AAV MUTANT THAT EFFICIENTLY INFECTS SUPPORTING CELLS

Abstract

Provided is an AAV mutant that efficiently infects supporting cells. Specifically, provided is a gene expression vector used for treating hearing disorders, and the described expression vector is an AAV mutant. In the AAV mutant, an expression cassette of a therapeutic gene used for treating hearing disorders is inserted or carried, and additionally, the AAV mutant contains a gene sequence that encodes a viral capsid protein mutant.

Claims

1. A viral capsid protein mutant, wherein the viral capsid protein mutant is a non-natural protein, and relative to the wild-type viral capsid protein, the viral capsid protein mutant has one or more amino acid mutations selected from the group consisting of: Serine (S) at position 670 is mutated to alanine (A), threonine (T) at position 251 is mutated to alanine (A), and lysine (K) at position 534 is mutated to arginine (R); wherein, positions 670, 251 and 534 correspond to positions 670, 251 and 534 of the sequence as shown in SEQ ID NO:1.

2. The viral capsid protein mutant of claim 1, wherein the viral capsid protein mutant has the activity of promoting the AAV vector to infect inner ear supporting cells.

3. The viral capsid protein mutant of claim 1, wherein the AAV vector is an AAV-DJ vector.

4. A gene expression vector for the treatment of the hearing disorders, wherein the expression vector is an AAV mutant, wherein in the AAV mutant, an expression cassette for a therapeutic gene for the treatment of the hearing disorders is inserted or carried; in addition, the AAV mutant contains a gene sequence encoding the viral capsid protein mutant of claim 1.

5. The gene expression vector of claim 4, wherein the AAV mutant contains a gene sequence encoding the amino acid sequence as shown in SEQ ID NO: 2, 3 or 4.

6. A pharmaceutical composition comprising: (i) the gene expression vector of claim 4; (ii) a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is an injection for intracochlear injection.

8. (canceled)

9. A method for treating hearing disorders by administering the gene expression vector of claim 4 to a subject in need.

10. (canceled)

Description

DESCRIPTION OF FIGURE

[0075] FIG. 1 shows the design of screening experiment of AAV-DJ and its mutants on supporting cell infection in mice.

[0076] Among them, the viruses of AAV-DJ and its mutants (10 species) are packaged separately, and then injected into the cochlea of P1 ICR mice. After 2-3 weeks, the samples are taken to perform fluorescence observation and phenotypic analysis of the infection of supporting cells.

[0077] FIG. 2 shows analysis of the efficiency of AAV-DJ and its mutants infecting HEK293T cells in vitro.

[0078] Among them, AAV-DJ and its mutant viruses are infected with HEK293T cells in vitro, and the expression of tdTomato is observed by fluorescence microscope after 48 hours. The titer and dosage of AAV-DJ and its mutant virus are shown in the figure.

[0079] FIG. 3 shows the infection results of AAV-DJ and its mutants on the supporting cells in the apical part of the mouse cochlea.

[0080] After injection of AAV-DJ and its mutant virus, the representative fluorescence image of the supporting cells at the top is shown in the figure. P1 ICR mice are collected 3 weeks after virus injection. Each mouse is injected with 4×10.sup.9 vg AAV virus.

[0081] FIG. 4 shows the infection results of AAV-DJ and its mutants on supporting cells in the middle part of the mouse cochlea.

[0082] After injection of AAV-DJ and its mutant virus, the representative fluorescence image of the supporting cells in the middle part is shown in the figure. P1 ICR mice are collected 3 weeks after virus injection. Each mouse is injected with 4×10.sup.9 vg AAV virus.

[0083] FIG. 5 shows the infection results of AAV-DJ and its mutants on supporting cells in the basal part of the mouse cochlea.

[0084] After injection of AAV-DJ and its mutant virus, the representative fluorescence image of the supporting cells in the basal part is shown in the figure. P1 ICR mice are collected 3 weeks after virus injection. Each mouse is injected with 4×10.sup.9 vg AAV virus.

[0085] FIG. 6 shows the statistical analysis results of infection of AAV-DJ and its mutants on mouse cochlear supporting cells.

[0086] (A-C) the infection efficiency is explained by counting the proportion of mCherry+ cells in the top (A), middle (B) and basal (C) supporting cells in a random 100 micron field of view. AAV-DJ and its mutant virus are collected 3 weeks after injection of P1 ICR mouse cochlea. Each group of mice is injected with 4×10.sup.9 vg AAV virus. 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.

DETAILED DESCRIPTION

[0087] After extensive and in-depth research, after a large number of screenings, the inventors have developed an AAV mutant capable of efficiently infecting supporting cells of the inner ear for the first time. Specifically, the inventors have modified the cap sequence of AAV-DJ; among them, the specific tyrosine, serine, threonine, or lysine sites in AAV-DJ are mutated to study whether the mutation site can further improve the infection efficiency in supporting cells, and screen a large number of different mutations. In the experiment, the mutant viruses expressing tdTomato are packaged separately and injected into the cochlea of mice. Three weeks after injection, immunofluorescence analysis is performed on the cells of cochlea (Apical, Middle, Basal). Experimental results show that in supporting cells, the AAV-DJ mutant (S670A) can significantly improve the infection efficiency of supporting cells. The present invention has been completed on this basis.

Viral Capsid Protein Mutant

[0088] In the present invention, a viral capsid protein mutant is provided, the viral capsid protein mutant is a non-natural protein, and the viral capsid protein mutant has one or more amino acid mutations selected from the group consisting of in the wild-type viral capsid protein:

[0089] Serine (S) at position 670 is mutated to alanine (A), threonine (T) at position 251 is mutated to alanine (A), and lysine (K) at position 534 is mutated to arginine (R);

[0090] wherein, the positions 670, 251 and 534 correspond to the positions 670, 251 and 534 of the sequence as shown in SEQ ID NO:1.

[0091] In one embodiment of the present invention, the amino acid sequence of the wild-type viral capsid protein is shown in SEQ ID NO:1.

TABLE-US-00001 (SEQ ID NO: 1) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVE HSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPS GVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTR TWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS PRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID QYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRV SKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQS GVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRG NRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMG GFGLKHPPPQILIKNTPVPADPPTTFNQSKLN FITQYSTGQVSVEIEW ELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTR NL*

[0092] In a preferred embodiment of the present invention, the amino acid sequence of the viral capsid protein mutant is shown in SEQ ID NO: 2, wherein the S670A mutation occurs corresponding to the wild-type viral capsid protein sequence.

TABLE-US-00002 (SEQ ID NO: 2) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVE HSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPS GVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTR TWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS PRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID QYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRV SKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQS GVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRG NRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMG GFGLKHPPPQILIKNTPVPADPPTTFNQSKLN FITQYSTGQVSVEIEW ELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTR NL*

[0093] In a preferred embodiment of the present invention, the amino acid sequence of the viral capsid protein mutant is shown in SEQ ID NO: 3, wherein the T251A mutation occurs corresponding to the wild-type viral capsid protein sequence.

TABLE-US-00003 (SEQ ID NO: 3) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVE HSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPS GVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTR TWALP YNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS PRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID QYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRV SKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQS GVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRG NRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMG GFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEW ELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTR NL*

[0094] In a preferred embodiment of the present invention, the amino acid sequence of the viral capsid protein mutant is shown in SEQ ID NO: 4, wherein a K534R mutation occurs corresponding to the wild-type viral capsid protein sequence.

TABLE-US-00004 (SEQ ID NO: 4) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVE HSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPS GVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTR TWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS PRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID QYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRV SKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEE FFPQS GVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRG NRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMG GFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEW ELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTR NL*

[0095] The present invention also includes polypeptides or proteins with the same or similar functions that have 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homology with the sequence as shown in SEQ ID NO: 2, 3 or 4 of the present invention.

[0096] The “same or similar function” mainly refers to: “promoting the activity of the AAV vector to infect the inner ear supporting cells”. Preferably, the AAV vector is an AAV-DJ vector.

[0097] It should be understood that the amino acid numbering in the viral capsid protein mutants of the present invention is based on SEQ ID NO: 2, 3 or 4. When the homology of a specific viral capsid protein mutant with the sequence as shown in SEQ ID NO: 2, 3 or 4 reaches 80% or more, the amino acid numbering of the viral capsid protein mutant may be misplaced relative to the amino acid numbering of SEQ ID NO: 2, 3, or 4, such as shifting positions 1-5 to the N-terminus or C-terminus of the amino acid, and using conventional sequence alignment techniques in the art, those skilled in the art can generally understand that such a misalignment is within a reasonable range, and should not be due to misplacement of amino acid numbering, mutants with the same or similar catalytic activity for producing viral capsid protein mutants that have homology to 80% (such as 90%, 95%, 98%) are not within the scope of the viral capsid protein mutants of the present invention.

[0098] The viral capsid protein mutant of the present invention is a synthetic protein or a recombinant protein, that is, it can be a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production protocol, the viral capsid protein mutant of the present invention may be glycosylated or non-glycosylated. The viral capsid protein mutants of the present invention may also include or not include the initial methionine residue.

[0099] The present invention also includes fragments, derivatives and analogs of the viral capsid protein mutants. As used herein, the terms “fragment”, “derivative” and “analog” refer to a protein that substantially retains the same biological function or activity as the viral capsid protein mutant.

[0100] The viral capsid protein mutant fragment, derivative or analogue of the present invention may be (i) a viral capsid protein mutant with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a viral capsid protein mutant with substitution groups in one or more amino acid residues, or (iii) a viral capsid protein mutant formed by fusion of a mature viral capsid protein mutant with another compound (such as a compound that extends the half-life of the viral capsid protein mutant, such as polyethylene glycol), or (iv) an additional amino acid sequence is fused to this viral capsid protein mutant sequence to form a viral capsid protein mutant (such as the leader sequence or secretory sequence or the sequence or the proprotein sequence used to purify the viral capsid protein mutant, or the fusion protein formed with the antigen IgG fragment). According to the teachings herein, these fragments, derivatives and analogs fall within the scope of those skilled in the art. In the present invention, conservatively substituted amino acids are preferably generated by amino acid substitutions according to Table I.

TABLE-US-00005 TABLE I Preferred Initial residues Representative substitution substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; phe Leu Leu (L) Ile; Val; Met; Ala; phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; phe; Ile Leu Phe (F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; phe Tyr Tyr (Y) Trp; phe; Thr; Ser Phe Val (V) Ile; Leu; Met; phe; Ala Leu

[0101] In addition, the capsid protein mutant of the virus of the present invention can also be modified. Modified (usually unchanged primary structure) forms include: chemically derived forms of viral capsid protein mutants in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing or during further processing steps of viral capsid protein mutants. This modification can be accomplished by exposing the viral capsid protein mutant to an enzyme that performs glycosylation (such as mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes mutants of the viral capsid protein that have been modified to improve their resistance to proteolysis or to optimize their solubility.

[0102] The term “polynucleotide encoding a viral capsid protein mutant” may include a polynucleotide encoding the viral capsid protein mutant of the present invention, or may also be a polynucleotide that also includes additional coding and/or non-coding sequences.

[0103] The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or viral capsid protein mutants having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the encoded viral capsid protein mutant.

[0104] The present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under strict conditions (or stringent conditions). In the present invention, “ strict conditions” refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60° C.; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42° C., etc.; or (3) Hybridization occurs only when the identity between the two sequences is at least 90% or more, and more preferably 95% or more.

[0105] The viral capsid protein mutants and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, are purified to homogeneity.

[0106] The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombination method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template, amplifying the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.

[0107] Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

[0108] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.

[0109] At present, the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.

[0110] The method of using PCR technology to amplify DNA/RNA is preferably used to obtain the polynucleotide of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA end rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and can be synthesized by conventional methods. The amplified DNA/RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.

Gene Expression Vector of the Present Invention

[0111] As used herein, the terms “gene expression vector” and “AAV mutant” can be used interchangeably and refer to the AAV mutant of the present invention, wherein in the AAV mutant, an expression cassette for a therapeutic gene for the treatment of hearing disorders is inserted or carried; and, the AAV mutant contains a gene sequence encoding the viral capsid protein mutant as described in the first aspect of the present invention.

[0112] In a preferred embodiment, the AAV mutant is an AAV-DJ mutant.

[0113] Preferably, the therapeutic gene includes: a hearing-related gene expressed in normal individuals (i.e., a wild-type hearing-related gene), or a related gene for gene editing.

[0114] In a preferred embodiment, the related gene used for gene editing includes: the coding gene of the viral capsid protein mutant and the guide RNA (sgRNA) targeting a specific site.

[0115] In another preferred embodiment, the hearing-related gene is a gene expressed in inner ear supporting cells.

[0116] In one embodiment of the present invention, the gene expressed in the inner ear supporting cells is selected from the group consisting of: GJB2, SCL26A4, GJB3, Brn4, etc., and a combination thereof.

[0117] In a preferred embodiment, the AAV mutant of the present invention contains a gene sequence encoding the amino acid sequence as shown in SEQ ID NO: 2.

Pharmaceutical Composition and Method of Administration

[0118] In the present invention, it further provides a pharmaceutical composition, which contains (i) a safe and effective amount of the gene expression vector according to the first aspect of the present invention; (ii) a pharmaceutically acceptable carrier.

[0119] As used herein, the term “including” includes “containing”, “consisting essentially of” and “consisting of”.

[0120] As used herein, 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.

[0121] As used herein, “pharmaceutically acceptable” ingredients are substances that are suitable for humans and/or mammals without excessive side effects (such as toxicity, irritation, and allergic reactions), that is, substances that have a reasonable benefit/risk ratio.

[0122] The term “pharmaceutically acceptable carrier” refers to a carrier used for the administration of a therapeutic agent, and includes various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients themselves, and there is no excessive toxicity after administration. Suitable carriers are well known to those of ordinary skill in the art.

[0123] 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 a combination thereof. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

[0124] 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 a combination thereof. Generally, 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. For example, 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.

[0125] In a preferred embodiment, the pharmaceutical composition is in a liquid dosage form.

[0126] In a more preferred embodiment, the dosage form of the pharmaceutical composition is an injection. Preferably, the pharmaceutical composition of the present invention is an injection form for intracochlear injection.

[0127] In one embodiment of the present invention, the carrier is an injection carrier. Preferably, the carrier is one or more carriers selected from the group consisting of physiological saline, dextrose saline, and a combination thereof.

[0128] In one embodiment of the present invention, the pharmaceutical composition can be used alone or in combination in the treatment of hearing disorders.

[0129] In the present invention, the used in combination includes: used in combination with other drugs for the treatment of hearing disorders.

[0130] In a more preferred embodiment, 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.

[0131] As used herein, the term “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.

[0132] 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 route of administration, etc. For example, due to the urgent request of the treatment condition, several divided doses can be given every day, or the dose can be reduced proportionally.

[0133] The main advantages of the present invention include:

[0134] 1) High efficiency: AAV-DJ mutant has a high infection efficiency on inner ear supporting cells.

[0135] 2) Ease of production: The AAV-DJ mutant vector of the present invention has a high virus detection rate and high stability, and it is easy to obtain high-titer and high-quality AAV during the production process.

[0136] 3) Good safety: AAV is a vector approved by the FDA for clinical treatment, and the AAV-DJ mutant vector of the present invention has no damage to inner ear tissue.

[0137] 4) High targeting: Compared with small molecule drugs, the AAV-DJ mutant vector of the present invention has the characteristics of tissue and cell-specific infection, and can target specific cell types.

[0138] The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

EXPERIMENTAL MATERIALS AND METHODS

Mouse

[0139] ICR mice (P1) were used for AAV virus injection. The use and care of animals were completed under the guidance of the animal ethics committee.

Cell Culture and Infection

[0140] HEK293T cells used 10% FBS medium, the ingredients were Dulbecco's modified Eagle medium (DMEM) (Gibco, 11965-02), 10% fetal bovine serum (FBS) (Gibco), 2 mM glutamine (Gibco), 1% penicillin/streptomycin (Thermo Fisher Scientific) and 0.1 mM non-essential amino acids (Gibco). All cells were cultured under 5% CO.sub.2, 37° C.

[0141] 48 hours after HEK293T cells were infected with AAV, the expression of tdTomato was observed by fluorescence microscope.

AAV Virus Packaging

[0142] The three-plasmid system was used to transfect 293T cells, and the medium was changed 4-6 hours after transfection. Collecting the supernatant and cells on the fourth day. The supernatant was precipitated with PEG overnight, centrifuged at 4200 rpm, 4° C. for 30 min, and then centrifuged at 4400 rpm for 10 min, and the supernatant was discarded. Dissolved with 1×GB. The cells were repeatedly frozen and thawed three times with liquid nitrogen. The supernatant and cells were digested with benzonase and 5M NaCl for 30 minutes. After digestion, centrifuged at 3000 g for 10 minutes and taking the supernatant. Density gradient ultracentrifugation, 68000 rpm 18° C. 1h 25 min. Taking the layer, diluted with PBS and concentrated with an ultrafiltration tube.

AAV Virus Injection

[0143] ICR P1 mice, there is no restriction on male and female, are randomly grouped according to different AAV serotypes, with 4 mice in each group. Under a stereomicroscope, cutting the skin 2 mm behind the posterior sulcus of auricle with ophthalmological scissors, and slightly separating the subcutaneous tissue. The facial nerve, the panes posterior of the auditory vesicle, and the posterior belly of the musculus digastricus can be seen. A glass microelectrode was used to puncture the cochlear sidewall ligament and injecting 1 microliter of virus into the mouse cochlea. After the injection, the glass electrode was gently pulled out, and the incision was sewn up. Three weeks after the injection, the samples were taken to analyze the phenotype.

Immunostaining Analysis

[0144] In the immunostaining experiment, the mice were anesthetized with Pentobarbital sodium (50 mg/Kg, Sigma), and peristaltic pump (Gilson) was used for cardiac perfusion with 0.9% saline and 4% paraformaldehyde, and then fixed in 4% paraformaldehyde at 4° C. overnight. The next day the tissue was decalcified in 10% EDTA. After the decalcification is completed, the basement membrane is separated under a dissecting microscope and cut into three sections (top, middle and base part). The separated basement membrane was washed three times with 0.1M phosphate buffer (PB), and then incubated with primary antibody diluted with 5% NGS at 4° C. overnight. The next day, the sections were washed three times with PB, and then incubated with the secondary antibody on a rotary shaker for two hours at room temperature. Finally, the sections were counter-stained with DAPI for 20 minutes, and then mounted on a glass slide with SlowFade Diamond Antifade Mountant (Life).

Antibody

Supporting Cells

[0145] Primary antibody: goat-anti-Sox2 (Santa Cruz Biotechnology, sc-17320)

[0146] Secondary antibody: Alexa Fluor® 488 AffiniPure Donkey Anti-Goat IgG (H+L) (Jackson ImmunoResearch, 705-545-003)

Hair Cells

[0147] Primary antibody: rabbit anti-Myosin-VI polyclonal (Proteus Biosciences, 25-6791)

[0148] Secondary antibody: Cy™ 5 AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson Immuno Research, 711-175-152)

Data Statistical Analysis and Software

[0149] The infection efficiency in supporting cells and hair cells was quantified, 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 results are obtained from at least 3 mice and are expressed as mean±standard deviation. *P<0.05, ***P<0.001, unpaired T test.

[0150] Snapgene: for plasmid map construction and design

[0151] Excel: Raw data processing

[0152] NIS-Elements Viewer 4.0: Experimental image processing

[0153] ImageJ: Experimental image processing

[0154] Adobe photoshop CS6: Experimental image processing

[0155] Adobe Illustrator CS4: Experimental image processing

Example 1: AAV-DJ Mutant Screening Experiment Design

[0156] In this example, the cap sequence of AAV-DJ was genetically modified to screen out AAV vectors that can efficiently infect supporting cells.

[0157] Specifically, the present inventors mutated specific tyrosine, serine, threonine or lysine sites in the cap sequence of AAV-DJ, and studied whether the mutants could further improve the infection efficiency in supporting cells (FIG. 1).

[0158] Among them, 7 different AAV-DJ mutants (K137A, T251A, S278A, S503A, K534R, K546R and S670A) were designed respectively.

Example 2: AAV-DJ Mutant Infects HEK293T Cells In Vitro

[0159] In this example, HEK293T cells were infected with AAV-DJ and its mutant viruses in vitro, respectively, and the expression of tdTomato was observed by fluorescence microscope after 48 hours.

[0160] The titer and dosage of AAV-DJ and its mutant virus are shown in the figure (FIG. 2).

[0161] The results show that AAV-DJ mutants (K137A, T251A, S670A) have a higher infection efficiency than AAV-DJ in HEK293T cells in vitro.

Example 3

[0162] In this example, AAV-DJ and its mutant virus were respectively injected into the cochlea of P1 ICR mice. Each mouse was injected with 4×10.sup.9 vg AAV virus. Similarly, 3 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 in the top, middle and basal parts were counted, respectively.

[0163] The experimental results show that in AAV-DJ mutants (T251A and S670A), 34.99±0.70% and 69.30±2.96% of the supporting cells (sox2 positive) in the top part of the AAV-DJ mutants are Tdtomato positive, which is significantly higher than the 24.64±3.79% of AAV-DJ. (FIG. 3, FIG. 6).

[0164] In AAV-DJ mutants (T251A, K534R and S670A), 28.01±5.01%, 31.84±10.63% and 27.38±4.61% of the supporting cells (sox2 positive) in the middle part of the AAV-DJ mutants are Tdtomato positive, which is higher than 16.1±3.00% of AAV-DJ, but there is no significant difference (FIG. 4, FIG. 6).

[0165] The AAV-DJ mutants (5503A, K534R and S670A) have 25.61±20.99%, 16.74±3.84% and 44.28±7.37% in the supporting cells (sox2 positive) of the basal part, respectively. 44.28±7.37% of the cells are Tdtomato positive. It is significantly higher than 5.61±3.22% of AAV-DJ (FIG. 5, FIG. 6).

[0166] The above experimental results show that in supporting cells, the AAV-DJ mutant (S670A) can significantly improve the infection efficiency of supporting cells, and T251A and K534R can also promote the infection of supporting cells by AAV-DJ.

Discussion

[0167] In the present invention, in order to screen out AAV vectors that can efficiently infect supporting cells, genetic modification is performed on the cap sequence of AAV-DJ.

[0168] The ubiquitin-proteasome degradation mechanism is considered to be the main obstacle to the efficiency of AAV infection. Among them, the point mutation at the specific site of the viral capsid protein cap is the simplest and most common method, which can make the viral vector avoid intracellular phosphorylation and subsequent ubiquitination and proteasome-mediated degradation. It has been reported in the literature that specific tyrosine, serine, threonine or lysine point mutations in AAV2 and AAV8 viruses can significantly increase their infection efficiency, but they have specific liver tropism in vivo.

[0169] In a specific embodiment of the present invention, specific tyrosine, serine, threonine or lysine sites in AAV-DJ are mutated to investigate whether the mutants can further improve the infection efficiency in supporting cells.

[0170] Specifically, 7 different AAV-DJ mutants (K137A, T251A, S278A, 5503A, K534R, K546R and S670A) were designed respectively. The inventors have separately packaged viruses expressing the AAV-DJ mutant of tdTomato and injected them into the cochlea of mice. Three weeks after the injection, we have performed immunofluorescence analysis on the cells of the cochlea (top, middle, and basal).

[0171] Through in vitro and in vivo experimental results, it is found that the AAV-DJ mutant (S670A) can significantly improve the infection efficiency of supporting cells. This provides great help for the study of gene function of ear supporting cells and gene therapy strategies.

[0172] In addition to point mutations at specific locations, there are other ways to modify the viral protein coats, such as inserting random sequences, random point mutations, disrupting rearrangements, and so on. All of these may further promote the improvement of the infection efficiency of mouse supporting cells, and more experiments are needed for verification.

[0173] All publications mentioned herein are incorporated by reference as if each individual document was cited as a reference, as in the present application. It should also be understood that, after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications, equivalents of which falls in the scope of claims as defined in the appended claims.