WO2023143366A1 - Variant de virus adéno-associé et son application dans le traitement de maladies - Google Patents

Variant de virus adéno-associé et son application dans le traitement de maladies 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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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

La présente invention concerne un variant de virus adéno-associé et son utilisation dans le traitement génique et la régénération cellulaire d'une cochlée médiée. Le variant de virus adéno-associé peut transduire efficacement des cellules ciliées et/ou des cellules de support dans la cochlée, favorise la régénération de cellules de type HC, et aide à restaurer l'audition. Le variant du virus adéno-associé est utilisé en tant que vecteur sûr, et présente un grand potentiel clinique dans le traitement d'une maladie induisant une déficience auditive provoquée par une lésion cochléaire, et en particulier dans le traitement de la perte auditive provoquée par la mort des cellules ciliées.
PCT/CN2023/073135 2022-01-30 2023-01-19 Variant de virus adéno-associé et son application dans le traitement de maladies WO2023143366A1 (fr)

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