Variant adeno-associated viruses and their application in disease treatment

Abstract

This invention relates to the field of biotechnology and discloses a variant adeno-associated virus (AAV) and its application in mediating gene therapy and hair cell regeneration in the cochlea. The variant AAV can efficiently transduce hair cells and / or supporting cells in the cochlea, promoting the regeneration of HC-like cells and helping to restore hearing. As a safe vector, the variant AAV has significant clinical potential in treating hearing impairment caused by cochlear damage, especially hearing loss caused by hair cell death.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, to a variant adeno-associated virus and its application in disease treatment, and particularly to AAV-ie-K558R and its application in mediating cochlear gene therapy and hair cell regeneration. Background Technology

[0002] The cochlea is composed of various cell types, including hair cells, supporting cells, and spiral ganglion neurons, responsible for converting mechanical energy into electrical signals to enable hearing. The cells that make up the cochlea are crucial for hearing. Genetic and environmental factors can lead to dysfunction of the cochlea and auditory system. While sensorineural hearing loss can be caused by gene mutations in cochlear hair cells (HCs) and supporting cells (SCs), non-genetic factors, such as noise, ototoxic drugs, or aging, can also induce hearing loss by damaging HCs. In either case, this damage is irreversible in mammals that lack the ability to regenerate cochlear cells. Although 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.

[0003] The hair cells (HCs) in the cochlea comprise two distinct types: outer hair cells (OHCs) responsible for amplifying sound and inner hair cells (IHCs) that convert mechanical energy into electrical signals. HCs anchor sensory epithelial cells to the basilar membrane, an environment crucial for maintaining normal HC function. Notably, because sensorineural cells (SCs) have the potential to transdifferentiate into HC-like cells, HC regeneration is considered a potential treatment for acquired hearing loss caused by non-genetic factors.

[0004] Gene therapy has become an important means of treating hereditary diseases. In fact, current research has demonstrated its potential in treating hearing loss. When certain genes, such as tmc1, clrn, and otof, are delivered to the cochlea, hearing function can be restored in animal models. Previous studies have shown that regeneration of HCs in the adult cochlea can restore hearing function in a deaf guinea pig model, but further research is needed to extend this finding to other animal models.

[0005] In recent years, gene therapy has emerged as a promising treatment for deafness. A major challenge in gene therapy for deafness is how to effectively deliver genes to specific cells in the cochlea. Adeno-associated viruses (AAVs) have demonstrated high safety in both animal models and humans and are widely used to deliver genetic material to cells for gene therapy of many different organs and diseases. In the field of hearing, early research found that Anc80L65 is a promising vector for delivering Harmonin to treat deafness caused by HCs dysfunction. However, its efficiency in transducing HCs needs improvement. This invention has developed a synthetic AAV, AAV-ie, that can target both SCs and HCs; and by delivering the transcription factor Atoh1, it transdifferentiates SCs into HC-like cells to regenerate HC-like cells. However, its targeting efficiency for either SCs or HCs needs further improvement, particularly in the basal region of the cochlea. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a variant adeno-associated virus and its application in mediating cochlear gene therapy and hair cell regeneration, in order to solve the problems in the prior art where AAV has low transduction efficiency for hair cells and supporting cells, and cannot efficiently utilize gene therapy or induce hair cell regeneration for the treatment of hearing impairment.

[0007] To achieve the above and other related objectives, the present invention provides a variant adeno-associated virus and its application in mediating cochlear gene therapy and hair cell regeneration.

[0008] One objective of this invention is to provide a variant adeno-associated virus (AAV) capsid protein, which, compared to the wild-type AAV-DJ capsid protein VP1, includes a mutation at amino acid K at position 558. Preferably, this mutation at amino acid K at position 558 is the K558R mutation.

[0009] Another object of the present invention is to provide a nucleic acid that encodes the nucleotide sequence of a variant adeno-associated virus capsid protein as described above.

[0010] Another object of the present invention is to provide a construct containing the nucleic acid as described above.

[0011] Another object of the present invention is to provide a host cell comprising the construct described above or having exogenous nucleic acids as described above integrated into its genome.

[0012] Another object of the present invention is to provide a variant adeno-associated virus, wherein the capsid structure of the variant adeno-associated virus contains the variant adeno-associated virus capsid protein as described above.

[0013] Another object of the present invention is to provide host cells transformed using the variant adeno-associated virus as described above.

[0014] Another object of the present invention is to provide a variant adeno-associated virus vector system comprising a packaging plasmid containing nucleic acid fragments as described above.

[0015] Another object of the present invention is to provide a variant adeno-associated virus obtained by viral packaging from the variant adeno-associated virus vector system described above.

[0016] Another object of the present invention is to provide a pharmaceutical composition comprising the variant adeno-associated virus as described above and a pharmaceutically acceptable vector.

[0017] Another object of the present invention is to provide the use of the variant adeno-associated virus, host cell, vector system, and pharmaceutical composition described above in the preparation of a medicament for treating diseases.

[0018] Compared with the prior art, the beneficial effects of the present invention include:

[0019] The variant adeno-associated virus (AAV) provided by this invention exhibits reduced ubiquitination or phosphorylation, enabling efficient transduction of hair cells and supporting cells in the cochlea (e.g., in newborn mice). It can partially restore hearing loss in Prestin knockout mice and deliver Atoh1 to cochlear supporting cells, generating HC-like cells. The variant AAV described in this invention is a safe vector; gene therapy using this vector for hearing loss-related diseases is safe and effective in humans, with no negative effects on the auditory and vestibular systems. For example, it does not significantly affect the morphology of HCs, does not cause hair cell loss, does not affect the hearing threshold, and does not cause gait or vestibular dysfunction. It has clinical potential in treating hearing loss caused by hair cell death. Attached Figure Description

[0020] Figure 1 Amino acid mutations in the capsid protein enhanced AAV transduction. (a) Amino acid mutation strategy. Serine (S) at the phosphorylation site was replaced with alanine (A), and lysine (K) at the ubiquitination site was replaced with arginine (R). The amino acids with the most similar structures were selected to avoid any significant changes in the properties of the capsid protein while preventing phosphorylation or ubiquitination modifications. (b) Schematic diagram of AAV mutant vector screening. P3 mice were injected with the AAV-ie mutant through the round window at a dose of 4.5 × 10⁻⁶ per ear. 9Each cochlea contained genome (particles) (GCs). Eleven days after viral injection, the cochlea was dissected into three parts as shown by the dashed lines, representing low frequency (apex), mid frequency (middle), and high frequency (basal). The cochlea was then fixed and immunohistochemically labeled, followed by confocal imaging. (c) AAV-ie and AAV-ie-S / K mutant vectors at the same dose (4.5 × 10⁻⁶) 9 Transduction efficiency of GCs in outer and inner hair cells. AAV-ie-K558R showed comparable transduction efficiency to AAV-ie. (d, e) Comparison of transduction efficiency between AAV-ie and AAV-ie-K558R. Imaging of virally expressed NLS-mNeonGreen (green) and staining for Myo7a (magenta) and SOX2 (red). Although both AAV-ie and AAV-ie-K558R effectively transduced hair cells and supporting cells throughout the cochlea, AAV-ie-K558R showed the same or even higher transduction efficiency in parts of the cochlea. (Scale bar: 50 μm) (f) Statistical analysis of transduction efficiency between AAV-ie and AAV-ie-K558R. Data shown here are mean ± SEM. Significance was tested between AAV-ie and AAV-ie-K558R, and p-values ​​were calculated using t-tests. *p<0.05, **p<0.01, and ***p<0.001.

[0021] Figure 2 AAV-ie-K558R is a safe vector. (a) Schematic diagram of the experimental setup used in the AAV-ie-K558R study, demonstrating the safety of the vector. (b) Representative SEM images of the apical, middle, and basal regions of the cochlea injected with AAV-ie-K558R-NLS-mNeonGreen from the WT control group and AAV-ie-K558R-NLS-mNeonGreen. (Scale bar: 10 μm) (c) Number of OHCs and IHCs per 100 μm in the apical, middle, and basal regions of the cochlea from uninjected WT mice and AAV-ie-K558R-NLS-mNeonGreen-injected mice. (d) Magnified SEM images of the outer hair cells (OHCs) and inner hair cells (IHCs) of the cochlea from the P14WT control group and AAV-ie-K558R-NLS-mNeonGreen-injected mice, in the apical, middle, and basal regions. (Scale bar: 1 μm)(e, f) ABR and DPOAE thresholds in the ear injected with AAV-ie-K558R-NLS-mNeonGreen on day 27 after virus injection at P3 and in the uninjected contralateral ear. Virus injection and AAV vector had no effect on hearing in mice.

[0022] Figure 3AAV-ie-K558R-Prestin restored auditory function in Prestin KO mice. (a) Schematic diagram of hearing recovery experiment in Prestin knockout mice. AAV-ie and AAV-ie-K558R vectors were used to package a single-stranded genome expressing Prestin driven by the CAG promoter. (b) ABR waveform families recorded at P28 in uninjected WT mice, untreated Prestin knockout mice, and Prestin knockout mice injected with AAV-ie-Prestin or AAV-ie-K558R-Prestin. ABRs were used to vary sound pressure levels at 16 kHz, with sound pressure levels increasing in 10 dB increments. Thresholds were determined by the presence of peak 1 and represented by color marks. Scale bars apply to all families. (c, d) ABR and DPOAE thresholds in Prestin knockout mice injected with AAV-ie-Prestin (black) or AAV-ie-K558R-Prestin (red) as a function of stimulation frequency. The contralateral ear of uninjected Prestin knockout mice was used as a negative control (blue), and the contralateral ear of uninjected WT mice was used as a positive control (purple). Data are presented as mean ± SEM. Significance (*P<0.05, **P<0.01, ***P<0.001) was calculated using multiple t-tests between the AAV injection group and the contralateral uninjected group.

[0023] Figure 4 AAV-ie-K558R-Atoh1 induced HC-like cell regeneration in newborn mice. (a) Schematic diagram of AAV-induced hair cell regeneration in WT C57BL / 6 newborn mice. (b) Immunofluorescence imaging of cochlea transduced with AAV-ie-Atoh1 and AAV-ie-K558R-Atoh1 at a dose of 1×10⁻⁶. 10 GCs. (Scale bar: 20 μm) (c) SEM images of the apical, middle, and basal regions of the cochlea injected with AAV-ie-K558R-Atoh1 at P14. Top row: Numbering of OHCs in three rows. Bottom row: Immature (white) and mature (yellow) HC-like cells regenerated by AAV-ie-K558R-Atoh1 shown in boxes. (Scale bar: 5 μm). (d) Enlarged SEM images of immature and mature regenerated HC-like cells, numbered. Stereoblasts were observed in these regenerated cells, while the stomatal cilia were artificially stained red. Immature regenerated cells did not show obvious polarity, while those regenerated cells with motile cilia (yellow arrows) and some polarity were considered more mature. (Scale bar: 1 μm)

[0024] Figure 5Transduction efficiency of the AAV-ie variant. Immunofluorescence images of the cochlea and hair cell layer transduced by the AAV-ie S / K mutant vector. All cochleas were harvested at P14 after microinjection with 1.5 μL of AAV stock solution at P3, stained with anti-Myo7a antibody (magenta), and imaged for NLS-mNeonGreen fluorescence (green). (Scale bar: 50μm) From left to right and top to bottom, they are K39R, K61R, K137R, K142R, K143R, K161R, K258R, K332R, K492R, K546R, K551R, K558R, K676R, K699R, K703R, K717R, S225A, S269A, S314A, S392A, S393A, S425A, S431A, S491A, S505A, S539A, S675A, and S679A.

[0025] Figure 6 AAV-ie-K558R was widely transduced into mouse cochlear and vestibular sensory epithelial cells. (a) Schematic diagram of the experimental setup for transducing AAV-ie-K558R in other cell types. P3 WT mice were injected with AAV-ie-K558R-NLS-mNeonGreen at a dose of 1×10⁻⁶. 10 GCs. Tissue was harvested 11 days post-injection. (b) Cryosection of cochlea injected with AAV-ie-K558R-NLS-mNeonGreen, stained with antibodies against SOX2 (red) and Myo7a (magenta), and imaged for the fluorescence (green) of NLS-mNeonGreen. The image shows that AAV-ie-K558R effectively transduces cochlear hair cells and various types of supporting cells. (Scale bar: 20 μm) (c) Left panel, representative confocal image of stained mouse supporting cells. Right panel, magnified view of the area within the box in the left panel. (d) Same as in c, elliptical hair cell layer. Scale bar (c and d): Left, 100 μm, Right: 10 μm.

[0026] Figure 7 AAV-ie-K558R does not affect vestibular function. (a) Narrow-beam walking test path trajectory (120 seconds) of P28 mice injected with AAV-ie-K558R at P2. (b) Same as a, but without injection. (c) Number of rotations per minute. (d) Speed ​​per second. (e) Mice injected with AAV-ie-K558R and control mice traversing an 80 cm narrow beam.

[0027] Figure 8Genesis and validation of Prestin knockout mice. (a) Prestin knockout mice were constructed using CRISPR base substitution. Two stop codons were simultaneously introduced into the coding sequences of Prestin exons 4 and 11 to induce early transcription termination. (b) The genotype of the Prestin knockout mice was validated by PCR amplification around the mutation site using primers described in the methods, followed by sequencing. (c) Immunohistochemical detection of Prestin-specific antibodies (green) in WT mice and Prestin knockout mice clearly showed the absence of Prestin expression in the OHCs of Prestin knockout mice. (Scale bar: 20 μm)

[0028] Figure 9 AAV-ie-K558R-Prestin enables Prestin expression in both OHCs and IHCs. (a) AAV-ie-K558R was used to package a single-stranded (ss) AAV genome that expresses Prestin via a constitutive CAG promoter. (b) Phalloidin (green) was used to label the morphology of F-actin and HCs. Dapi (blue) was used to label the cell nucleus. Prestin knockout mice do not express Prestin. (Scale bar: 10 μm) (c) AAV-ie-K558R-Prestin enables Prestin expression in HCs and other cell types. (Scale bar: 10 μm) (d) AAV-ie-Prestin induces Prestin expression in HCs and other cell types, but to a lesser extent. (Scale bar: 10 μm)

[0029] Figure 10 AAV-ie-Atoh1-induced HC-like cells in newborn mice. Scanning images of the apical, middle, and basal regions of the P14 cochlea injected with AAV-ie-Atoh1 (1×10¹⁰ GCs). (Scale bar: 10 μm)

[0030] Figure 11 .AAV-ie-K558R-Atoh1 induces HC-like cells in the GER region. (a) AAV-ie-Atoh1 and AAV-ie-K558R-Atoh1 at 1×10 10Immunofluorescence imaging of the cochlear GER after dose-transduction by GCs. Both AAV-ie-Atoh1 and AAV-ie-K558R-Atoh1 generated Myo7a-positive cells in the GER region. (Scale bar: 5 μm) (b) Scanning image of P14 in the GER region after cochlear injection of AAV-ie-Atoh1 and AAV-ie-K558R-Atoh1. SEM images confirmed vector-induced HC-like cell regeneration, forming ciliary tracts. (Scale bar: left, 20 μm, right, 1 μm.)

[0031] Figure 12 Flowchart of the construction process of the AAV-ie-K558R vector in Example 1. Detailed Implementation

[0032] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0033] Adeno-associated virus (AAV) is a single-stranded DNA virus containing two open reading frames (rep and cap). The rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for genome replication, and the cap gene expresses three structural proteins (VP1-3) that assemble to form the viral capsid. This invention is based on wild-type adeno-associated virus AAV-DJ. An amino acid fragment as shown in SEQ ID NO:1 is inserted between N589 and R590 of the capsid protein VP1, as shown in SEQ ID NO:3, to obtain the variant adeno-associated virus AAV-ie (see patent document CN110437317 A). Then, a series of mutations are generated in the amino acid sequence of the AAV-ie capsid protein VP1 to manipulate phosphorylation or ubiquitination of AAV-ie in cells, constructing various mutants. Through multiple research and screening processes, this invention has yielded a variant adeno-associated virus (AAV-ie-K558R) containing a specific capsid protein amino acid mutant. This variant can efficiently transduce HCs and SCs, and is suitable for correcting gene mutations that cause functional disorders and for the regeneration of HC-like cells.

[0034] The present invention provides a variant adeno-associated virus capsid protein, which, compared with the wild-type adeno-associated virus AAV-DJ capsid protein VP1, includes a mutation at amino acid K at position 558.

[0035] Preferably, the mutation at amino acid K at position 558 is a K558R mutation. The mutation at amino acid K at position 558 includes the deletion or substitution of the amino acid. The substitution refers to replacing amino acid residue K at position 558 with another non-K amino acid or its derivative, such as replacing it with amino acid residues G, A, V, L, I, P, F, W, M, Y, S, T, C, N, Q, D, E, R, or H.

[0036] Preferably, an amino acid fragment as 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 adeno-associated virus AAV-ie capsid protein VP1. Then, the 558th amino acid K is mutated to R to obtain adeno-associated virus AAV-ie-K558R capsid protein VP1 as shown in SEQ ID NO.5.

[0037] DGTLAVPFK (SEQ ID NO.1).

[0038] In some embodiments of the present invention, the amino acid sequence of the wild-type AAV-DJ capsid protein VP1 is shown in SEQ ID NO.3.

[0039] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO. 3).

[0040] In some embodiments of the present invention, the amino acid sequence of the adeno-associated virus AAV-ie capsid protein VP1 is as shown in SEQ ID NO. 4.

[0041] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNDGTLAVPFKRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO. 4).

[0042] The amino acid sequence of the adeno-associated virus AAV-ie-K558R capsid protein VP1 is shown in SEQ ID NO. 5.

[0043] (SEQ IDNO.5).

[0044] The present invention also provides a nucleic acid that encodes the variant adeno-associated virus capsid protein as described above.

[0045] The present invention also provides a construct containing the aforementioned nucleic acid. The construct can typically be obtained by inserting the aforementioned nucleic acid into a suitable expression vector, and those skilled in the art can select a suitable expression vector.

[0046] The present invention also provides a host cell comprising the above-described construct or genome in which exogenous nucleic acids described above are integrated.

[0047] Representative examples of suitable host cells include mammalian cells (such as CHO or COS), plant cells, human cells (human embryonic kidney cells such as HEK293FT), bacterial cells (such as Escherichia coli, Streptomyces, and Salmonella typhimurium), fungal cells (such as yeast), and insect cells (such as Sf9). Those skilled in the art can select a suitable host based on the teachings herein. Preferably, the host cell is an animal cell, and more preferably a human cell. The host cell can be a cultured cell or a primary cell, i.e., directly isolated from an organism (such as a human). The host cell can be an adhesive cell or a suspension cell, i.e., a cell growing in suspension.

[0048] The present invention also provides a variant adeno-associated virus (AAV) containing the variant AAV capsid protein VP1 as described above. In some preferred embodiments, the variant AAV is AAV-ie-K558R.

[0049] The variant adeno-associated virus AAV-ie-K558R, compared to the wild-type adeno-associated virus AAV-DJ, contains an amino acid fragment as shown in SEQ ID NO.1 and a mutation at amino acid position 558 between N589 and R590 of the capsid protein VP1. The variant adeno-associated virus can be obtained by replacing the nucleotides encoding the capsid protein VP1 in the vector system used to produce the wild-type adeno-associated virus AAV-DJ with the nucleotides encoding the capsid protein VP1 of the variant adeno-associated virus AAV-ie-K558R, followed by packaging.

[0050] The adeno-associated virus AAV-ie-K558R, compared to adeno-associated virus AAV-ie (see patent document CN110437317A), contains a mutation at amino acid position 558. This variant adeno-associated virus can be obtained by replacing the nucleotide encoding the capsid protein VP1 in the vector system used to produce adeno-associated virus AAV-ie with the nucleotide encoding the capsid protein VP1 of the variant adeno-associated virus AAV-ie-K558R, followed by packaging; or by mutating adeno-associated virus AAV-ie.

[0051] In some embodiments, the Rep-Cap plasmid sequence used in the packaging process of the AAV-ie is shown in SEQ ID NO.2 (the same as SEQ ID NO.6 of AAV-ie patent document CN 110437317A).

[0052] SEQ ID NO.2

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] Furthermore, the variant adeno-associated virus also includes a heterologous nucleotide sequence encoding the target product, which can be carried by various capsid proteins. The aforementioned heterologous nucleotide sequence encoding the target product is typically a construct, which usually contains nucleic acid encoding the target product. The construct can typically be obtained by inserting the nucleic acid encoding the target product into a suitable expression vector. Those skilled in the art can select suitable expression vectors; for example, the expression vectors may include, but are not limited to, pAAV-CAG, pAAV-TRE, pAAV-EF1a, pAAV-GFAP promoter, pAAV-Lgr5 promoter, and pAAV-Sox2 promoter expression vectors. In this invention, when the variant adeno-associated virus encodes a heterologous nucleotide sequence of the target product, the variant adeno-associated virus contains a capsid, and the viral vector carries a transgene encoding the gene product, which is regulated by a regulatory sequence that directs its expression within the host cell; in some preferred embodiments, the amino acid sequence of the capsid protein is shown in SEQ ID NO:5.

[0059] Furthermore, the target product can be a nucleic acid or a protein, wherein the nucleic acid can be a small guide RNA (sgRNA), interfering RNA (RNAi), etc., and the protein-coding gene can be Prestin or Atoh1.

[0060] The adeno-associated virus AAV-ie-K558R can be used as a vector material to introduce exogenous genes into the cells of test individuals. Compared with the parental wild-type AAV-DJ and adeno-associated virus AAV-ie, the transduction efficiency of AAV-ie-K558R in hair cells and supporting cells is significantly improved.

[0061] The present invention also provides an engineered host cell obtained by transformation using the variant adeno-associated virus described above. The engineered host cell comprises the aforementioned variant adeno-associated virus. The host cell may be a eukaryotic cell and / or a prokaryotic cell.

[0062] Representative examples of suitable host cells include mammalian cells (such as CHO or COS), plant cells, human cells (human embryonic kidney cells such as HEK293FT), bacterial cells (such as Escherichia coli, Streptomyces, and Salmonella typhimurium), fungal cells (such as yeast), and insect cells (such as Sf9). Those skilled in the art can select a suitable host based on the teachings herein. Preferably, the host cell is an animal cell, and more preferably a human cell. The host cell can be a cultured cell or a primary cell, i.e., directly isolated from an organism (such as a human). The host cell can be an adhesive cell or a suspension cell, i.e., a cell growing in suspension.

[0063] The present invention also provides a variant adeno-associated virus vector system, the vector system comprising a packaging plasmid containing the nucleic acid fragments described above.

[0064] Furthermore, the packaging plasmid also contains a fragment of the adeno-associated virus (AAV) rep gene. The rep gene contains introns, and the introns contain transcription termination sequences.

[0065] Furthermore, the adeno-associated virus vector system also includes an expression plasmid containing heteronucleotides responsible for encoding the target product.

[0066] Furthermore, the adeno-associated virus vector system also includes helper viral plasmids.

[0067] Furthermore, the adeno-associated virus vector system also includes a host cell.

[0068] The packaging plasmid, expression plasmid, and helper virus plasmid are transferred into host cells, whereby all nucleic acid sequences are integrated into the host cells to produce the variant adeno-associated virus. In some embodiments, all nucleic acid sequences are integrated together at a single locus within the host cell genome. In some embodiments, the nucleic acid sequences encoding various genes exist as separate expression cassettes, preventing any risk of recombination leading to the formation of a replicating virus; the nucleic acid sequences encoding the rep and cap genes are contained in the same expression cassette.

[0069] The present invention also provides a variant adeno-associated virus, obtained by viral packaging from the variant adeno-associated virus vector system described above.

[0070] The present invention also provides a pharmaceutical composition comprising the adeno-associated virus as described above and a pharmaceutically acceptable vector.

[0071] Acceptable carriers include, for example, sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), and binders. Furthermore, they may also contain other low-molecular-weight peptides; proteins such as serum albumin, gelatin, or immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine, and lysine; sugars or carbohydrates such as polysaccharides and monosaccharides; and sugar alcohols such as mannitol or sorbitol. When preparing aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other adjuvant drugs, such as D-sorbitol, D-mannose, D-mannitol, or sodium chloride, appropriate solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), and nonionic surfactants (Tween 80, HCO-50, etc.) may be used.

[0072] In the pharmaceutical composition provided by this invention, AAV-ie-K558R can be a single active ingredient, or it can be combined with one or more other active ingredients useful for hearing loss to form a combined formulation. The other active ingredients can be various other drugs that can be used to treat hearing loss. The content of the active ingredient in the composition is generally a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of AAV-ie-K558R and the active ingredient in the pharmaceutical composition generally depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the bifunctional compound as the active ingredient can generally be 1–1000 mg / kg / day, 20–200 mg / kg / day, 1–3 mg / kg / day, 3–5 mg / kg / day, etc. 5~10mg / kg / day, 10~20mg / kg / day, 20~30mg / kg / day, 30~40mg / kg / day, 40~60mg / kg / day, 60~80mg / kg / day, 80~100m g / kg / day, 100~150mg / kg / day, 150~200mg / kg / day, 200~300mg / kg / day, 300~500mg / kg / day, or 500~1000mg / kg / day.

[0073] The variant adeno-associated virus provided by this invention can be adapted to suitable administration methods, such as injection into the cochlea, eyes, muscles, nervous system, or circulatory system. Those skilled in the art can select an appropriate dosage based on the administration method.

[0074] The present invention also provides the use of the above-mentioned variant adeno-associated virus, host cell, vector system or pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of diseases; preferably, the use in the preparation of medicaments for the prevention and / or treatment of gene-therapy diseases; said diseases include, but are not limited to, one or more of hearing impairment diseases, inflammation, tumors, metabolic diseases, pain, neurodegenerative inflammatory diseases, etc.

[0075] The hearing impairment diseases mentioned are selected from hearing loss, deafness, and tinnitus.

[0076] The inflammation is selected from skin inflammation, vasculitis, allergic reaction, autoimmune disease, fibrosis, scleroderma, or graft rejection; the autoimmune disease is selected from one or more of rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, xerostomia syndrome, polymyositis, etc.

[0077] The cancers mentioned are selected from lymphomas, hematologic malignancies, or solid tumors; specifically, they are selected from adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, lymphoid tumors, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, and oocytes. One or more of the following tumor cells: ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma, multiple myeloma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, T-cell lymphoma, and B-cell lymphoma; preferably, the tumor is one or more of colorectal cancer and / or melanoma.

[0078] The metabolic diseases are selected from diabetes, including type I and type II diabetes, as well as diseases and conditions related to diabetes; the metabolic diseases include, but are not limited to, one or more of the following: atherosclerosis, cardiovascular disease, nephropathy, neuropathy, retinopathy, β-cell dysfunction, dyslipidemia, hyperglycemia, insulin resistance, chronic obstructive pulmonary disease, etc.

[0079] In some preferred embodiments, the gene therapy refers to the treatment of hearing impairment. The variant adeno-associated virus or pharmaceutical composition can achieve the treatment of hearing impairment by delivering the target product to the hair cells and / or supporting cells of an individual.

[0080] In the use of the variant adeno-associated virus, host cell, vector system, or pharmaceutical composition described in this invention for delivering a target product to hair cells and / or supporting cells of an individual, the delivery of the target product may be for non-diagnostic or therapeutic purposes, for example, it may be in vitro, delivering the target product to isolated hair cells and / or supporting cells. The hair cells typically include outer hair cells and / or inner hair cells.

[0081] Furthermore, the target product is a nucleic acid or a protein, and the nucleic acid can be a small guide RNA (sgRNA), interfering RNA (RNAi), etc.

[0082] In this invention, the hearing impairment can be caused by cochlear damage due to environmental factors. Therefore, this invention also provides the use of the aforementioned variant adeno-associated virus in a medicament for treating environmentally induced hearing impairment in individuals.

[0083] Furthermore, the hearing impairment is a hair cell and / or supporting cell and / or spiral neuron cell related disease.

[0084] Furthermore, the hearing impairment disease is a disease related to gene defects, environmental damage, or aging. For example, it may be a disease caused by gene mutations, or a disease caused by noise or drugs, or a disease caused by aging.

[0085] Furthermore, hearing impairment can be related to cell damage, specifically cochlear hair cell damage, supporting cell damage, etc. More specifically, it can be cochlear hair cell damage caused by gene mutation, supporting cell damage caused by gene mutation, noise-induced cell damage, drug-induced cell damage, or cell damage caused by aging.

[0086] Furthermore, the variant adeno-associated virus serves as a vector for delivering the target product.

[0087] The present invention also provides a method for treating hearing impairment, the method comprising administering to a subject in need an effective amount of the variant adeno-associated virus described herein, the host cell described herein, a vector system, or a pharmaceutical composition. Typically, physicians can determine the most suitable practical dose for a single patient, and this dose varies depending on the individual's age, weight, and response.

[0088] In this invention, the variant adeno-associated virus, host cell, vector system, or pharmaceutical composition of this invention may be administered to a patient. Those skilled in the art can determine the appropriate route of administration and dosage.

[0089] The variant adeno-associated virus described in this invention delivers one or more therapeutic genes, which can be used alone or in combination with other therapeutic methods or components.

[0090] The present invention also provides a conjugate comprising a variant adeno-associated virus or a linked bioactive polypeptide as described above.

[0091] The variant adeno-associated virus of the present invention is used to infect cells, thereby delivering genes and / or linked (e.g., but not limited to, covalently linked) bioactive polypeptides into the cells. Therefore, the present invention provides a method for delivering transgenes into cells, the method being used to infect cells by introducing one or more variant adeno-associated viruses or conjugates of the present invention into the cells, wherein the variant adeno-associated virus or conjugate contains one or more transgenes.

[0092] The present invention also provides a method for producing a stable variant adeno-associated virus vector production cell line, comprising:

[0093] (a) Introducing a variant adeno-associated virus vector, as defined herein, into a culture of mammalian host cells; and

[0094] (b) Selecting mammalian host cells within the culture that have a nucleic acid sequence encoded on a vector integrated into the endogenous chromosome of the mammalian host cell.

[0095] The AAV vector production cells are mammalian cells. In some embodiments, the mammalian cells are selected from HEK293 cells, CHO cells, Jurkat cells, K562 cells, PerC6 cells, HeLa cells, or derivatives thereof. In some embodiments, the mammalian host cell is a HEK293 cell or a cell derived from a HEK293 cell. In some embodiments, the HEK293 cell is a HEK293T cell.

[0096] The genomic sequences of various serotypes of AAV, as well as the sequences of the native ITR, Rep protein, and capsid subunit, are known in the art. These sequences are available in the literature or in public databases such as GenBank. Their public information is incorporated herein by reference for the purpose of teaching AAV nucleic acid and amino acid sequences.

[0097] In the compounds of this invention and their applications, when the variant adeno-associated virus is used in combination with other therapeutic agents, the active compound is administered co-administered with the other therapeutic agents. "Co-administered" means administered simultaneously in the same formulation or in two different formulations via the same or different routes, or administered sequentially via the same or different routes. "Sequentially administered" means a time difference, measured in seconds, minutes, hours, or days, between the administration of two or more different compounds.

[0098] In some embodiments, the variant adeno-associated virus and method of the present invention can be used to prevent hearing loss, and can be administered as a preventive treatment before hearing loss or some time after exposure to an environment that may cause hearing loss.

[0099] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0100] As used in this article, "vector" refers to a macromolecule or combination of macromolecules containing or bound to a polypeptide that can be used to mediate the delivery of the polypeptide to cells. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, or other gene delivery vectors.

[0101] This article mentions that "AAV" is an abbreviation for adeno-associated virus, which can be used to refer to the virus itself or its derivatives.

[0102] The term "recombinant AAV vector" as used in this article refers to an AAV vector containing a heterologous polynucleotide sequence, typically the sequence of interest used for genetically transformed cells. Generally, the heterologous polynucleotide is flanked by at least one, usually two, AAV terminal inverted repeat (ITR) sequences.

[0103] The terms "AAV virus," "AAV virus particle," or "AAV vector particle" as used in this article refer to a viral particle containing at least one AAV capsid protein and an encapsulating polynucleotide. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes to be delivered to mammalian cells), it is generally referred to as an "AAV vector particle" or "AAV vector."

[0104] The term "packaging" in this article refers to a series of intracellular processes that lead to the assembly and encapsulation of AAV particles.

[0105] This article refers to the AAV "rep" and "cap" genes as polynucleotide sequences encoding the replication and packaging proteins of adeno-associated virus (AAV). In this article, AAV rep and cap refer to the AAV "packaging genes".

[0106] The term "helper virus" as used in this article refers to a virus that enables AAV to be replicated and packaged by mammalian cells. Various such AAV helper viruses are known in the art, including adenoviruses, herpesviruses, and poxviruses (e.g., cowpox).

[0107] This article refers to "infectious" viruses or viral particles as those capable of delivering polynucleotide components into cells to which the virus species is tropism. This term does not imply any replication capability of the virus.

[0108] The term "production cell" refers to a cell line that has AAV packaging genes (rep and cap genes) stably integrated into the host cell genome, the required helper viral genes, and the DNA genome of a recombinant AAV vector (e.g., a target transgene flanked by two AAV inverted terminal repeats (ITRs)).

[0109] The terms "include" and "contain" in this article should be understood as inclusive, without the meaning of exclusivity or exhaustion; that is, "including but not limited to".

[0110] The term "individual" as used in this article generally includes humans and non-human primates such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., who may benefit from treatment using the aforementioned formulations, kits, or combinations thereof.

[0111] The term "therapeutic effective dose" as used in this article generally refers to a dose that, after an appropriate period of administration, can achieve the therapeutic effect for the diseases listed above.

[0112] The terms "therapeutic" and "preventative" used in this article should be understood in their broadest sense. The term "therapeutic" does not necessarily imply that a mammal receives treatment until it is fully recovered. Similarly, "preventative" does not necessarily mean that the subject will ultimately not contract the disease. Therefore, treatment and prevention include alleviating the symptoms of a specific condition or preventing or reducing the risk of developing a specific condition. The term "prevention" can be understood as reducing the severity of a specific condition's onset. Treatment can also reduce the severity of an existing condition or the frequency of acute attacks.

[0113] In this invention, the object or individual for therapeutic or preventative treatment is preferably a mammal, such as, but not limited to, humans, primates, livestock (e.g., sheep, cattle, horses, donkeys, pigs), pets (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), or captured wild animals (e.g., foxes, deer). The object is preferably a primate. The most preferred object is a human.

[0114] This article uses the terms "transfection," "transformation," and "transduction" to describe the insertion of non-mammalian or viral vectors into target cells. Insertion of vectors is commonly referred to as transformation of bacterial cells and transfection of eukaryotic cells, although insertion of viral vectors can also be called transduction. Those skilled in the art will recognize the various non-viral transfection methods commonly used, including but not limited to those employing physical methods (e.g., electroporation, cell squeezing, acoustic perforation, optical transfection, protoplast fusion, impalefection, magnetic transfection, gene gun, or particle bombardment), chemical reagents (e.g., calcium phosphate, highly branched organic compounds, or cationic polymers), or cationic lipids (e.g., lipid transfection). Many transfection methods require contacting the vector DNA solution with cells, followed by growth and selection for marker gene expression.

[0115] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0116] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0117] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0118] Materials and Methods:

[0119] Materials and sources:

[0120] Young mice: Shanghai Lingchang Biotechnology Co., Ltd.

[0121] Various AAVs: Synthesized and constructed by Nanjing GenScript Company

[0122] Donkey serum: Shanghai Yisheng Biotechnology Co., Ltd.

[0123] The dilution ratios of each antibody to those used during staining are as follows:

[0124] Primary antibody: myosin 7A (Myo7a, #25-6790Proteus 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).

[0125] Secondary antibodies: three different marker colors (TRIC, FITC, Cy5) for each of the secondary antibody markers anti-rat, mouse, rabbit, and goat, sourced from Invitrogen.

[0126] Cell and tissue culture reagents: DMEM (Hyclone), fetal bovine serum (Lensa), N2 additive (ThermoFisher), ampicillin (ThermoFisher), penicillin and antibiotics (ThermoFisher).

[0127] Cell and tissue culture consumables: including various culture dishes, centrifuge tubes, pipettes, disposable filters and other commonly used consumables, were purchased from Corning.

[0128] Example 1: Mutations in the amino acids on the surface of the capsid protein improved the cochlear transduction efficiency of AAV-ie in vivo.

[0129] (1.1) Protein ubiquitination is related to AAV transduction efficiency; blocking ubiquitination can improve AAV transduction efficiency in vitro and in vivo. Previous studies in this invention have shown that AAV-ie can transduce cochlear cells in rodents and humans. However, the transduction efficiency of AAV-ie in the basal region of the cochlea is very low. This invention proposes that mutating capsid protein amino acids on the exposed side of AAV-ie can improve its transduction efficiency. Figure 1 a). To verify this hypothesis, this invention constructed 28 AAV-ie variants with different capsid protein sequences (Table 1).

[0130] (1.2) Construction of AAV-ie variant

[0131] The following description uses the construction of AAV-ie-K558R as an example; the construction steps for other AAV-ie variants are similar.

[0132] Building AAV-ie-K558R:

[0133] AAV packaging requires three plasmids: a genomic plasmid containing the target gene, a Capsid plasmid (Rep-Cap plasmid), and a Helper plasmid.

[0134] The sequence of the Cap protein in the Capsid plasmid determines the different serotypes of AAV, thus affecting the cell preference of AAV infection. Therefore, modifying the Cap protein can yield new AAV serotypes.

[0135] AAV-ie-K558R is generated by mutating a single amino acid K to R at position 558 of the Cap protein sequence of the parent AAV-ie.

[0136] (a) Linearization of AAV-ie capsid vector

[0137] First, the AAV-ie capsid plasmid was linearized and cut using the Sma1 and Xho1 restriction enzyme sites to generate the AAV-ie capsid vector. The linearized AAV-ie capsid fragment (Axygen: AP-GX-250G) was then recovered. The concentration of the recovered fragment was detected using Nanodrop2000.

[0138] (b) Construction of mutant fragments

[0139] First, primers were designed targeting the fragment from the Xho1 restriction site to amino acid 558. A forward primer was designed at the Xho1 restriction site, with a 15-20 nt homologous arm (Primer 1) added to the 5' end of the primer to bind to the linearized AAV-ie capsid vector. Then, a reverse primer was designed at amino acid 558, with a 15-20 nt homologous arm (Primer 2) added to the 5' end to bind to recombinant fragment 2. A K558R mutation was introduced into Primer 2. Recombinant fragment 1 was generated by polymerase chain reaction (PCR). Recombinant fragment 1 (Axygen: AP-GX-250G) was recovered. The concentration of the recovered fragment was detected using Nanodrop 2000.

[0140] Primers were designed targeting the 558 amino acid fragment to the Sma1 restriction site. A forward primer was designed at amino acid 558 of the Cap protein sequence, introducing the K558R mutation, and a 15-20 nt homologous arm (Primer 3) was added to the 5' end of the primer to bind to recombinant fragment 1. A reverse primer was designed at the Sma1 restriction site, and a 15-20 nt homologous arm (Primer 4) was added to the 5' end of the primer to bind to the linearized AAV-iecapsid vector. Recombinant fragment 2 was generated by polymerase chain reaction (PCR). Recombinant fragment 2 (Axygen: AP-GX-250G) was recovered. The concentration of the recovered fragment was detected using Nanodrop 2000.

[0141] The primers are designed as follows, with gray indicating the introduction of the K558R mutant base in the primers:

[0142] Forward primer Primer 1: 5'-TTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGAC-3' (SEQ ID NO.6);

[0143] Reverse primer Primer 2:

[0144] Forward primer Primer 3:

[0145] Reverse primer Primer 4: 5'-CGCCCGCTGTTTAAACGCCCGGGCTGTAGTTAATGATTAACCCG-3' (SEQ ID NO.9).

[0146] (c) Construction and purification of the AAV-ie-K558R vector

[0147] Using a multi-fragment homologous recombination method, linearized AAV-ie capsid vector and recombinant fragments 1 and 2 obtained from PCR were recombinated to generate AAV-ie-K558R. The recombination system was as follows: linearized AAV-ie capsid vector, 50 ng; recombinant fragment 1, 30 ng; recombinant fragment 2, 30 ng; recombinant ligase, 1 μL; recombinant ligation buffer, 5 μL; ddH2O, to a final volume of 20 μL; reaction at 50 °C for 20 minutes to produce the recombinant product.

[0148] Flowchart as follows Figure 12 As shown.

[0149] (d) Vector transformation and plasmid extraction

[0150] The transformation steps are as follows: Thaw 100 μL of competent cells (TransGen: CD201) on ice; mix 10 μL of recombinant product with competent cells and place on ice for 20 minutes; heat shock at 42℃ for 60 seconds; place on ice for 2 minutes, add 400 μL of LB medium (MDBio: L001-1kg), and shake for 30 minutes; spread 70 μL on an ampicillin plate (50 μg / ml, incubate at 37℃ for 14 hours).

[0151] Single clones were selected and cultured in 4 ml of liquid LB medium. After 14 hours, the plasmid (Axygene:AP-MN-P-250G) was extracted.

[0152] The steps are as follows: Centrifuge the bacterial culture at 4000 rpm for 10 minutes, discard the supernatant; add 350 μL of buffer S1, disperse the bacterial cells, and transfer to a 2 ml centrifuge tube; add 250 μL of buffer S2, invert 8 times; add 250 μL of buffer S3, invert 6 times to mix, and a precipitate will form; centrifuge at 12000 rpm for 10 minutes, and pass the supernatant through a column; centrifuge for 1 minute, discard the waste liquid, add 500 μL of W1, centrifuge for 1 minute, discard the waste liquid; add 750 μL of W2, centrifuge, and discard the supernatant; add 500 μL of W2, centrifuge, and discard the supernatant; run empty for 1 minute; add 50 μL of elution buffer, let stand for 2 minutes, and centrifuge. The obtained plasmid was analyzed for concentration, and 10 μL was sent for sequencing. Positive plasmids were stored at -20℃. Sequencing results showed that the obtained plasmid encodes the variant capsid protein VP1.

[0153] Further experimental results showed that the prepared plasmid could express the variant capsid protein VP1. The polynucleotide coding sequence of AAV-ie-K558R capsid VP1 is shown in SEQ ID NO.10, and the full sequence of the constructed Rep-Cap plasmid is shown in SEQ ID NO.11.

[0154] (e) Packaging and purification of the AAV variant (named AAV-ie-K558R) virus

[0155] The obtained Rep-Cap plasmid, the genomic plasmid pAAV-CAG-mNeonGreen expressing a green fluorescent protein mNeonGreen (GenBank: LC279210.1) (the full sequence of the plasmid is shown in SEQ ID NO.12 (same as SEQ ID NO.11 of AAV-ie patent document CN 110437317A)), and the pHelper plasmid (the 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)) were co-transfected into HEK-293T cells in appropriate amounts. The AAV virus was purified by gradient ultracentrifugation using iododialkylol. The appropriate concentration was measured to be 1E+12-1E+13 GC / mL. The cells were then stored at -80℃ for later use.

[0156] HEK 293T cells cultured in DMEM with 10% fetal bovine serum were inoculated with AAV viruses (AAV-ie and AAV-DJ, respectively) generated from capsid protein variants and their parental capsid proteins. The MOI value for virus inoculation was 1000. After 48 hours, the expression of green fluorescent protein mNeonGreen was observed using a fluorescence microscope.

[0157] The results showed that the proportion of AAV variants infected with HEK 293T cells was similar to that of their parent AAV variants.

[0158] SEQ ID NO.10(AAV-ie-K558R capsid VP1):

[0159]

[0160]

[0161] SEQ ID NO.11 (AAV-ie-K558R Rep-Cap plasmid):

[0162]

[0163] SEQ ID NO.12

[0164]

[0165]

[0166]

[0167] SEQ ID NO.13

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] (1.3) Screening for AAV-ie variants

[0175] To improve the accuracy of screening, this invention will use an equal amount (4.5 × 10) 9 The genome-containing AAV-ie-K558R particles were injected into the cochlea of ​​newborn P3 mice via a round window membrane (RWM) to screen for these AAV-ie variants in (1.1) in vivo. Figure 1 b). Immunostaining was performed on cochlea samples from the P14 stage: hair cells were labeled with Myo7a antibody, and nuclear localization signal (NLS)-mNeonGreen signaling indicated the nuclear localization of transduced cells in the cochlea. The transduction efficiency of these AAV-ie variants was analyzed and compared with AAV-ie under the same conditions. Figure 1 As shown in c, compared to AAV-ie, although most AAV-ie variants do not show an increase in transduction efficiency in the basal cochlea region ( Figure 5 Some even showed a decrease, but the variant AAV-ie-K558R exhibited significantly improved transduction efficiency in both OHCs and IHCs in the basal region of the cochlea. Further comparison of the transduction properties of AAV-ie-K558R and AAV-ie revealed that AAV-ie-K558R significantly improved transduction efficiency in both OHCs in the intermediate region of the cochlea and SCs in the basal region. Figure 1 This invention also evaluated the targeting efficiency of AAV-ie-K558R in the vestibular system responsible for detecting linear motion and sensing gravity, observing NLS-mNeonGreen expression throughout the mouse sensory epithelium, and AAV-ie-K558R transduction efficiency approaching 100% in HCs and SCs. Figure 6 ).

[0176] These experimental results collectively demonstrate that AAV-ie-K558R can effectively target different types of cells in the cochlea, revealing its potential as a suitable carrier to mediate gene correction of hearing diseases or regenerate HC-like cells in the cochlea or vestibular system.

[0177] Table 1. Sequences of amino acid mutations in AAV-ie capsid protein (uppercase letters represent codons encoding amino acids, and red letters represent mutated bases).

[0178]

[0179]

[0180]

[0181] Example 2: Security Verification of AAV-ie-K558R

[0182] Our group's earlier research indicated that AAV-ie is a safe carrier and has not shown any toxic effects on HCs or auditory function. This invention investigates whether AAV-ie-K558R possesses similar safety characteristics to AAV-ie.

[0183] 2.1 Experimental Grouping and Treatment

[0184] AAV-ie-K558R injection group (n=3 per group): AAV-ie-K558R-NLS-mNeonGreen (1×10⁻⁶) was injected into each ear of mice via the round window membrane (RWM). 10 GCs);

[0185] WT control group (3 mice per group): Each ear of mice was injected with AAV-ie-NLS-mNeonGreen (1×10⁻⁶) via the round window membrane (RWM). 10 GCs).

[0186] 2.2 Effects on the morphology and quantity of HCs

[0187] Two weeks after the treatment in step 2.1, the cochlea was analyzed by scanning electron microscopy (SEM). The SEM results showed that the HCs showed little or no morphological change after injection, and the hair tufts remained in the correct orientation. Figure 2 b, 2d). This embodiment also calculated the number of HCs in the field of view and observed no difference between the AAV injection group and the control group, indicating no significant hair cell loss. Figure 2 c).

[0188] 2.3 Determining the effect of AAV-ie-K558R on hearing

[0189] This embodiment verifies whether AAV-ie-K558R injection affects hearing by measuring auditory brainstem responses (ABRs), which reflect auditory function in live animals, and aberration product otoacoustic emissions (DPOAEs), which reflect the integrity of outer hair cells. Similarly, no significant difference in threshold values ​​was observed between the injection group and the control group. Figure 2 e, 2f).

[0190] 2.4 Determining the effects of AAV-ie-K558R on the auditory and vestibular systems

[0191] This embodiment then examines whether AAV-ie-K558R has any side effects on the behavior of animals exhibiting severe vestibular dysfunction, such as circling or other gait abnormalities.

[0192] Four weeks after the treatment in step 2.1, no abnormalities in gait or circulatory behavior were observed in the AAV-ie-K558R injection group during the open path tracking test.

[0193] In further narrow-beam walking tests, no significant differences were observed between mice injected with the virus (AAV-ie-K558R) and mice in the control group that were not injected with the virus. Figure 7 ).

[0194] Based on these results, the present invention concludes that AAV-ie-K558R has no negative effects on the auditory and vestibular systems.

[0195] Example 3: Generation and verification of Prestin knockout mice.

[0196] Constructing Prestin knockout mice: Using the CRISPR-Cas system, two stop codons were simultaneously introduced into the coding sequences of Prestin exon 4 and exon 11, causing premature cessation of prestin protein translation, thereby knocking out the expression of prestin in the outer hair of the cochlea, thus constructing Prestin knockout mice.

[0197] like Figure 8 As shown in b, PCR amplification was performed around the mutation site using 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') to verify the genotype of the Prestin knockout mouse and to perform sequencing detection.

[0198] like Figure 8As shown in c, immunohistochemical detection of Prestin-specific antibodies (green) in WT mice and Prestin knockout mice clearly shows that there is no Prestin expression in the OHCs of Prestin knockout mice.

[0199] Example 4: AAV-ie-K558R-Prestin can restore partial hearing loss in Prestin knockout mice.

[0200] Gene therapy in OHC gene deletion-induced hearing loss models is challenging due to the lack of AAVs that can efficiently transduce OHC. To test whether AAV-ie-K558R could potentially restore hearing loss caused by OHC gene dysfunction, this example uses Prestin knockout mice, in which the Prestin protein expression gene is removed from OHCs. Prestin is a key molecule located exclusively in OHCs and plays a crucial role in mediating the electrokinetics of OHCs. In Prestin knockout mice, the hearing threshold is significantly higher than in wild-type (WT) mice. Figure 3 ab). Prestin knockout mice did not exhibit circling behavior or other gait abnormalities (data not shown).

[0201] 4.1 Mouse and virus construction

[0202] Construction of AAV-ie-K558R-Prestin: Driven by the promoter CAG, the prestin gene slc26a5 was packaged into the AAV-ie-K558R vector.

[0203] Construction of AAV-ie-Prestin: Driven by the promoter CAG, the prestin gene slc26a5 was packaged into the AAV-ie vector.

[0204] 4.2 Experimental Grouping and Processing

[0205] In this embodiment, AAV-ie-K558R-Prestin virus was injected into P1-2 ("P1-2" means 1 or 2 days after birth) Prestin knockout mice to study its effect on treating hereditary hearing diseases caused by gene function defects in OHCs. To test whether AAV-mediated overexpression of Prestin in HCs could restore hearing function in Prestin knockout mice, the present invention conducted ABR experiments on different groups of mice.

[0206] 4.3 Experimental Results

[0207] One month after treatment in step 4.2, the ABR threshold of mice in each group was measured, and a partial decrease in the ABR threshold was observed in the AAV-ie-K558R-Prestin experimental group. Figure 3 b, 3c); DPOAE results show that AAV-ie-K558R-Prestin has a 10dB mitigation effect at 16kHz.

[0208] Two months after treatment in step 4.2, the expression of Prestin in the cochlea was detected, and the expression of Prestin in the hair cells of Prestin knockout mice was also examined. It was observed that in the AAV-ie-K558R-Prestin experimental group, Prestin was highly expressed in the cochlear OHCs of mice two months after viral injection. Figure 9 In the AAV-ie-Prestin experimental group, after Prestin was integrated into the AAV-ie vector and injected into newborn Prestin knockout mice, the ABR threshold did not decrease, and no large expression of Prestin in cochlear OHCs was observed one month after injection. Figure 3 c, 3d).

[0209] The results of this embodiment demonstrate that AAV-ie-K558R can serve as a potential vector to deliver genes to OHCs and be used for gene therapy in a mouse model of deafness with hair cell defects.

[0210] Example 5: AAV-ie-K558R-Atoh1-mediated in vivo HC-like cell regeneration

[0211] 5.1 Construction of the AAV-ie-K558R-Atoh1 virus: Driven by the CAG promoter, Atoh1 is packaged into the AAV-ie-K558R vector.

[0212] AAV-ie-Atoh1 virus construction: Driven by the CAG promoter, Atoh1 is packaged into the AAV-ie vector.

[0213] 5.2 Experimental Grouping and Processing

[0214] Studies have shown that HC regeneration may help restore hearing loss caused by aging, noise, or ototoxic drugs. The transcription factor Atoh1 has been shown to induce transdifferentiation of SCs into HCs. Earlier research in this invention demonstrated that AAV-ie can deliver Atoh1 to SCs and cause them to transdifferentiate into HC-like cells.

[0215] To evaluate the potential of the AAV-ie-K558R vector for HC regeneration, this invention uses AAV-ie-K558R-Atoh1 to deliver mouse Atoh1 into the cochlea of ​​newborn mice in vivo. Figure 4a).

[0216] AAV-ie-Atoh1 injection group: AAV-ie-Atoh1 (1×10⁻⁶ per ear) was administered via RWM at P3. 10 GCs are injected into the cochlea.

[0217] AAV-ie-K558R-Atoh1 injection group: AAV-ie-K558R-Atoh1 virus (1×10⁻¹⁰ per ear) was administered via RWM at P3. 10 GCs are injected into the cochlea.

[0218] The cochlea was harvested at P14. In the AAV-ie-Atoh1 injection group, some Myo7a-expressing HC-like cells appeared in the sensory area. Figure 4 b). In the AAV-ie-K558R-Atoh1 injection group, a large number of new HCs expressing Myo7a were observed in the epithelial ridge (GER) region. Notably, some new HC-like cells in both the AAV-ie-Atoh1 and AAV-ie-K558R-Atoh1 injection groups showed sustained expression of Sox2, suggesting that these new regenerated HCs may be at different developmental stages. Figure 4 (b) Compared with other Atoh1 overexpression methods, such as the genetic methods used in previous studies, the number of regenerated HCs cells in the AAV-ie-K558R-Atoh1 injection group in this embodiment was not significantly different, indicating that AAV-ie-K558R is a highly efficient viral vector for HCs regeneration.

[0219] Finally, in this embodiment, the morphology of the newly regenerated HCs cells was quantified using SEM. AAV-ie-K558R-Atoh1 induced the regeneration of numerous HC-like cells, with hair tufts growing from the HC-like cells beneath the IHC region. Figure 4 c). Consistent with earlier studies of this invention, AAV-ie-Atoh1 can also induce HC-like cell regeneration ( Figure 10 However, SEM analysis showed that AAV-ie-K558R-Atoh1 could induce the production of some HC-like cells in the GER region. Figure 11 AAV-ie-Atoh1, however, does not produce a similar effect. This invention observes that newly regenerated HC-like cells produce cilia (motiles) representing hair cells entering a more mature developmental stage. Figure 4 d). These results indicate that AAV-ie-K558R can effectively induce HC-like cell regeneration in the cochlea by delivering Atoh1.

[0220] This invention optimizes AAV-ie by reducing ubiquitination or phosphorylation of the exposed side capsid protein amino acids. The results of this invention demonstrate that the newly generated AAV variant, AAV-ie-K558, is safe and beneficial for hair cell regeneration and gene therapy. Most AAV-ie variants did not exhibit high targeting efficiency, but rather low targeting efficiency, indicating that reduction in ubiquitination or phosphorylation does not necessarily lead to higher transduction efficiency. Therefore, the exact mechanism of transduction efficiency requires further investigation. These results also highlight the importance of conducting screening experiments to determine the optimal AAV for different tissues. Nevertheless, the results of this invention demonstrate that peptide insertion and amino acid mutation are feasible for modifying to produce suitable AAVs for efficient transduction of cells in the cochlea and other tissues.

[0221] This invention reveals that AAV-ie-K558R can partially restore auditory function in Prestin knockout mice. Since effectively delivering genes to IHCs (intracorporeal cholangiform ducts) has always been a major challenge, this represents a significant advance in gene therapy for hearing diseases. In this study, AAV-ie-K558R only achieved partial restoration of auditory function. Several factors can explain this partial restoration. First, AAV-ie-K558R can also transduce IHCs, and overexpression of Prestin in IHCs may lead to IHC dysfunction, thus preventing complete restoration of auditory function. Second, to fully restore the function of OHCs, the expression level of Prestin in OHCs may need to be precisely controlled. In this study, a widely used gene regulator promoter, CAG, was used, which may not be the optimal choice for controlling Prestin expression in OHCs. Further research is needed to identify the optimal gene regulatory element for the cochlea in AAV delivery pathways. Developing a method for regulating gene expression in the cochlea and other tissues using AAVs will be crucial.

[0222] Early studies used several viral vectors, such as lentiviruses and adenoviruses, to deliver Atoh1 into the cochlea. Overexpression of Atoh1 was reported to induce the regeneration of HC-like cells, even achieving partial hearing recovery in deaf guinea pigs. The FDA approved a human clinical trial (NCT02132130) using an adenovirus vector to deliver the human Atoh1 gene into the cochlea; however, the efficiency and effectiveness of adenovirus-mediated HC regeneration require further refinement. This invention demonstrates that AAV-ie-K558R-Atoh1 can induce the generation of numerous new HCs in the sensory cortex with efficiency comparable to previous genetically modified mice. After measuring the regeneration efficiency of adenoviruses and the strength of the immune response, AAV-ie-K558R shows greater advantages in HC regeneration.

[0223] In summary, AAV-ie-K558R is the first AAV vector that can be used for gene therapy in deaf mouse models and induce the regeneration of HC-like cells in newborn mice. Further development and refinement of AAV-ie-K558R will play a crucial role in improving the efficacy of gene therapy or HC cell regeneration for various hearing diseases. AV-ie-K558R-mediated gene therapy can not only restore hearing function in mouse models of deafness caused by genetic dysfunction of HCs or SCs, but also alleviate environmental and age-induced deafness, demonstrating significant potential for the treatment of hearing impairment.

[0224] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention. sequence list <110> Shanghai Weimei Gene Technology Co., Ltd. <120> Variant adeno-associated viruses and their application in disease treatment <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <400> 1 Asp Gly Thr Leu Ala Val Pro Phe Lys 1 5 <210> 2 <211> 7360 <212> DNA <213> Artificial Sequence <400> 2 gcgcgccgat atcgttaacg ccccgcgccg gccgctctag aactagtgga tcccccggaa 60 gatcagaagt tcctattccg aagttcctat tctctagaaa gtataggaac ttctgatctg 120 cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga 180 gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt 240 gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga 300 gaagctgcag cgcgactttc tgacggaatg gcgccgtgtt agtaaggccc cggaggccct 360 tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggagaac 420 caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat 480 tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcaaaagac 540 cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt 600 gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag 660 cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc 720 gcagacgcag gagcgaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag 780 atcaaaaact tcagccaggt acatggagct gtcgggtgg ctcgtggaca agggattac 840 ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc 900 caactcgcgg tcccaatca aggctgcctt ggacaatgcg ggaagatta tgagcctgac 960 taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt ccccaatcg 1020 gatttataa attttggac taaacgggta cgatccccaa tatgcggctt ccgtctttct 1080 gggatgggcc acgaaaagt tcggcaagg gaacaccac tggctgttg ggcctgcaac 1140 taccgggaag accacacatcg cggaggccat agcccacact gtgccctct acgggtgcgt 1200 aaactggacc atgagaact tccctcaa cgactgtgtc gataggg tgatctggtg 1260 ggaggagggg aagatgaccg ccaggtcgt ggagtcggcc aaagccattc tcggaggag 1320 caagtgcgc gtggaccaga atgcaagtc ctcggcccag atagacccga ctcccgtgat 1380 cgtcacctcc aacaccaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca 1440 ccagcagccg tgcagacc ggatgttca atttgactc acccgccgtc tggatcatga 1500 ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg aatctgatgc tgtttccctg cagacaatgc cagagaatga atcagaattc aaatatctgc ttcactcacg gacagaaaga ctgtttagg tgctttcccg tgtcagaatc tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt ggctcgagga cactctctct gacagtggtg gagctcaa cctggcccac 2160. gcccgcagag cggcataagg acgacagcag gggtcttgtg cttcctgggt acaagtacct cggacccttc aacggactcg acaagggaga gccggtcaac gaggcagacg ccgcggccct cgagcacgac aaagcctacg accggcagct cgacagcgga gacaacccgt 2280 acctcaagta caaccacgcc gacgccgagt tccaggagcg gctcaaagaa gatacgtctt 2340 ttggggcaa cctcgggcga gcagtcttcc aggccaaaaa gaggcttctt gaacctcttg 2400 gtctggttga ggaagcggct aagacggctc ctggaaagaa gaggcctgta gagcactctc 2460 ctgtggagcc agactctcc tcgggaaccg gaaaggcggg ccagcagcct gcaagaaaaa 2520 gattgaattt tggtcagact ggagacgcag actcagtccc agaccctcaa ccaatcggag 2580 aacctcccgc agccccctca ggtgtgggat ctcttacaat ggctccaggc ggtggcgcac 2640 caatggcaga caataacgag ggcgccgacg gagtgggtaa ttcctcggga aattggcatt 2700 gcgattccac atggatgggc gacagagtca tcaccaccag cacccgaacc tgggccctgc 2760 ccacctacaa caaccacctc tacaagcaaa tctccaacag cacatctgga ggatcttcaa 2820 atgacaacgc ctacttcggc tacagcaccc cctggggta ttttgacttt aacagattcc 2880 actgccactt ttcaccacgt gactggcagc gactcatcaa caacaactgg ggattccggc 2940 ccaagagact cagcttcaag ctcttcaaca tccaggtcaa ggaggtcacg cagaggtcaag 3060. gcaccaagac catcgccat aacctcacca gcaccatcca ggtgtttacg gactcggagt 3120. accagctgcc gtacgttctc ggctctgccc accagggctg cctgcctccg ttcccggcgg acgtgttcat gattccccag tacggctacc taacactcaa caacggtagt caggccgtgg gacgctcctc cttctactgc ctggaatact ttccttcgca gatgctgaga accggcaaca acttccagtt tacttacacc ttcgaggacg tgccttttcca cagcagctac gcccacagcc 3300. agagcttgga ccggctgatg aatcctctga ttgaccagta cctgtactac ttgtctcgga 3360 ctcaaacaac aggaggcacg acaatacgc agactctggg cttcagccaa ggtgggccta atacaatggc caatcaggca aagaactggc tgccaggacc ctgttaccgc cagcagcgag tatcaaagac atctgcggat aacaacaaca gtgaatactc gtggactgga gctaccaagt accacctcaa tggcagagac tctctggtga atccggggccc ggccatggca agccacaagg acgatgaaga aaagtttttt cctcagagcg gggttctcat ctttgggaag caaggctcag agaaaacaaa tgtggacatt gaaaaggtca tgattacaga cgaagaggaa atcaggacaa 3720 ccaatcccgt ggctacggag cagtatggtt ctgtatctac caacctccag agaggcaacg 3780 atgggacttt ggcggtgcct tttaagagac aagcagctac cgcagatgtc aacacacaag 3840 gcgttcttcc aggcatggtc tggcaggaca gagatgtgta ccttcagggg cccatctggg 3900 caaagattcc acacacggac ggacattttc acccctctcc cctcatgggt ggattcggac 3960 ttaaacaccc tccgcctcag atcctgatca agaacacgcc tgtacctgcg gatcctccga 4020 ccaccttcaa ccagtcaaag ctgaactctt tcatcaccca gtattctact ggccaagtca 4080 gcgtggagat cgagtgggag ctgcagaagg aaaacagcaa gcgctggaac cccgagatcc 4140 agtacacctc caactactac aaatctacaa gtgtggactt tgctgttaat acagaaggcg 4200 tgtactctga accccgcccc attggcaccc gttacctcac ccgtaatctg taattgcttg 4260 ttaatcaata aaccgtttaa ttcgtttcag ttgaactttg gtctctgcgt atttctttct 4320 tatctagttt ccatggctac gtagataagt agcatggcgg gttaatcatt aactacagcc 4380 cgggcgttta aacagcgggc ggaggggtgg agtcgtgacg tgaattacgt catagggtta 4440 gggaggtcct gtattagagg tcacgtgagt gttttgcgac attttgcgac accatgtggt 4500 ctcgctgggg ggggggccc gagtgagcac gcagggtctc cattttgaag cgggaggttt 4560 gaacgagcgc tggcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct 4620 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtataagc 4680 gaaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa 4740 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt 4800 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata 4860 gggttgagtg ttgttccagt ttggaacaag agtccactat taagaacgtg gactccaacg 4920 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat 4980 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc 5040 gatttagagc ttgacggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga 5100 aagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac 5160 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cactttttcgg ggaaatgtgc 5220 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 5280 aataaccctg ataaatgctt aataatatt gaaaaaggaa gagtatgagt attcaacatt 5340 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 5400 aaacgctggt gaagtaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 5460 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 5520 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 5580 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 5640 tcacagaaaa gcatcttacg gatggcatga cattaagaga attatgcagt gctgccataa 5700 5760 taaccgcttt tttgcacaac atggggatc atgtaactcg ccttgatcgt tgggaaccgg 5820 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 5880 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 5940 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 6000 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 6060 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 6120 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 6180 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 6240 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 6300 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 6360 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 6420 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 6480 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 6540 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 6600 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 6660 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 6720 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 6780 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 6840 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 6900 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 6960 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 7020 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 7080 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 7140 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg 7200 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 7260 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 7320 aatttcacac aggaaacagc tatgaccatg attacgccaa 7360 <210> 3 <211> 737 <212> PRT <213> Artificial Sequence <400> 3 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ala Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr Thr 405 410 415 Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Arg Thr Gln Thr Thr Gly Gly Thr Thr Asn Thr Gln Thr Leu Gly Phe 450 455 460 Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp Leu 465 470 475 480 Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp 485 490 495 Asn Asn Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu 500 505 510 Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His 515 520 525 Lys Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe 530 535 540 Gly Lys Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met 545 550 555 560 Ile Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu 565 570 575 Gln Tyr Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala 580 585 590 Ala Thr Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp 595 600 605 Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro 610 615 620 His Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly 625 630 635 640 Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro 645 650 655 Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe Ile 660 665 670 Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu 675 680 685 Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser 690 695 700 Asn Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala Val Asn Thr Glu Gly 705 710 715 720 Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn 725 730 735 Leu <210> 4 <211> 746 <212> PRT <213> Artificial Sequence <400> 4 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ala Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr Thr 405 410 415 Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Arg Thr Gln Thr Thr Gly Gly Thr Thr Asn Thr Gln Thr Leu Gly Phe 450 455 460 Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp Leu 465 470 475 480 Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp 485 490 495 Asn Asn Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu 500 505 510 Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His 515 520 525 Lys Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe 530 535 540 Gly Lys Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met 545 550 555 560 Ile Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu 565 570 575 Gln Tyr Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Asp Gly Thr 580 585 590 Leu Ala Val Pro Phe Lys Arg Gln Ala Ala Thr Ala Asp Val Asn Thr 595 600 605 Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu 610 615 620 Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His 625 630 635 640 Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln 645 650 655 Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Thr Phe 660 665 670 Asn Gln Ser Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln 675 680 685 Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg 690 695 700 Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Thr Ser 705 710 715 720 Val Asp Phe Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro 725 730 735 Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 740 745 <210> 5 <211> 746 <212> PRT <213> Artificial Sequence <400> 5 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ala Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr Thr 405 410 415 Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Arg Thr Gln Thr Thr Gly Gly Thr Thr Asn Thr Gln Thr Leu Gly Phe 450 455 460 Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp Leu 465 470 475 480 Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp 485 490 495 Asn Asn Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu 500 505 510 Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His 515 520 525 Lys Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe 530 535 540 Gly Lys Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Arg Val Met 545 550 555 560 Ile Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu 565 570 575 Gln Tyr Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Asp Gly Thr 580 585 590 Leu Ala Val Pro Phe Lys Arg Gln Ala Ala Thr Ala Asp Val Asn Thr 595 600 605 Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu 610 615 620 Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His 625 630 635 640 Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln 645 650 655 Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Thr Phe 660 665 670 Asn Gln Ser Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln 675 680 685 Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg 690 695 700 Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Thr Ser 705 710 715 720 Val Asp Phe Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro 725 730 735 Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 740 745 <210> 6 <211> 47 <212> DNA <213> Artificial Sequence <400> 6 ttatcttcca gattggctcg aggacactct ctctgaagga ataagac 47 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <400> 7 ctgtaatcat gacccgttca atgtcc 26 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <400> 8 ggacattgaa cgggtcatga ttacag 26 <210> 9 <211> 44 <212> DNA <213> Artificial Sequence <400> 9 cgcccgctgt ttaaacgccc gggctgtagt taatgattaa cccg 44 <210> 10 <211> 2241 <212> DNA <213> Artificial Sequence <400> 10 atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60 cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120 gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180 aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240 cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300 caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360 gccaaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420 ggaaaga ggcctgtaga gcactctcct gtggagccag actctctctc gggaaccgga 480 aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540 tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600 cttacaatgg ctgcaggcgg tggcgcacca atggcagaca ataacgaggg cgccgacgga 660 gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720 accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caagcaaatc 780 tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcacccc 840 tggggtatt ttgactttaa cagattccac tgccactttt caccacgtga ctggcagcga 900 ctcatcaaca acaactgggg attccggccc aagagactca gcttcaagct cttcaacatc 960 caggtcaagg aggtcacgca gaatgaaggc accaagacca tcgccaataa cctcaccagc 1020 accatccagg tgtttacgga ctcggagtac cagctgccgt acgttctcgg ctctgcccac 1080 cagggctgcc tgcctccgtt cccggcggac gtgttcatga ttccccagta cggctaccta 1140 acactcaaca acggtagtca ggccgtggga cgctcctcct tctactgcct ggaatacttt 1200 ccttcgcaga tgctgagaac cggcaacaac ttccagttta cttacacctt cgaggacgtg 1260 cctttccaca gcagctacgc ccacagccag agcttggacc ggctgatgaa tcctctgatt 1320 gaccagtacc tgtactactt gtctcggact caaaacag gaggcacgac aaatacgcag 1380 actctgggct tcagccaagg tgggcctaat acaatggcca atcaggcaaa gaactggctg 1440 ccaggaccct gttaccgcca gcagcgagta tcaaagacat ctgcggataa caacaacagt 1500 gaatactcgt ggactggagc taccaagtac cacctcaatg gcagagactc tctggtgaat 1560 ccgggcccgg ccatggcaag ccacaaggac gatgaagaaa agttttttcc tcagagcggg 1620 gttctcatct ttgggaagca aggctcagag aaaacaaatg tggacattga acgggtcatg 1680 attacagacg aaggaaat caggacaacc aatcccgtgg ctacggagca gtatggttct 1740 gtatctacca acctccagag aggcaacgat gggactttgg cggtgccttt taagagacaa 1800 gcagctaccg cagatgtcaa cacacaaggc gttcttccag gcatggtctg gcaggacaga 1860 gatgtgtacc ttcaggggcc catctgggca aagattccac acacggacgg acattttcac 1920 ccctctcccc tcatgggtgg attcggactt aaacaccctc cgcctcagat cctgatcaag 1980 aacacgcctg tacctgcgga tcctccgacc accttcaacc agtcaaagct gaactctttc 2040 atcacccagt attctactgg ccaagtcagc gtggagatcg agtgggagct gcagaaggaa 2100 aacagcaagc gctggaaccc cgagatccag tacacctcca actactacaa atctacaagt 2160 gtggactttg ctgttaatac agaaggcgtg tactctgaac cccgccccat tggcacccgt 2220 tacctcaccc gtaatctgta a 2241 <210> 11 <211> 1866 <212> DNA <213> Artificial Sequence <400> 11 atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc 60 ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat 120 tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 180 cgcgactttc tgacggaatg gcgccgtgtg agtaggccc cggaggccct tttctttgtg 240 caatttgaga agggagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg 300 aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt 360 taccgcggga tcgagccgac ttgccaac tggttcgcgg tcacaagac cagaatggc 420 gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctcccaaa 480 acccagcctg agctccagtg gggcgtggact atatggaac agtatttaag cgcctgtttg 540 aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 600 gagcagaca agagaatca gatcccaat tctgatgcgc cggtgatcag atcaaaact 660 tcagccaggt acatggagct ggtcgggtgg ctcgtggaca agggattac ctcggagaag 720 cagtggatcc agggacca ggcctac atctccttca atgcggccctc caactcgcgg 780 tcccaatca aggctgcctt ggacaatgcg ggaagatta tgagcctgac taaaaccgcc 840 cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg gatttataaa 900 attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatggggcc 960 acgaaaaagt tcggcaag gaacaccatc tggctgttg ggcctgcaac taccgggag 1020 accacatcg cggaggccat agcccacact gtgccctct acgggtgcgt aactggacc 1080 aatgagaact ttccctcaa cgactgtgtc ugaragatgg tgatctggtg ggaggagggg 1140 aagatgaccg ccaggtcgt ggagtcggcc aaagccattc tcggaggag caggtgcgc 1200 gtggaccaga atgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc 1260 aacaccaaca tgtgcgccgt gattgacggg aaccaacga ccttcgaaca ccagcagccg 1320 ttgcaagacc ggatgttca atttgaacctc acccgccgtc tggatcatga ctttgggaag 1380 gtcaccaagc aggaagtca agactttc cggtgggcaa aggatcacgt ggttgaggtg 1440 gagcatgaat tctacgtca aaagggtgga gccaagaaa gacccgcccc cagtgacgca 1500 gatatagtg agcccaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg 1560 gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg 1620 aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc 1680 ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt 1740 tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg 1800 ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa 1860 caataa 1866 <210> 12 <211> 5704 <212> DNA <213> Artificial Sequence <400> 12 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt 180 ttaaacagag aggaatctct cgagccattg acgtcaataa tgacgtatgt tcccatagta 240 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 300 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 360 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 420 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc 480 actctcccca tctcccccc ctccccaccc ccaattttgt atttattat tttttaatta 540 ttttgtgcag cgatgggggc gggggggggg ggggggggc ggggcgaggc ggagaggtgc 600 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg 660 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg 720 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt 780 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt 840 ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg 900 ccctttgtgc ggggggagcg gctcggggct gtccgcgggg ggacggctgc cttcgggggg 960 gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta 1020 accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc 1080 tgtctcatca ttttggcaaa gaattggatc gaatcgcca ccatgggctc cggagccacc aacttttctc tgctgagca ggctggcgac gtggaggaga acccaggacc tatggtgtct aagggcgagg aggataacat ggccagcctg cctgctacac acgagctgca catcttcgga agcatcaacg gcgtggactt tgatatggtg ggccaggga ccggcaaccc aaacgacgga 1380. 1380. 1380. 1380. 1380. 1380. 1380. 1380. 1380 gtgcctcaca tcggatacgg ctttcaccag tacctgccat acccagacgg aatgagccca ttccaggctg ctatggtgga tggcagcggc taccaggtgc acaggaccat gcagtttgag gacggcgcct ctctgaccgt gaactacaga tacacatacg agggagcca catcaagggc gaggcccagg tgaagggac cggcttccct gctgacggac cagtgatgac caactccctg acagccgctg actggtgccg gtctaagaaa acatacccta acgataagac catcatctct acattcaagt ggagctacac cacaggaac ggcaagaggt acagatccac cgcccggacc acatacacat ttgctaagcc aatggccgct aactacctga agaaccagcc catgtacgtg 1800 ttccgcaaga ccgagctgaa gcactccaag acagagctga acttcaagga gtggcagaag 1860 gcctttaccg acgtgatggg aatggatgag ctgtacaagg gatccgacta caaggaccac 1920 gatggcgact acaaggatca cgacatcgat tacaaggacg atgacgataa gaagcggaca 1980 gctgatggca gcgagttcga gtcccccaag areas aggtggagtg agctagcgat 2040 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt 2100 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat 2160 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct 2220 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct 2280 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc 2340 gctttcccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg 2400 acaggggctc ggctggtggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc 2460 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac 2520 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg 2580 cctcttccgc gacttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc 2640 ccgcagatct aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 2700 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 2760 caatgtatct tatcatgtct ggctagacac gtggccgcta ccccgaccac atgaagcagc 2820 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca 2880 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga 2940 accgcacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc 3000 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt 3060 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg 3120 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag 3180 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 3240 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 3300 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 3360 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 3420 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 3480 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta 3540 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 3600 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc 3660 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 3720 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 3780 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 3840 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 3900 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 3960 4020 agtatgagta ttcaacattt ccgtgtcgcc cttatccct tttttgcggc attttgcctt 4080 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 4140 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagtttttcg 4200 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 4260 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 4320 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 4380 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 4440 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 4500 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 4560 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 4620 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 4680 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 4740 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 4800 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 4860 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 4920 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 4980 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 5040 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 5100 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 5160 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 5220 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 5280 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 5340 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 5400 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 5460 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 5520 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 5580 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 5640 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 5700 atgt 5704 <210> 13 <211> 11635 <212> DNA <213> Artificial Sequence <400> 13 ggtacccaac tccatgctta acagtcccca ggtacagccc accctgcgtc gcaaccagga 60 acagctctac agcttcctgg agcgccactc gccctacttc cgcagccaca gtgcgcagat 120 taggagcgcc acttcttttt gtcacttgaa aaacatgtaa aaataatgta ctaggagaca 180 ctttcaataa aggcaaatgt ttttatttgt acactctcgg gtgattattt accccccacc 240 cttgccgtct gcgccgttta aaaatcaaag gggttctgcc gcgcatcgct atgcgccact 300 ggcagggaca cgttgcgata ctggtgttta gtgctccact taaactcagg caaaccatc 360 cgcggcagct cggtgaagtt ttcactccac aggctgcgca ccatcaccaa cgcgtttagc 420 aggtcgggcg ccgatatctt gaagtcgcag ttggggcctc cgccctgcgc gcgcgagttg 480 cgatacacag ggttgcagca ctggaacact atcagcgccg ggtggtgcac gctggccagc 540 acgctcttgt cggagatcag atccgcgtcc aggtcctccg cgttgctcag ggcgaacgga 600 gtcaactttg gtagctgcct tcccaaaag ggtgcatgcc caggctttga gttgcactcg 660 caccgtagtg gcatcagaag gtgaccgtgc ccggtctggg cgttaggata cagcgcctgc 720 atgaaagcct tgatctgctt aaaagccacc tgagcctttg cgccttcaga gaagaacatg 780 ccgcaagaact tgccggaaaa ctgattggcc ggacaggccg cgtcatgcac gcagcacctt 840 gcgtcggtgt tggagatctg caccacattt cggccccacc ggttcttcac gatcttggcc 900 ttgctagact gctccttcag cgcgcgctgc ccgttttcgc tcgtcacatc catttcaatc 960 acgtgctcct tatttatcat aatgctcccg tgtagacact taagctcgcc ttcgatctca 1020 gcgcagcggt gcagccacaa cgcgcagccc gtgggctcgt ggtgcttgta ggttacctct 1080 gcaaacgact gcaggtacgc ctgcaggaat cgccccatca tcgtcacaaa ggtcttgttg 1140 ctggtgaagg tcagctgcaa cccgcggtgc tcctcgttta gccaggtctt gcatacggcc 1200 gccagagctt ccacttggtc aggcagtagc ttgaagtttg cctttagatc gttatccacg 1260 tggtacttgt ccatcaacgc gcgcgcagcc tccatgccct tctcccacgc agacacgatc 1320 ggcaggctca gcgggtttat caccgtgctt tcactttccg cttcactgga ctcttccttt 1380 tcctcttgcg tccgcatacc ccgcgccact gggtcgtctt cattcagccg ccgcaccgtg 1440 cgcttacctc ccttgccgtg cttgattagc accggtgggt tgctgaaacc caccatttgt 1500 agcgccacat cttctctttc ttcctcgctg tccacgatca cctctgggga tggcgggcgc 1560 tcgggcttgg gagaggggcg cttctttttc tttttggacg caatggccaa atccgccgtc 1620 gaggtcgatg gccgcgggct gggtgtgcgc ggcaccagcg catcttgtga cgagtcttct 1680 tcgtcctcgg actcgagacg ccgcctcagc cgcttttttg ggggcgcgcg gggaggcggc 1740 ggcgacggcg acggggacga cacgtcctcc atggttggtg gacgtcgcgc cgcaccgcgt 1800 ccgcgctcgg gggtggtttc gcgctgctcc tcttcccgac tggccatttc cttctcctat 1860 aggcagaaaa agatcatgga gtcagtcgag aaggaggaca gcctaaccgc cccctttgag 1920 ttcgccacca ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca 1980 cccccgcttg aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac 2040 gacgaggatc gctcagtacc aacagaggat aaaaagcaag accaggacga cgcagaggca 2100 aacgaggaac aagtcgggcg gggggaccaa aggcatggcg actacctaga tgtgggagac 2160 gacgtgctgt tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag 2220 cgcagcgatg tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctgttc 2280 tcaccgcgcg taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc 2340 aacttctacc ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa 2400 aactgcaaga tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc 2460 ttgcggcagg gcgctgtcat acctgatatc gcctcgctcg acgaagtgcc aaaaatcttt 2520 gagggtcttg gacgcgacga gaaacgcgcg gcaaacgctc tgcaacaaga aaacagcgaa 2580 aatgaaagtc actgtggagt gctggtggaa cttgagggtg acaacgcgcg cctagccgtg 2640 ctgaaacgca gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag 2700 gttatgagca cagtcatgag cgagctgatc gtgcgccgtg cacgacccct ggagagggat 2760 gcaaacttgc aagaacaaac cgaggagggc ctacccgcag ttggcgatga gcagctggcg 2820 cgctggcttg agacgcgcga gcctgccgac ttggaggagc gacgcaagct aatgatggcc 2880 gcagtgcttg ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg 2940 cagcgcaagc tagaggaaac gttgcactac acctttcgcc agggctacgt gcgccaggcc 3000 tgcaaaattt ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa 3060 aaccgcctcg ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac 3120 gtccgcgact gcgtttactt atttctgtgc tacacctggc aaacggccat gggcgtgtgg 3180 cagcaatgcc tggaggagcg caacctaaag gagctgcaga agctgctaaa gcaaaacttg 3240 aggacctat ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacattatc 3300 ttccccgaac gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc 3360 atgttgcaaa actttaggaa ctttatccta gagcgttcag gaattctgcc cgccacctgc 3420 tgtgcgcttc ctagcgactt tgtgcccatt aagtaccgtg aatgccctcc gccgctttgg 3480 ggtcactgct accttctgca gctagccaac taccttgcct accactccga catcatggaa 3540 gacgtgagcg gtgacggcct actggagtgt cactgtcgct gcaacctatg caccccgcac 3600 cgctccctgg tctgcaattc gcaactgctt agcgaaagtc aaattatcgg tacctttgag 3660 ctgcagggtc cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg 3720 ctgtggacgt cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt 3780 aggttctacg aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc 3840 cagggccaca tccttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta 3900 cgaaagggac ggggggttta cctggacccc cagtccggcg aggagctcaa cccaatcccc 3960 ccgccgccgc agccctatca gcagccgcgg gcccttgctt cccaggatgg cacccaaaaa 4020 gaagctgcag ctgccgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc 4080 agaggaggtt ttggacgagg aggaggagat gatggaagac tgggacagcc tagacgaagc 4140 ttccgaggcc gaagaggtgt cagacgaaac accgtcaccc tcggtcgcat tcccctcgcc 4200 ggcgccccag aaattggcaa ccgttcccag catcgctaca acctccgctc ctcaggcgcc 4260 gccggcactg cctgttcgcc gacccaaccg tagatgggac accactggaa ccagggccgg 4320 taagtctaag cagccgccgc cgttagccca agagcaacaa cagcgccaag gctaccgctc 4380 gtggcgcggg cacaagaacg ccatagttgc ttgcttgcaa gactgtgggg gcaacatctc 4440 cttcgcccgc cgctttcttc tctaccatca cggcgtggcc ttcccccgta acatcctgca 4500 ttactaccgt catctctaca gcccctactg caccggcggc agcggcagcg gcagcaacag 4560 cagcggtcac acagaagcaa aggcgaccgg atagcaagac tctgacaaag cccaagaaat 4620 ccacagcggc ggcagcagca ggaggaggag cgctgcgtct ggcgcccaac gaacccgtat 4680 cgacccgcga gcttagaaat aggatttttc ccactctgta tgctatattt caacaaagca 4740 ggggccaaga acaagagctg aaaataaaaa acaggtctct gcgctccctc acccgcagct 4800 gcctgtatca caaaagcgaa gatcagcttc ggcgcacgct ggaagacgcg gaggctctct 4860 tcagcaaata ctgcgcgctg actcttaagg actagtttcg cgccctttct caaatttaag 4920 cgcgaaaact acgtcatctc cagcggccac acccggcgcc agcacctgtc gtcagcgcca 4980 ttatgagcaa ggaaattccc acgccctaca tgtggagtta ccagccacaa atgggacttg 5040 cggctggagc tgcccaagac tactcaaccc gaataaacta catgagcgcg ggaccccaca 5100 tgatatcccg ggtcaacgga atccgcgccc accgaaaccg aattctcctc gaacaggcgg 5160 ctattaccac cacacctcgt aataacctta atccccgtag ttggcccgct gccctggtgt 5220 accaggaaag tcccgctccc accactgtgg tacttcccag agacgcccag gccgaagttc 5280 agatgactaa ctcaggggcg cagcttgcgg gcggctttcg tcacagggtg cggtcgcccg 5340 ggcgttttag ggcggagtaa cttgcatgta ttgggaattg tagttttttt aaaatgggaa 5400 gtgacgtatc gtgggaaaac ggaagtgaag atttgaggaa gttgtgggtt ttttggcttt 5460 cgtttctggg cgtaggttcg cgtgcggttt tctgggtgtt ttttgtggac tttaaccgtt 5520 acgtcatttt ttagtcctat atatactcgc tctgtacttg gcccttttta cactgtgact 5580 gattgagctg gtgccgtgtc gagtggtgtt ttttaatagg tttttttact ggtaaggctg 5640 actgttatgg ctgccgctgt ggaagcgctg tatgttgttc tggagcggga gggtgctatt 5700 ttgcctaggc aggagggttt ttcaggtgtt tatgtgtttt tctctcctat taattttgtt 5760 atacctccta tgggggctgt aatgttgtct ctacgcctgc gggtatgtat tcccccgggc 5820 tatttcggtc gctttttagc actgaccgat gttaaccaac ctgatgtgtt taccgagtct 5880 tacattatga ctccggacat gaccgaggaa ctgtcggtgg tgctttttaa tcacggtgac 5940 cagttttttt acggtcacgc cggcatggcc gtagtccgtc ttatgcttat aagggttgtt 6000 tttcctgttg taagacaggc ttctaatgtt taaatgtttt tttttttgtt attttatttt 6060 gtgtttaatg caggaacccg cagacatgtt tgagagaaaa atggtgtctt tttctgtggt 6120 ggttccggaa cttacctgcc tttatctgca tgagcatgac tacgatgtgc ttgctttttt 6180 gcgcgaggct ttgcctgatt ttttgagcag caccttgcat tttatatcgc cgcccatgca 6240 acaagcttac ataggggcta cgctggttag catagctccg agtatgcgtg tcataatcag 6300 tgtgggttct tttgtcatgg ttcctggcgg ggaagtggcc gcgctggtcc gtgcagacct 6360 gcacgattat gttcagctgg ccctgcgaag ggacctacgg gatcgcggta tttttgttaa 6420 tgttccgctt ttgaatctta tacaggtctg tgaggaacct gaatttttgc aatcatgatt 6480 cgctgcttga ggctgaaggt ggagggcgct ctggagcaga tttttacaat ggccggactt 6540 aatattcggg atttgcttag agacatattg ataaggtggc gagatgaaaa ttatttgggc 6600 atggttgaag gtgctggaat gtttatagag gagattcacc ctgaagggtt tagcctttac 6660 gtccacttgg acgtgagggc agtttgcctt ttggaagcca ttgtgcaaca tcttacaaat 6720 gccattatct gttctttggc tgtagttt gccacgcca ccggagggga gcgcgttcac 6780 ttatagatc ttcattttga ggttttggat aatcttttgg aataaaaaa aaaaaacatg gttcttccag ctcttcccgc tcctcccgtg tgtgactcgc agaacgaatg tgtaggttgg 6900. ctgggtgtgg cttattctgc ggtggtggat gttatcaggg cagcggcgca tgaaggagtt 6960 7020. ccgaagccag ggggcgcctg gatgctttga gagagtggat attack tactacacag agcgagctaa gcgacgagac cggagacgca gatctgtttg tcacgcccgc 7080. acctggtttt gcttcagga atatgactc gtccggcgtt cctttggca tgacactacg accaacacga tctcggttgt ctcggcgcac tccgtacagt agggatcgcc tacctccttt tgagacagag acccgcgcta ccatactgga ggatcatccg ctgctgcccg aatgtaacac tttgacaatg cacaacgtga gttacgtgcg aggtcttccc tgcagtgtgg gatttacgct 7380. gattcagga tggggttgttc cctgggatat ggttctgacg cgggaggagc ttgtaatcct gaggaagtgt atgcacgtgt gcctgtgttg tgccaacatt gatatcatga cgagcatgat gatccatggt tacgagtcct gggctctcca ctgtcattgt tccagtcccg gttccctgca 7500 gtgcatagcc ggcgggcagg ttttggccag ctggtttagg atggtggtgg atggcgccat 7560 gtttaatcag aggtttatat ggtaccggga ggtggtgaat tacaacatgc caaaagaggt 7620 aatgtttatg tccagcgtgt ttatgagggg tcgccactta atctacctgc gcttgtggta 7680 tgatggccac gtgggttctg tggtccccgc catgagcttt ggatacagcg ccttgcactg 7740 tgggattttg aacaatattg tggtgctgtg ctgcagttac tgtgctgatt taagtgagat 7800 cagggtgcgc tgctgtgccc ggaggacaag gcgtctcatg ctgcgggcgg tgcgaatcat 7860 cgctgaggag accactgcca tgttgtattc ctgcaggacg gagcggcggc ggcagcagtt 7920 tattcgcgcg ctgctgcagc accaccgccc tatcctgatg cacgattatg actctacccc 7980 catgtaggcg tggacttccc cttcgccgcc cgttgagcaa ccgcaagttg gacagcagcc 8040 tgtggctcag cagctggaca gcgacatgaa cttaagcgag ctgcccgggg agtttattaa 8100 tatcactgat gagcgtttgg ctcgacagga aaccgtgtgg aatataacac ctaagaatat 8160 gtctgttacc catgatatga tgctttttaa ggccagccgg ggagaaagga ctgtgtactc 8220 tgtgtgttgg gagggaggtg gcaggttgaa tactagggtt ctgtgagttt gattaaggta 8280 cggtgatcaa tataagctat gtggtggtgg ggctatacta ctgaatgaaa aatgacttga 8340 aattttctgc aattgaaaaa taaacacgtt gaaacataac atgcaacagg ttcacgattc 8400 tttattcctg ggcaatgtag gagaaggtgt aagagttggt agcaaaagtt tcagtggtgt 8460 attttccact ttcccaggac catgtaaaag acatagagta agtgcttacc tcgctagttt 8520 ctgtggattc actagaatcg atgtaggatg ttgcccctcc tgacgcggta ggagaagggg 8580 agggtgccct gcatgtctgc cgctgctctt gctcttgccg ctgctgagga ggggggcgca 8640 tctgccgcag caccggatgc atctgggaaa agcaaaaaag gggctcgtcc ctgtttccgg 8700 aggaatttgc aagcggggtc ttgcatgacg gggaggcaaa cccccgttcg ccgcagtccg 8760 gccggcccga gactcgaacc gggggtcctg cgactcaacc cttggaaaat aaccctccgg 8820 ctacagggag cgagccactt aatgctttcg ctttccagcc taaccgctta cgccgcgcgc 8880 ggccagtggc caaaaaagct agcgcagcag ccgccgcgcc tggaaggaag ccaaaaggag 8940 cgctcccccg ttgtctgacg tcgcacacct gggttcgaca cgcgggcggt aaccgcatgg 9000 atcacggcgg acggccggat ccggggttcg aaccccggtc gtccgccatg atacccttgc 9060 gaatttatcc accagaccac ggaagagtgc ccgcttacag gctctccttt tgcacggtct 9120 agagcgtcaa cgactgcgca cgcctcaccg gccagagcgt cccgaccatg gagcactttt 9180 tgccgctgcg caacatctgg aaccgcgtcc gcgactttcc gcgcgcctcc accaccgccg 9240 ccggcatcac ctggatgtcc aggtacatct acggattacg tcgacgttta aaccatatga 9300 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 9360 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 9420 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 9480 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 9540 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 9600 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 9660 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 9720 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 9780 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 9840 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 9900 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 9960 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 10020 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 10080 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 10140 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 10200 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 10260 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 10320 cgagacccac gctcaccgg tccagattta tcagcaataa accagccagc cggaagggcc 10380 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 10440 gaagctagag tagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 10500 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 10560 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 10620 ccgatcgttg tcaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 10680 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 10740 accaagtcat tctgagata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 10800 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 10860 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 10920 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 10980 aaggaaggc aaaatgccgc aaaaaaggga ataagggcga cagggaaatg ttgaatactc 11040 atactcttcc tttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 11100 tacatatttg aatgtattta gaaaaataa aaatagggg ttccgcgcac atttccccga 11160 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta atttttgtt 11220 aaatcagctc atttttaac caataggccg aaatcggcaa atcccttat aaatcaaag 11280 atagaccga gataggttg agtgttgttc cagttttggaa caagtcca ctattaaaga 11340 acgtggactc caacgtcaa gggcgaaaaa ccgtctca gggcgatggc ccactacgtg 11400 aaccatcacc ctaatcaagt ttttggtt cgaggtgccg taaagcacta aatcggaacc 11460 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg 11520 aagggaagaa agcgaaagga gcggcgcta gggcgctggc aagtgtagcg gtcacgctgc 11580 gcgtaaccac cacaccccgcc gcgctatg cgccgctaca gggcgcgatg gatcc 11635

Claims

1. A variant adeno-associated virus capsid protein, characterized in that, The amino acid sequence of the variant adeno-associated virus capsid protein is shown in SEQ ID NO.

5.

2. A nucleic acid, characterized in that, The nucleic acid encodes the variant adeno-associated virus capsid protein as described in claim 1.

3. A construct comprising the nucleic acid as described in claim 2.

4. A host cell, characterized in that, The host cell comprises the construct of claim 3 or the genome in which exogenous nucleic acids of claim 2 are integrated.

5. A variant adeno-associated virus, characterized in that, The capsid structure of the variant adeno-associated virus contains the variant adeno-associated virus capsid protein as described in claim 1.

6. The variant adeno-associated virus as described in claim 5, characterized in that, The variant adeno-associated virus also includes a heterologous nucleotide sequence encoding the target product.

7. Using host cells transformed with the variant adeno-associated virus as described in claim 5 or 6.

8. A variant adeno-associated virus vector system, characterized in that, It includes a packaging plasmid containing the nucleic acid fragment of claim 2.

9. The variant adeno-associated virus vector system as described in claim 8, characterized in that, The packaging plasmid also contains a fragment of the rep gene from adeno-associated virus.

10. The variant adeno-associated virus vector system according to claim 8, characterized in that, The adeno-associated virus vector system further includes an expression plasmid containing heterologous nucleotides responsible for encoding the target product.

11. The variant adeno-associated virus vector system according to any one of claims 8-10, characterized in that, The adeno-associated virus vector system further includes helper viral plasmids, and / or, the adeno-associated virus vector system further includes host cells.

12. A variant adeno-associated virus, obtained by viral packaging of the variant adeno-associated virus vector system according to any one of claims 8-11.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a variant of adeno-associated virus as described in claim 5, 6 or 12, and a pharmaceutically acceptable vector.

14. Use of the variant adeno-associated virus as described in claim 5, 6 or 12, or the host cell as described in claim 7, the variant adeno-associated virus vector system as described in any one of claims 8-11, or the pharmaceutical composition as described in claim 13 in the preparation of a medicament for treating hearing impairment.

15. The use as described in claim 14, characterized in that, Includes at least one of the following 1) to 5): 1) The hearing impairment described is caused by cochlear damage; 2) The hearing impairment described is a cell damage-related disease; 3) The hearing impairment described is a disease caused by a gene defect; 4) The hearing impairment is a disease caused by environmental factors; the environmental factors are selected from noise or ototoxic drugs; 5) The hearing impairment disease mentioned is an age-related disease.

16. The use as described in claim 14, characterized in that, The drug has the function of inducing HC cell regeneration or HC-like cell regeneration.

17. The use as described in claim 16, characterized in that, The regenerated HC cells or HC-like cells express Myo7a and / or Sox2.

18. The use as described in claim 14, characterized in that, The drug includes the ability to induce the production of HC cells or HC-like cells in the GER region.

19. The use as described in claim 14, characterized in that, The drug is expressed through overexpression Atoh1 Other regeneration factors induce HC-like cell regeneration in the cochlea.

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