WO2020215968A1 - Vecteur aav pour infecter efficacement des cellules de soutien et des cellules ciliées - Google Patents

Vecteur aav pour infecter efficacement des cellules de soutien et des cellules ciliées Download PDF

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WO2020215968A1
WO2020215968A1 PCT/CN2020/081210 CN2020081210W WO2020215968A1 WO 2020215968 A1 WO2020215968 A1 WO 2020215968A1 CN 2020081210 W CN2020081210 W CN 2020081210W WO 2020215968 A1 WO2020215968 A1 WO 2020215968A1
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aav
cells
gene
hearing
vector
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PCT/CN2020/081210
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Chinese (zh)
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杨辉
姚璇
胡新德
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中国科学院脑科学与智能技术卓越创新中心
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to an AAV vector for efficiently infecting support cells and hair cells.
  • the cochlea of the inner ear is our peripheral sound perception organ.
  • the auditory cells of the cochlea play a very important role in our perception of peripheral sounds. They convert external sound waves into electrophysiological signals, which are then gradually transmitted to the auditory center of the brain through the spiral ganglion cells of the inner ear.
  • hereditary deafness is caused by mutations in certain genes in the inner ear.
  • the low efficiency of gene transfer in inner ear cells not only affects the study of inner ear gene function, but also hinders gene therapy for hereditary deafness.
  • GJB2 which accounts for about 50% of hereditary deafness
  • SLC26A4 gene mutation
  • Myo15A and OTOF genes are expressed in hair cells and account for 5-8% of hereditary deafness.
  • Adeno-associated virus has broad application prospects in the field of human gene therapy. Because of its long-term gene expression ability and non-pathogenicity, it has been widely used in various studies in the liver, Muscle, heart, brain, eye, kidney and other tissues.
  • the Acn80L65 virus has a certain infection effect on inner ear hair cells. But this virus is very difficult to obtain, and the virus production rate is extremely low when the virus is packaged, and it is very difficult to reach a titer of 1.0 ⁇ 10 12 vg/ml. Therefore, although this virus can infect inner ear hair cells, it is impossible to achieve large-scale applications.
  • AAV of various serotypes such as AAV1-AAV9 hardly infect inner ear Sertoli cells.
  • AAV2.7m8 virus can infect Sertoli cells, the infection efficiency is less than 20%, and a higher titer is required to achieve an infection efficiency of about 20%.
  • the purpose of the present invention is to provide an AAV vector that is easily available and can efficiently infect hair cells and/or supporting cells of inner ear cells.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the AAV vector provided by the present invention.
  • Another object of the present invention is to provide the use of the AAV vector of the present invention for preparing preparations or pharmaceutical compositions for the treatment of hearing disorders.
  • a gene expression vector for the treatment of hearing disorders is provided.
  • the expression vector is an AAV vector, wherein the AAV vector is inserted or carried for the treatment of hearing disorders.
  • the AAV carrier is selected from the following group: AAV-PHP.eB, AAV-DJ, or a combination thereof.
  • the therapeutic gene includes: hearing-related genes expressed in normal individuals (ie, wild-type hearing-related genes), or related genes for gene editing.
  • the related genes for gene editing include: genes encoding gene editing enzymes and guide RNAs (sgRNAs) targeting specific sites.
  • sgRNAs guide RNAs
  • the AAV vector is AAV-PHP.eB
  • the therapeutic gene is a gene expressed in inner ear hair cells.
  • the genes expressed in hair cells are selected from the group consisting of Myo15A, OTOF, Myo6, vGlut3, Tmc1, Myo7A, KCNQ4, SLC26A5, Pou4f3, etc., or a combination thereof.
  • the AAV vector is AAV-DJ
  • the therapeutic gene is a gene expressed in inner ear supporting cells.
  • the gene expressed in the supporting cell is selected from the group consisting of GJB2, SCL26A4, GJB3, Brn4, etc., or a combination thereof.
  • composition comprising:
  • the component (i) accounts for 0.1-99.9 wt% of the total weight of the pharmaceutical composition, preferably 10-99.9 wt%, more preferably 70-99 wt%.
  • the pharmaceutical composition is in a liquid dosage form.
  • the dosage form of the pharmaceutical composition is an injection.
  • the pharmaceutical composition is an injection form for intracochlear injection.
  • the carrier is an injection carrier.
  • the carrier is one or more carriers selected from the group consisting of physiological saline, dextrose saline, or a combination thereof.
  • the pharmaceutical composition can be used alone or in combination in the treatment of hearing disorders.
  • the combined use includes: combined use with other drugs for the treatment of hearing disorders.
  • the other drugs for the treatment of hearing disorders include: anti-infective antibiotic drugs, neurotrophic factor drugs, ion channel modulator drugs, vitamin drugs, etc., or a combination thereof.
  • the use of the gene expression vector as described in the first aspect of the present invention is provided to prepare a preparation or pharmaceutical composition for the treatment of hearing disorders.
  • the preparation or pharmaceutical composition is used to treat patients with hearing disorders caused by gene mutations in inner ear hair cells or supporting cells.
  • the hearing impairment disease is selected from the group consisting of hereditary deafness, non-hereditary deafness, or a combination thereof.
  • the hereditary deafness includes deafness caused by factors selected from the following group: gene mutation, gene deletion, or a combination thereof.
  • the non-hereditary deafness includes deafness caused by factors selected from the following group: drug use, trauma, infection, aging, or a combination thereof.
  • the fourth aspect of the present invention there is provided a method for treating hearing disorders, by administering the gene expression vector as described in the first aspect of the present invention to a subject in need.
  • the method of administration is intracochlear injection.
  • the AAV vector is AAV-PHP.eB or AAV-DJ, preferably The place is AAV-PHP.eB.
  • the dosage of AAV-PHP.eB is 1 ⁇ 10 11 -2 ⁇ 10 12 vg, preferably 3 ⁇ 10 11 -1.2 ⁇ 10 12 vg, more It is preferably 5 ⁇ 10 11 -1 ⁇ 10 12 vg.
  • the AAV vector in the gene expression vector is AAV-DJ.
  • the dosage of AAV-DJ is 1 ⁇ 10 11 -5 ⁇ 10 12 vg, preferably 5 ⁇ 10 11 -4 ⁇ 10 12 vg, more preferably It is 1 ⁇ 10 12 -3 ⁇ 10 12 vg.
  • the fifth aspect of the present invention there is provided a method for preparing the gene expression vector of the first aspect of the present invention.
  • the expression cassette of the therapeutic gene for the treatment of hearing disorders is connected to the AAV vector to obtain One aspect of the gene expression vector.
  • a method for transfecting hearing-related cells in vitro which includes the steps:
  • the AAV vector is selected from the following group: AAV-PHP.eB, AAV-DJ, or a combination thereof; the hearing-related cells are hair cells or supporting cells.
  • Figure 1 shows the design of screening experiments for different subtypes of AAV on mouse hair cell infections.
  • viruses of different AAV subtypes were packaged, and then injected into the cochlea of P1 ICR mice. After 2-3 weeks, the materials were taken for fluorescence observation and observation of hair cell infection. Phenotypic analysis.
  • Figure 2 shows the infection results of different subtypes of AAV on the top hair cells of the mouse cochlea.
  • Figure 3 shows the infection results of different subtypes of AAV on the hair cells of the middle part of the mouse cochlea.
  • Figure 4 shows the infection results of different subtypes of AAV on the hair cells of the basal part of the mouse cochlea.
  • Figure 5 shows the infection results of different doses of AAV-PHP.eB on the hair cells at the top of the mouse cochlea.
  • Figure 6 shows the infection results of different doses of AAV-PHP.eB on the hair cells of the middle part of the mouse cochlea.
  • Figure 7 shows the infection results of different doses of AAV-PHP.eB on the hair cells of the basal part of the mouse cochlea.
  • Figure 8 shows the design of screening experiments for different serotypes of AAV on murine Sertoli cell infection.
  • viruses of different AAV serotypes were packaged, and then injected into the cochlea of P1 ICR mice. After 2-3 weeks, the materials were taken to observe and express the infection of supporting cells by fluorescence. Type analysis.
  • Figure 9 shows the infection results of different serotypes of AAV on the supporting cells of the apical part of the mouse cochlea.
  • (B) Illustrate the infection efficiency by counting the proportion of mCherry+ cells in the top part of the supporting cells in a random 100 micron field of view. The results are obtained from at least 3 mice and are expressed as mean ⁇ standard deviation. *P ⁇ 0.05, ***P ⁇ 0.001, unpaired T test.
  • Figure 10 shows the infection results of different serotypes of AAV on Sertoli cells in the middle part of the mouse cochlea.
  • (B) Illustrate the infection efficiency by counting the proportion of mCherry+ cells in the supporting cells in the middle part of the random 100 micron field of view. The results are obtained from at least 3 mice and are expressed as mean ⁇ standard deviation. *P ⁇ 0.05, ***P ⁇ 0.001, unpaired T test.
  • Figure 11 shows the infection results of different serotypes of AAV on Sertoli cells in the basal part of the mouse cochlea.
  • (B) Illustrate the infection efficiency by counting the proportion of mCherry+ cells in the supporting cells of the basal part in a random 100-micron field of view. The results are obtained from at least 3 mice and are expressed as mean ⁇ standard deviation. *P ⁇ 0.05, ***P ⁇ 0.001, unpaired T test.
  • Figure 12 shows the infection results of different doses of AAV-DJ on Sertoli cells in the apical part of the mouse cochlea.
  • (B) Illustrate the infection efficiency by counting the proportion of mCherry+ cells in the supporting cells of the basal part in a random 100-micron field of view. The results are obtained from at least 3 mice and are expressed as mean ⁇ standard deviation. *P ⁇ 0.05, ***P ⁇ 0.001, unpaired T test.
  • Figure 13 shows the infection results of different doses of AAV-DJ on Sertoli cells in the middle part of the mouse cochlea.
  • (B) Illustrate the infection efficiency by counting the proportion of mCherry+ cells in the supporting cells of the basal part in a random 100-micron field of view. The results are obtained from at least 3 mice and are expressed as mean ⁇ standard deviation. *P ⁇ 0.05, ***P ⁇ 0.001, unpaired T test.
  • Figure 14 shows the infection results of different doses of AAV-DJ on Sertoli cells in the basal part of the mouse cochlea.
  • (B) Illustrate the infection efficiency by counting the proportion of mCherry+ cells in the supporting cells of the basal part in a random 100-micron field of view. The results are obtained from at least 3 mice and are expressed as mean ⁇ standard deviation. *P ⁇ 0.05, ***P ⁇ 0.001, unpaired T test.
  • the present inventors targeted different subtypes of AAV (AAV-2, AAV-8, AAV-9, AAV-DJ and AAV-eB), respectively packaged the virus expressing tdTomato and injected it into mice.
  • AAV-2, AAV-8, AAV-9, AAV-DJ and AAV-eB the virus expressing tdTomato
  • mice In the cochlea. Three weeks after injection, the cells of the cochlea (Apical, Middle, Basal) were subjected to immunofluorescence analysis. The experimental results show that in hair cells, AAV-PHP.eB has a higher infection efficiency than other AAV vectors in parallel experiments.
  • AAV-PHP.eB and AAV-9 can reach 100% infection in inner hair cells, AAV-8 infection efficiency is relatively low, while AAV-2 and AAV-DJ hardly infect; in outer hair cells, AAV-PHP.eB can almost reach 100% infection, AAV-8 has relatively low infection efficiency, while AAV-2 and AAV-DJ have almost no infection.
  • the infection efficiency of hair cells has gradually increased.
  • the injected dose reaches 3 ⁇ 10 9 vg, the ratio of mCherry+ cells in the inner hair cells and outer hair cells of the top, middle and base of the cochlea (Apical, Middle, Basal) is almost close to 100%, achieving complete infection.
  • AAV-DJ has a higher infection efficiency than AAV-2/8/9, which can reach an infection efficiency of about 50%.
  • the present invention has been completed on this basis.
  • a gene expression vector for the treatment of hearing disorders which is characterized in that the expression vector is an AAV vector, wherein the AAV vector is inserted or carried for the treatment of hearing An expression cassette of a therapeutic gene for a disordered disease; wherein the AAV vector is selected from the group consisting of AAV-PHP.eB, AAV-DJ, or a combination thereof.
  • the therapeutic gene includes: hearing-related genes expressed in normal individuals (ie, wild-type hearing-related genes), or related genes for gene editing.
  • the related genes used for gene editing include: genes encoding gene editing enzymes and guide RNAs (sgRNAs) targeting specific sites.
  • the therapeutic gene is a hearing-related gene expressed in a normal individual.
  • Hearing impaired diseases are very widespread. About 500 million people in the world have hearing impairments of varying degrees, and most of them are people over 60 years old. The incidence of neonatal deafness is about two to three per thousand, half of which are congenital deafness caused by genetic factors.
  • GJB2 which accounts for about 50% of hereditary deafness
  • SLC26A4 gene mutation
  • Myo15A and OTOF genes are expressed in hair cells and account for 5-8% of hereditary deafness.
  • an AAV vector capable of efficiently infecting inner ear supporting cells and hair cells is provided.
  • the AAV vector is AAV-PHP.eB
  • the therapeutic gene is a gene expressed in inner ear hair cells.
  • the genes expressed in hair cells are selected from the group consisting of Myo15A, OTOF, Myo6, vGlut3, Tmc1, Myo7A, KCNQ4, SLC26A5, Pou4f3, etc., or a combination thereof.
  • the AAV vector is AAV-DJ
  • the therapeutic gene is a gene expressed in inner ear supporting cells.
  • the gene expressed in the supporting cell is selected from the group consisting of GJB2, SCL26A4, GJB3, Brn4, etc., or a combination thereof.
  • a pharmaceutical composition which contains (i) a safe and effective amount of the gene expression vector of the first aspect of the present invention; (ii) a pharmaceutically acceptable carrier.
  • the term “including” includes “containing”, “consisting essentially of” and “consisting of”.
  • the term “consisting essentially of” means that in addition to the effective active ingredient or auxiliary ingredient, the pharmaceutical composition may also contain a small amount of minor ingredients and/or impurities that do not affect the active ingredient.
  • pharmaceutically acceptable ingredients are substances that are suitable for humans and/or mammals without excessive adverse reactions (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • the term refers to such pharmaceutical carriers: they are not essential active ingredients themselves, and they do not have excessive toxicity after administration. Suitable carriers are well known to those of ordinary skill in the art.
  • the pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or Its combination.
  • the choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the dosage form of the pharmaceutical composition of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, and sustained-release agent.
  • it can be prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition should be manufactured under aseptic conditions.
  • the pharmaceutical composition is in a liquid dosage form.
  • the dosage form of the pharmaceutical composition is an injection.
  • the pharmaceutical composition of the present invention is an injection form for intracochlear injection.
  • the carrier is an injection carrier.
  • the carrier is one or more carriers selected from the group consisting of physiological saline, glucose saline, or a combination thereof.
  • the pharmaceutical composition can be used alone or in combination in the treatment of hearing disorders.
  • the combined use includes: combined use with other drugs for the treatment of hearing disorders.
  • the other drugs for the treatment of hearing disorders include: anti-infective antibiotics, neurotrophic factor drugs, ion channel modulator drugs, vitamin drugs, etc., or a combination thereof.
  • an effective amount or “effective dose” refers to an amount that can produce function or activity on humans and/or animals and can be accepted by humans and/or animals.
  • the effective amount of the active ingredient of the present invention can vary with the mode of administration and the severity of the disease to be treated.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, through clinical trials).
  • the factors include, but are not limited to: the pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, and administration Way etc. For example, due to the urgent need to treat the condition, several divided doses can be given every day, or the dose can be reduced proportionally.
  • the two AAV vectors of the present invention have high toxicity and high stability, and it is easy to obtain high-titer and high-quality AAV in the production process.
  • AAV is a carrier approved by the FDA for clinical treatment, and the AAV carrier of the present invention has no damage to inner ear tissue.
  • the AAV vector of the present invention has the characteristics of tissue and cell-specific infection, and can target specific cell types;
  • ICR mice (P1) were used for AAV virus injection. The use and care of animals are under the guidance of the animal ethics committee.
  • HEK293T cells use 10% FBS medium, the ingredients are Dulbecco's modified Eagle medium (DMEM) (Gibco, 11965-02), 10% fetal bovine serum (FBS) (Gibco), 2mM glutamine (Gibco) ), 1% penicillin/streptomycin (Thermo Fisher Scientific) and 0.1 mM non-essential amino acids (Gibco). All cells were cultured at 5% CO 2 and 37°C.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • Gibco 2mM glutamine
  • penicillin/streptomycin Thermo Fisher Scientific
  • All cells were cultured at 5% CO 2 and 37°C.
  • the three-plasmid system was used to transfect 293T cells, and the medium was changed 4-6 hours after transfection. Collect the supernatant and cells on the fourth day. The supernatant was precipitated with PEG overnight, centrifuged at 4200 rpm and 4°C for 30 min, and then at 4400 rpm for 10 min, and the supernatant was discarded. Dissolve with 1xGB. The cells were frozen and thawed three times with liquid nitrogen. The supernatant and cells were digested with benzonase and 5M NaCl for 30 minutes. After digestion, centrifuge at 3000g for 10 minutes and take the supernatant. Density gradient ultracentrifugation, 68000rpm 18°C1h 25min. Take the layer, dilute with PBS and concentrate with an ultrafiltration tube.
  • ICR P1 mice both male and female, are randomly grouped according to different AAV serotypes, with 4 mice in each group.
  • Under a stereomicroscope cut the skin 2mm behind the ear groove with ophthalmological scissors, and separate the subcutaneous tissue slightly. The facial nerve, the posterior wall of the auditory bubble and the posterior abdomen of the digastric muscle can be seen.
  • the cochlear lateral ligament was punctured with a glass microelectrode and 1 microliter of virus was injected into the mouse cochlea. After the injection, the glass electrode was gently pulled out and the incision was sutured. After 3 weeks of injection, samples were taken to analyze the phenotype.
  • mice were anesthetized with sodium pentobarbital (50mg/Kg, Sigma), and then perfused with 0.9% normal saline and 4% paraformaldehyde through a peristaltic pump (Gilson), and then in 4% poly Fix in formaldehyde at 4°C overnight.
  • the basement membrane is separated under a dissecting microscope and cut into three sections (top, middle and base). The separated basement membrane was washed three times with 0.1M phosphoric acid buffer (PB), and then incubated with primary antibody diluted with 5% NGS at 4°C overnight.
  • PB 0.1M phosphoric acid buffer
  • the infection efficiency in Sertoli cells and hair cells was quantified, and the infection efficiency was explained by counting the proportion of mCherry+ cells in the Sertoli cells in a random 100-micron field of view. The results are obtained from at least 3 mice and are expressed as mean ⁇ standard deviation. *P ⁇ 0.05, ***P ⁇ 0.001, unpaired T test.
  • Example 1 Screening of different subtypes of AAV on mouse hair cell infection
  • AAV-2, AAV-8, AAV-9, AAV-PHP.eB, and AAV-DJ were used to package CAG-Tdtomato-polyA, and by observing the infection rate of hair cells .
  • Figure 1 To judge the infection efficiency of different AAV subtypes (Figure 1), to screen out AAV subtypes that can efficiently infect mouse hair cells in vivo.
  • AAV-2, AAV-8, AAV-9, AAV-PHP.eB and AAV-DJ AAV capsid packaged CAG-Tdtomato-polyA virus were injected into the cochlea of P1 ICR mice. Each mouse was injected with 0.5-1 ⁇ 10 10 vg AAV virus. Three weeks after the injection, the cochlear basement membrane of the mouse was stripped and stained, and the infection efficiency was demonstrated by counting the proportion of mCherry+ cells in the hair cells in a random 100 micron field of view.
  • the hair cells of the apical, middle, and basal (apical, middle, and basal) parts were counted separately.
  • AAV-PHP.eB 100% of the inner hair cells (Myo6 positive) in the basal part are Tdtomato positive, while only 0%, 77.64 ⁇ in each group of AAV-2, AAV-8, AAV-9, and AAV-DJ 9.89%, 60.76 ⁇ 7.35%, 0% of hair cells were Tdtomato positive ( Figure 4).
  • AAV-PHP.eB 97.11 ⁇ 1.78% of the outer hair cells (Myo6 positive) in the basal part were Tdtomato positive, while only 0% in each group of AAV-2, AAV-8, AAV-9, and AAV-DJ. 8.94 ⁇ 5.09%, 32.28 ⁇ 4.96%, 0% of the hair cells were Tdtomato positive ( Figure 4, Table 1).
  • AAV-PHP.eB and AAV-9 can achieve 100% infection in inner hair cells, AAV-8 infection efficiency is relatively low, while AAV-2 and AAV-DJ almost do not infect.
  • AAV-PHP.eB can almost achieve 100% infection, AAV-9 and AAV-8 have relatively low infection efficiency, while AAV-2 and AAV-DJ hardly infect.
  • the infection of hair cells by AAV-PHP.eB virus at different doses was tested.
  • the AAV-PHP.eB virus was divided into dose gradient groups, and each group of mice was injected with 5 ⁇ 10 8 , 1 ⁇ 10 9 , 3 ⁇ 10 9 , 5 ⁇ 10 9 and 1 ⁇ 10 10 vg virus.
  • the cochlear basement membrane of the mouse was stripped and stained, and the proportion of mCherry+ cells in the hair cells of the top, middle and base were counted.
  • Example 3 Screening of mouse Sertoli cell infections by different serotypes of AAV
  • AAV-2, AAV-8, AAV-9 and AAV-DJ Different AAV capsid packaged CAG-Tdtomato-polyA virus were injected into the cochlea of P1 ICR mice. Each mouse was injected with 0.5-1 ⁇ 10 10 vg AAV virus.
  • the cochlear basement membrane of the mouse was stripped and stained, and the infection efficiency was explained by counting the proportion of mCherry+ cells in the supporting cells in a random 100 micron field of view.
  • the supporting cells in the apical, middle, and basal (apical, middle, and basal) parts were counted separately.
  • Tdtomato positive 50.84 ⁇ 1.55% of the supporting cells (sox2 positive) in the middle part of AAV-DJ are Tdtomato positive, while only 0%, 2.78 ⁇ 0.83%, 10.85 ⁇ in each group of AAV-2, AAV-8, and AAV-9 2.57% of Sertoli cells were positive for Tdtomato ( Figure 10, Table 1).
  • AAV-DJ has a higher infection efficiency than AAV-2/8/9.
  • the infection of Sertoli cells by AAV-DJ virus at different doses was determined.
  • the AAV-DJ virus was divided into dose gradient groups, and each group of mice was injected with 5 ⁇ 10 8 , 1 ⁇ 10 9 , 3 ⁇ 10 9 , 5 ⁇ 10 9 and 1 ⁇ 10 10 vg virus.
  • the cochlear basement membrane of the mouse was stripped and stained, and the proportion of mCherry+ cells in the supporting cells of the top, middle and base were counted.
  • mice Three weeks after the injection of different subtypes of AAV viruses, the cochlear basement membrane of the mice was stripped and stained, and the proportion of mCherry+ cells in the supporting cells of the top, middle and basal parts were counted. The results are obtained from at least 3 mice and are expressed as mean ⁇ standard deviation.
  • the AAV--PHP.eB and AAV--DJ viruses were divided into dose gradient groups. Each group of mice was injected with 5 ⁇ 10 8 , 1 ⁇ 10 9 , 3 ⁇ 10 9 , 5 ⁇ 10 9 and 1 ⁇ 10 10 vg. virus. Three weeks after the injection, the cochlear basement membrane of the mouse was stripped and stained, and the proportion of mCherry+ cells in the supporting cells of the top, middle and base were counted. The results are obtained from at least 3 mice and are expressed as mean ⁇ standard deviation.
  • the present invention is the first study of the infection performance of AAV-PHP.EB in the inner ear.
  • the viruses used in existing researches are AAV1-AAV9, Rh10 and other viruses, and PHP.EB is a new artificially modified virus serotype.
  • Previous studies have shown that AAV-PHP.eB has a strong ability to infect the central nervous system in vivo, but it has not been studied for the inner ear system. This study proved for the first time that AAV-PHP.eB has a very high infection rate on inner ear hair cells, and can still maintain an infection efficiency of about 95% when the titer is 5.0x10 12 vg/ml.
  • the newly published Acn80L65 virus also has a high infection rate for inner ear hair cells, but this virus is very difficult to obtain, and the virus production rate is extremely low when the virus is packaged, and it is very difficult to reach a titer of 1.0x10 12 vg/ml, so this virus Although the infection rate is high, it is impossible to achieve large-scale applications.
  • the PHP.EB used in this study is very easy to obtain, and can easily reach a titer of 3.0x10 13 vg/ml, which can easily exceed the titer of the Acn80L65 virus by 30 times.
  • AAV-PHP.eB has great potential to become an AAV virus widely used in inner ear hair cell infection.
  • the AAV-DJ used in the present invention has not been studied in the inner ear.
  • the present invention is the first time to explore the infection of the inner ear by AAV-DJ in the inner ear.
  • the inventors found that AAV-DJ has a high infection performance on inner ear Sertoli cells, and can reach more than 50% infection.
  • Previous studies have found that AAV of various serotypes such as AAV1-AAV9 hardly infect inner ear supporting cells. Although the newly published AAV2.7m8 virus can infect Sertoli cells, the infection efficiency is less than 20%, and a higher titer is required to achieve an infection efficiency of about 20%.
  • the AAV-DJ used in the present invention can reach an infection efficiency of more than 50% when the titer is 1.0x10 13 vg/ml, which far exceeds the infection efficiency of AAV2.7m8.
  • AAV-DJ is easy to package and has a very high toxin production rate. It is easy to obtain high-quality and high-titer viruses (in our research we can often obtain high-titer viruses of 5.0x10 13 vg/ml).
  • AAV-DJ has great potential to be widely used in AAV infected by inner ear Sertoli cells.

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Abstract

L'invention concerne un vecteur d'expression génique pour traiter une maladie de déficience auditive, le vecteur d'expression étant un vecteur AAV dans lequel une cassette d'expression d'un gène thérapeutique pour traiter la maladie de déficience auditive est insérée ou transportée; et le vecteur AAV est choisi dans le groupe des AAV-PHP.eB, des AAV-DJ ou une combinaison correspondante. Le vecteur AAV a une efficacité élevée pour infecter des cellules ciliées et des cellules de soutien de l'oreille interne, est facile à produire, et a une bonne sécurité et un niveau élevé de ciblage.
PCT/CN2020/081210 2019-04-22 2020-03-25 Vecteur aav pour infecter efficacement des cellules de soutien et des cellules ciliées WO2020215968A1 (fr)

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