，验证Prestin敲除小鼠的基因型，并进行测序检测。As shown in Figure 8b, the primers described in the method (SEQ ID NO.42-45: forward primer 1: 5'-CCACCACGTTTAGTAGCATC-3', reverse primer 1: 5'-ACTGTGATGAACATGAGCCA-3', forward primer 2: 5'-AGAGCACACCTGCGCTCTTC-3', reverse primer 2: 5'-AGTGTGGATGTCAGGCAGAGTA-3') Perform PCR amplification around the mutation site to verify the genotype of the Prestin knockout mice, and perform sequencing detection.
如图8c所示，通过对WT小鼠和Prestin敲除小鼠的Prestin特异性抗体(绿色)进行免疫组织化学检测，清楚地显示Prestin敲除小鼠的OHCs中没有Prestin表达。As shown in Figure 8c, the absence of Prestin expression in OHCs of Prestin knockout mice was clearly shown by immunohistochemical detection of Prestin-specific antibodies (green) from WT mice and Prestin knockout mice.
实施例4 AAV-ie-K558R-Prestin能使Prestin敲除小鼠的耳聋部分恢复Example 4 AAV-ie-K558R-Prestin can partially restore the deafness of Prestin knockout mice
由于缺乏可以高效转导OHC的AAV，在OHC基因缺失引起的耳聋模型中进行基因治疗是具有挑战性的。为了测试AAV-ie-K558R是否有可能恢复OHC基因功能障碍引发的听力损失，本实施例采用了在OHCs中除去Prestin蛋白表达基因的Prestin敲除小鼠。Prestin是一个只定位在OHCs的关键分子，在介导OHCs的电动性方面起着关键作用。在Prestin敲除小鼠中，听觉阈值明显高于野生型(WT)(图3a-b)。Prestin敲除小鼠没有出现转圈行为或其他步态异常(数据未显示)。 Due to the lack of AAVs that can efficiently transduce OHC, it is challenging to perform gene therapy in a deafness model caused by OHC gene deletion. In order to test whether AAV-ie-K558R may restore hearing loss caused by OHC gene dysfunction, this example uses Prestin knockout mice in which the Prestin protein expression gene is deleted in OHCs. Prestin is a key molecule localized only in OHCs and plays a key role in mediating the electromotive force of OHCs. In Prestin knockout mice, the auditory threshold was significantly higher than in wild type (WT) (Fig. 3a-b). Prestin knockout mice did not exhibit circling behavior or other gait abnormalities (data not shown).
4.1小鼠及病毒构建4.1 Mice and virus construction
构建AAV-ie-K558R-Prestin：在启动子CAG的驱动下，prestin基因slc26a5包装到AAV-ie-K558R载体。Construction of AAV-ie-K558R-Prestin: Driven by the promoter CAG, the prestin gene slc26a5 was packaged into the AAV-ie-K558R vector.
构建AAV-ie-Prestin：在启动子CAG的驱动下，prestin基因slc26a5包装到AAV-ie载体。Construction of AAV-ie-Prestin: Driven by the promoter CAG, the prestin gene slc26a5 was packaged into the AAV-ie vector.
4.2实验分组及处理4.2 Experimental grouping and processing
本实施例将AAV-ie-K558R-Prestin病毒注射到P1-2(“P1-2”的含义为小鼠出生后1天或者2天)的Prestin敲除小鼠体内，以研究它在治疗OHCs中基因功能缺陷引发的遗传性听觉疾病的效果。为了测试由AAV介导的Prestin在HCs中的过表达是否能恢复Prestin敲除小鼠的听力功能，本发明对不同组的小鼠中进行了ABR实验。In this example, the AAV-ie-K558R-Prestin virus was injected into P1-2 ("P1-2" means 1 day or 2 days after birth) Prestin knockout mice to study its role in the treatment of OHCs Effects of genetic hearing disorders triggered by defective gene function in the medium. In order to test whether the AAV-mediated overexpression of Prestin in HCs can restore the hearing function of Prestin knockout mice, the present invention conducted ABR experiments on different groups of mice.
4.3实验结果4.3 Experimental results
在步骤4.2处理后1个月，测定各组小鼠的ABR阈值，观察到AAV-ie-K558R-Prestin实验组的ABR阈值的部分降低(图3b，3c)；DPOAE结果表明，AAV-ie-K558R-Prestin在16kHz有10dB的缓解作用。One month after step 4.2 was processed, the ABR threshold of each group of mice was measured, and a partial reduction of the ABR threshold of the AAV-ie-K558R-Prestin experimental group was observed (Fig. 3b, 3c); DPOAE results showed that AAV-ie- The K558R-Prestin has 10dB relief at 16kHz.
在步骤4.2处理后两个月，检测耳蜗中Prestin的表达，并检测Prestin敲除小鼠的毛细胞中Prestin的表达情况。观察到AAV-ie-K558R-Prestin实验组在病毒注射两个月后，Prestin在小鼠的耳蜗OHCs中大量表达(图9)。AAV-ie-Prestin实验组中，将Prestin整合到AAV-ie载体并注射到新生Prestin敲除小鼠体内后，ABR阈值没有降低，注射一个月后，也没有观察到Prestin在耳蜗OHCs的大量表达(图3c，3d)。Two months after the treatment in step 4.2, the expression of Prestin in the cochlea was detected, and the expression of Prestin in the hair cells of the Prestin knockout mice was detected. It was observed that in the AAV-ie-K558R-Prestin experimental group, two months after virus injection, Prestin was massively expressed in the cochlear OHCs of the mice ( FIG. 9 ). In the AAV-ie-Prestin experimental group, after Prestin was integrated into the AAV-ie vector and injected into the neonatal Prestin knockout mice, the ABR threshold did not decrease, and a large amount of Prestin expression in cochlear OHCs was not observed after one month of injection. (Fig. 3c, 3d).
本实施例的结果表明，AAV-ie-K558R可以作为一个潜在的载体，将基因传递到OHCs，并用于毛细胞缺陷的耳聋小鼠模型的基因治疗。The results of this example show that AAV-ie-K558R can be used as a potential carrier to deliver genes to OHCs and be used for gene therapy in a deaf mouse model with hair cell deficiency.
实施例5 AAV-ie-K558R-Atoh1介导的体内类HC细胞再生Example 5 AAV-ie-K558R-Atoh1-mediated regeneration of HC-like cells in vivo
5.1 AAV-ie-K558R-Atoh1病毒构建：在启动子CAG的驱动下，Atoh1包装到AAV-ie-K558R载体5.1 AAV-ie-K558R-Atoh1 virus construction: Driven by the promoter CAG, Atoh1 is packaged into the AAV-ie-K558R vector
AAV-ie-Atoh1病毒构建：在启动子CAG的驱动下，Atoh1包装到AAV-ie载体 AAV-ie-Atoh1 virus construction: Driven by the promoter CAG, Atoh1 is packaged into the AAV-ie vector
5.2实验分组及处理5.2 Experimental grouping and processing
研究表明，HC的再生可能有助于恢复由老化、噪音或耳毒性药物引起的听力损失。转录因子Atoh1被证明能够使SCs转分化为HCs。本发明早期的研究表明，AAV-ie能将Atoh1传递给SCs并使其转分化为类HC细胞。Research suggests that HC regeneration may help restore hearing loss caused by aging, noise, or ototoxic drugs. The transcription factor Atoh1 was shown to enable the transdifferentiation of SCs into HCs. Earlier studies of the present invention showed that AAV-ie can deliver Atoh1 to SCs and make them transdifferentiate into HC-like cells.
为了评估AAV-ie-K558R载体用于HC再生的潜力，本发明使用AAV-ie-K558R-Atoh1在体内将小鼠Atoh1送入新生小鼠耳蜗(图4a)。To assess the potential of AAV-ie-K558R vectors for HC regeneration, we used AAV-ie-K558R-Atoh1 to deliver mouse Atoh1 into neonatal mouse cochlea in vivo (Fig. 4a).
AAV-ie-Atoh1注射组：在P3时通过RWM将AAV-ie-Atoh1(每只耳朵1×1010GCs)注入耳蜗。AAV-ie-Atoh1 injection group: AAV-ie-Atoh1 (1×10 10 GCs per ear) was injected into the cochlea by RWM at P3.
AAV-ie-K558R-Atoh1注射组：在P3时通过RWM将AAV-ie-K558R-Atoh1病毒(每只耳朵1×1010GCs)注入耳蜗。AAV-ie-K558R-Atoh1 injection group: AAV-ie-K558R-Atoh1 virus (1×10 10 GCs per ear) was injected into the cochlea by RWM at P3.
在P14时取耳蜗。在AAV-ie-Atoh1注射组中，感觉区出现了一些表达Myo7a的类HC细胞(图4b)。在AAV-ie-K558R-Atoh1注射组，观察到在上皮脊(GER)区域有大量新的表达Myo7a的HCs。值得注意的是，在AAV-ie-Atoh1注射组和AAV-ie-K558R-Atoh1注射组中一些新的类HC细胞有Sox2的持续表达，这意味着这些新的再生HCs细胞可能处于不同的发育阶段(图4b)。与其他Atoh1过表达方法，如以前研究中使用的遗传方法相比，本实施例AAV-ie-K558R-Atoh1注射组的再生HCs细胞数量差别不显著，这表明AAV-ie-K558R是一种高效的HCs再生病毒载体。The cochlea was harvested at P14. In the AAV-ie-Atoh1-injected group, some HC-like cells expressing Myo7a appeared in the sensory area (Fig. 4b). In the AAV-ie-K558R-Atoh1 injected group, a large number of new Myo7a-expressing HCs in the epithelial ridge (GER) region was observed. Notably, some new HC-like cells in the AAV-ie-Atoh1-injected group and AAV-ie-K558R-Atoh1-injected group had sustained expression of Sox2, implying that these newly regenerated HCs might be in different developmental stages stage (Fig. 4b). Compared with other Atoh1 overexpression methods, such as the genetic method used in previous studies, there was no significant difference in the number of regenerated HCs in the AAV-ie-K558R-Atoh1 injection group in this example, which indicates that AAV-ie-K558R is a highly efficient HCs regenerate viral vectors.
最后，本实施例用SEM对新再生的HCs细胞的形态进行了定量。AAV-ie-K558R-Atoh1引起了很多类HC细胞的再生，毛束从IHC区域下面的类HC细胞生长出来(图4c)。与本发明早期的研究一致，AAV-ie-Atoh1也能诱导类HC细胞再生(图10)。然而SEM证明AAV-ie-K558R-Atoh1能在GER区域诱导产生一些类HC细胞(图11)，而AAV-ie-Atoh1不能产生类似的效果。本发明观察到新再生的类HC细胞产生了代表毛细胞步入更成熟发育阶段的动纤毛(图4d)。这些结果表明，AAV-ie-K558R可以通过传递Atoh1有效诱导耳蜗中的类HC细胞再生。Finally, this example quantifies the morphology of newly regenerated HCs cells by SEM. AAV-ie-K558R-Atoh1 induced the regeneration of many HC-like cells, and hair bundles grew out of the HC-like cells below the IHC region (Fig. 4c). Consistent with earlier studies of the present invention, AAV-ie-Atoh1 was also able to induce regeneration of HC-like cells (Fig. 10). However, SEM proved that AAV-ie-K558R-Atoh1 could induce some HC-like cells in the GER region (Figure 11), while AAV-ie-Atoh1 could not produce similar effects. We observed that newly regenerated HC-like cells produced kinetocilia representing hair cells entering a more mature developmental stage (Fig. 4d). These results suggest that AAV-ie-K558R can efficiently induce HC-like cell regeneration in the cochlea by delivering Atoh1.
本发明通过突变AAV-ie暴露侧衣壳蛋白氨基酸来减少泛素化或磷酸化从而 优化AAV-ie。本发明的研究结果表明，新产生的AAV变体AAV-ie-K558是安全的，而且对毛细胞再生和基因治疗都有益。大多数AAV-ie变体都未表现出高靶向效率，反而表现出较低的靶向效率，这表明泛素化或磷酸化的减少并不一定能引发更高的转导效率。因此，转导效率的确切机制有待进一步研究。这些结果也揭示了进行筛选实验对确定不同组织最佳AAV的重要性。尽管如此，本发明的结果表明，肽的插入和氨基酸的突变对于改造产生合适的AAVs以有效地转导细胞在耳蜗和其他组织中是可行的。The present invention reduces ubiquitination or phosphorylation by mutating the amino acids of the AAV-ie exposed side capsid protein Optimized for AAV-ie. The findings of the present invention show that the newly generated AAV variant AAV-ie-K558 is safe and beneficial for both hair cell regeneration and gene therapy. Instead of high targeting efficiency, most AAV-ie variants showed lower targeting efficiency, suggesting that reduced ubiquitination or phosphorylation does not necessarily lead to higher transduction efficiency. Therefore, the exact mechanism of transduction efficiency awaits further investigation. These results also reveal the importance of performing screening experiments to determine the optimal AAV for different tissues. Nonetheless, the results of the present invention demonstrate that peptide insertion and amino acid mutations are feasible for engineering suitable AAVs to efficiently transduce cells in the cochlea and other tissues.
本发明发现AAV-ie-K558R能使Prestin敲除小鼠的听觉功能部分恢复，因为将基因有效传递到OHCs一直是一个重大挑战，所以这是针对听觉疾病的基因治疗领域的重大进展。在这项研究中，本发明用AAV-ie-K558R只实现了部分听觉功能的恢复。有以下几个因素来解释这种部分恢复的现象。首先，AAV-ie-K558R也能转导IHCs，Prestin在IHCs中的过表达可能导致IHCs功能障碍，从而阻止听觉功能的完全恢复。其次，想要完全恢复OHCs的功能，Prestin在OHCs中的表达水平可能要被精确控制。在这项研究中，本发明使用了一个广泛使用的基因调控启动子，CAG，这可能不是控制Prestin在OHCs中表达的最佳选择，需要进一步的研究来发现AAV传递方式中用于耳蜗的最佳基因调节元件。开发一种用AAVs控制耳蜗和其他组织的基因表达的调节方法将是很重要的。The present invention found that AAV-ie-K558R can partially restore the auditory function of Prestin knockout mice, because it has always been a major challenge to effectively deliver genes to OHCs, so this is a major progress in the field of gene therapy for auditory diseases. In this study, the present invention achieved only partial restoration of auditory function with AAV-ie-K558R. There are several factors to explain this partial recovery phenomenon. First, AAV-ie-K558R can also transduce IHCs, and the overexpression of Prestin in IHCs may lead to IHCs dysfunction, thereby preventing the full recovery of auditory function. Second, in order to fully restore the function of OHCs, the expression level of Prestin in OHCs may have to be precisely controlled. In this study, the present invention used a widely used gene regulatory promoter, CAG, which may not be the best choice for controlling the expression of Prestin in OHCs, and further studies are needed to find the optimal AAV delivery mode for the cochlea. optimal gene regulatory elements. It will be important to develop a regulatory approach to control gene expression in the cochlea and other tissues with AAVs.
早期的研究使用了几种病毒载体，如慢病毒和腺病毒，将Atoh1递送到耳蜗，据报道，Atoh1的过表达能引起类HC细胞的再生，甚至实现了聋豚鼠听力的部分恢复。FDA批准了一项使用腺病毒载体将人Atoh1基因递送到耳蜗的人体临床试验(NCT02132130)，然而，腺病毒介导的HC再生的效率和效果需要精进。在此，本发明表明AAV-ie-K558R-Atoh1能在感觉区诱导产生许多新的HC，而且效率与以前的遗传小鼠相当。衡量腺病毒的再生效率和强烈的免疫反应后，AAV-ie-K558R在HC再生方面有更多的优势。Earlier studies used several viral vectors, such as lentivirus and adenovirus, to deliver Atoh1 to the cochlea, and it was reported that overexpression of Atoh1 could cause the regeneration of HC-like cells and even achieve partial restoration of hearing in deaf guinea pigs. The FDA approved a human clinical trial (NCT02132130) using an adenoviral vector to deliver the human Atoh1 gene to the cochlea, however, the efficiency and effectiveness of adenovirus-mediated HC regeneration need to be refined. Here, the present invention demonstrates that AAV-ie-K558R-Atoh1 induces many new HCs in sensory regions with comparable efficiency to previously genetic mice. After measuring the regeneration efficiency of adenovirus and strong immune response, AAV-ie-K558R has more advantages in HC regeneration.
总之，AAV-ie-K558R是第一个可用于耳聋小鼠模型基因治疗且能诱导新生小鼠的类HC细胞再生的AAV载体。AAV-ie-K558R的进一步发展和完善将对不同听觉疾病的基因治疗或HC细胞再生的更好疗效起到至关重要的作用。AV-ie-K558R介导的基因治疗不仅可以恢复由HCs或SCs的遗传功能障碍引起的耳聋小鼠模型的听力功能，而且还可以缓解环境和年龄引发的耳聋，对于听力损伤 的治疗具有较大应用潜力。In conclusion, AAV-ie-K558R is the first AAV vector that can be used for gene therapy in a mouse model of deafness and can induce the regeneration of HC-like cells in neonatal mice. The further development and improvement of AAV-ie-K558R will play a crucial role in gene therapy of different auditory diseases or better efficacy of HC cell regeneration. AV-ie-K558R-mediated gene therapy not only restores hearing function in a deaf mouse model caused by genetic dysfunction of HCs or SCs, but also relieves environmental and age-induced deafness, which is especially important for hearing impairment The treatment has great application potential.
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法、组合物的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。 The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. In addition, various modifications set forth herein, as well as changes in the method and composition of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in connection with various specific preferred embodiments of the invention, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as mentioned above which are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

Claims (21)

  1. 一种变异型腺相关病毒衣壳蛋白，其特征在于，所述衣壳蛋白相比野生型腺相关病毒AAV-DJ衣壳蛋白VP1，包括第558位氨基酸K的突变。A mutated adeno-associated virus capsid protein, characterized in that, compared with wild-type adeno-associated virus AAV-DJ capsid protein VP1, the capsid protein includes a mutation of the 558th amino acid K.
  2. 如权利要求1所述的变异型腺相关病毒衣壳蛋白，其特征在于，所述第558位氨基酸K的突变为K558R突变。The mutant adeno-associated virus capsid protein according to claim 1, wherein the mutation of the 558th amino acid K is a K558R mutation.
  3. 如权利要求2所述的变异型腺相关病毒衣壳蛋白，其特征在于，所述衣壳蛋白相比野生型AAV-DJ衣壳蛋白VP1，还插入有SEQ ID NO:1所示的氨基酸片段，插入位点为野生型AAV-DJ衣壳蛋白VP1的N589与R590之间。The mutant adeno-associated virus capsid protein according to claim 2, wherein the capsid protein is also inserted with an amino acid fragment shown in SEQ ID NO:1 compared to the wild-type AAV-DJ capsid protein VP1 , the insertion site is between N589 and R590 of the wild-type AAV-DJ capsid protein VP1.
  4. 如权利要求3所述的变异型腺相关病毒衣壳蛋白，其特征在于，所述野生型AAV-DJ衣壳蛋白VP1的序列如SEQ ID NO.3所示。The mutant adeno-associated virus capsid protein according to claim 3, wherein the sequence of the wild-type AAV-DJ capsid protein VP1 is as shown in SEQ ID NO.3.
  5. 如权利要求1所述的变异型腺相关病毒衣壳蛋白，其特征在于，所述变异型腺相关病毒衣壳蛋白的氨基酸序列如SEQ ID NO.5所示。The variant adeno-associated virus capsid protein according to claim 1, wherein the amino acid sequence of the variant adeno-associated virus capsid protein is as shown in SEQ ID NO.5.
  6. 一种核酸，其特征在于，所述核酸编码如权利要求1～5任一项所述的变异型腺相关病毒衣壳蛋白。A nucleic acid, characterized in that the nucleic acid encodes the mutant adeno-associated virus capsid protein according to any one of claims 1-5.
  7. 一种构建体，所述构建体含有如权利要求6所述的核酸。A construct comprising the nucleic acid of claim 6.
  8. 一种宿主细胞，其特征在于，所述宿主细胞包含如权利要求7所述的构建体或基因组中整合有外源的如权利要求6所述的核酸。A host cell, characterized in that the host cell comprises the construct according to claim 7 or the exogenous nucleic acid according to claim 6 integrated in the genome.
  9. 一种变异型腺相关病毒，其特征在于，所述变异型腺相关病毒的衣壳结构含有如权利要求1～5任一项所述的变异型腺相关病毒衣壳蛋白。A mutant adeno-associated virus, characterized in that the capsid structure of the mutant adeno-associated virus contains the mutant adeno-associated virus capsid protein according to any one of claims 1-5.
  10. 如权利要求9所述的变异型腺相关病毒，其特征在于，所述变异型腺相关病毒还包括编码目的产物的异源核苷酸序列。The mutated adeno-associated virus according to claim 9, characterized in that, the mutated adeno-associated virus further comprises a heterologous nucleotide sequence encoding a target product.
  11. 使用如权利要求9或10所述的变异型腺相关病毒转化的宿主细胞。A host cell transformed with the mutant adeno-associated virus according to claim 9 or 10.
  12. 一种变异型腺相关病毒载体系统，其特征在于，包含包装质粒，所述包装质粒中包含权利要求6所述的核酸片段。A mutant adeno-associated virus vector system, characterized in that it comprises a packaging plasmid, and the packaging plasmid comprises the nucleic acid fragment according to claim 6.
  13. 如权利要求12所述的变异型腺相关病毒载体系统，其特征在于，所述包装质粒中还包含腺相关病毒的rep基因片段。The mutant adeno-associated virus vector system according to claim 12, characterized in that, the packaging plasmid further comprises a rep gene fragment of the adeno-associated virus.
  14. 根据权利要求12所述的变异型腺相关病毒载体系统，其特征在于，所述腺相关病毒载体系统还包括表达质粒，所述表达质粒包含负责编码目的产物的异源核苷酸。The mutant adeno-associated virus vector system according to claim 12, characterized in that, the adeno-associated virus vector system further comprises an expression plasmid, and the expression plasmid contains heterologous nucleotides responsible for encoding the target product.
  15. 根据权利要求12-14任一项所述的变异型腺相关病毒载体系统，其特征在于， 所述腺相关病毒载体系统还包括辅助病毒质粒，和/或，所述腺相关病毒载体系统还包括宿主细胞。The mutant adeno-associated virus vector system according to any one of claims 12-14, characterized in that, The adeno-associated virus vector system also includes a helper virus plasmid, and/or, the adeno-associated virus vector system also includes host cells.
  16. 一种变异型腺相关病毒，由权利要求12-15任一权利要求所述变异型腺相关病毒载体系统经病毒包装获得。A mutated adeno-associated virus, which is obtained by packaging the mutated adeno-associated virus vector system according to any one of claims 12-15.
  17. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求9、10或16所述的变异型腺相关病毒以及药学上可接受的载体。A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the mutant adeno-associated virus according to claim 9, 10 or 16 and a pharmaceutically acceptable carrier.
  18. 如权利要求9、10或16所述的变异型腺相关病毒或如权利要求11所述的宿主细胞、权利要求12-15任一所述的变异型腺相关病毒载体系统，或如权利要求17所述的药物组合物在制备治疗疾病的药物中的用途；优选地，在制备基因治疗疾病的药物中的用途。The mutated adeno-associated virus as claimed in claim 9, 10 or 16 or the host cell as claimed in claim 11, the mutated adeno-associated virus vector system as claimed in any one of claims 12-15, or as claimed in claim 17 The use of the pharmaceutical composition in the preparation of medicines for treating diseases; preferably, the use in the preparation of medicines for gene therapy of diseases.
  19. 如权利要求18所述的用途，其特征在于，所述疾病选自听力障碍疾病、炎症、肿瘤、代谢疾病、疼痛、神经退行性炎症疾病中的一种或几种。The use according to claim 18, wherein the disease is selected from one or more of hearing impairment, inflammation, tumor, metabolic disease, pain, and neurodegenerative inflammatory disease.
  20. 如权利要求19所述的用途，其特征在于，包括以下1)～5)中的至少一项：The use according to claim 19, characterized in that it comprises at least one of the following 1) to 5):
    1)所述听力障碍疾病由耳蜗损伤导致；1) The hearing impairment disease is caused by cochlear damage;
    2)所述听力障碍疾病为细胞损伤的相关疾病；2) The hearing impairment disease is a disease related to cell damage;
    3)所述听力障碍疾病为由基因缺陷导致的相关疾病；3) The hearing impairment disease is a related disease caused by a gene defect;
    4)所述听力障碍疾病为由环境因素导致的相关疾病；所述环境因素选自噪声或耳毒性药物；4) The hearing impairment disease is a related disease caused by environmental factors; the environmental factors are selected from noise or ototoxic drugs;
    5)所述听力障碍疾病为由衰老导致的相关疾病。5) The hearing impairment disease is a related disease caused by aging.
  21. 如权利要求18所述的用途，其特征在于，所述药物包括以下1)～4)中的至少一项的功能：The use according to claim 18, characterized in that the drug includes the functions of at least one of the following 1) to 4):
    1)诱导HC细胞再生或类HC细胞再生；1) Inducing HC cell regeneration or HC-like cell regeneration;
    2)诱导HC细胞再生或类HC细胞再生，所述再生的HC细胞或类HC细胞表达Myo7a和/或Sox2；2) Inducing the regeneration of HC cells or HC-like cells, the regenerated HC cells or HC-like cells express Myo7a and/or Sox2;
    3)在GER区域诱导产生HC细胞或类HC细胞；3) Inducing HC cells or HC-like cells in the GER region;
    4)通过过表达Atoh1以及其它再生因子诱导耳蜗中的类HC细胞再生。 4) Induce the regeneration of HC-like cells in the cochlea by overexpressing Atoh1 and other regeneration factors.
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