WO2025162267A1 - Dual-vector system for expressing myo7a protein, and use thereof - Google Patents

Abstract

The present invention relates to a dual-vector system for expressing an MYO7A protein. The dual-vector system comprises a first nucleic acid vector and a second nucleic acid vector, wherein the first nucleic acid vector comprises a first nucleotide sequence, and the second nucleic acid vector comprises a second nucleotide sequence. The first nucleotide sequence comprises an expression cassette inserted between two first ITR sequences, and the second nucleotide sequence comprises an expression cassette inserted between two second ITR sequences. The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A, an N-terminal coding sequence of intein, and polyA; and the expression cassette of the second nucleotide sequence comprises a promoter, a C-terminal coding sequence of intein, a C-terminal coding sequence of MYO7A, and polyA. The present invention also relates to a packaging vector system of an adeno-associated virus, a method for packaging an adeno-associated virus, and an adeno-associated virus obtained thereby. The dual-vector system for expressing the MYO7A protein, or the adeno-associated virus can be used for gene therapy, especially for the treatment of hearing loss, such as for the treatment of MYO7A-mutation-related USH1B, autosomal recessive hereditary deafness DFNB2 and autosomal dominant hereditary deafness DFNA11.

Description

Dual vector system for expressing MYO7A protein and its use Technical Field

The present invention relates to a dual-vector system for expressing MYO7A protein and its application in gene therapy, in particular to the treatment of hearing loss.

Background Art

The Myo7a gene encodes the MYO7A protein (also known as "myosin 7A"). Since the Myo7a gene was first reported in 1996, more than 500 mutation sites have been confirmed to be associated with hereditary deafness. The Myo7a gene is located on chromosome 11q13.5 and encodes an unconventional myosin VIIA composed of 2215 amino acids. It is a motor molecule widely expressed in the retinal epithelium and inner ear hair cells. Mutations in the Myo7a gene lead to abnormal morphology of inner ear hair cell stereocilia, as well as hair cell and neuronal death, and are associated with Usher syndrome (USH) (also known as hereditary deafness-retinitis pigmentosa syndrome) type 1B, autosomal recessive deafness DFNB2, and autosomal dominant deafness DFNA11. Therefore, neither hearing aids nor cochlear implants are perfect and effective treatment options for related hearing loss.

Rapidly evolving gene therapy strategies offer a potential treatment for hearing loss caused by Myo7a mutations. First, the cochlea's highly compartmentalized structure and blood-cochlear barrier (BCB) isolate it from other organs, significantly reducing medication dosage and the risk of drug penetration. Second, the cells in the cochlea are highly specialized; hair cells and supporting cells no longer divide, resulting in a stable cellular structure that favors sustained transgene expression via non-integrating viral vectors (such as AAV). Furthermore, successful gene therapy has been demonstrated in the eye, also considered "immune-privileged," and AAV has been used as a viral vector to treat OTOF mutations in the cochlea. This virus has been used in three investigator-initiated clinical trials (IITs) targeting OTOF mutations (two at Dingxin Pharmaceuticals and one at Suzhou Xingaotowei Biotechnology Co., Ltd.). Currently, there is no evidence that AAV vectors cause any adverse reactions in cochlear gene therapy. This significantly enhances the safety of AAV as a vector for delivering gene therapy drugs.

Most importantly, the hearing loss caused by the Myo7a gene mutation is due to the loss of a single gene protein. Therefore, in theory, as long as the exogenous Myo7a gene is transduced into the inner ear hair cells through AAV to express a sufficient amount of MYO7A protein, the abnormal morphology of the stereocilia in the inner ear hair cells can be repaired, the normal survival of the hair cells and neurons can be maintained, and the hearing function can be protected.

Although the small size of AAV has transduction advantages, its loading capacity is limited. Since the DNA length of the coding region of Myo7a is 6.65kb, it exceeds the packaging capacity of an AAV (4.5kb). This makes it impossible for the AAV vector to directly carry the complete Myo7a coding sequence (CDS) to express the complete Myo7a in hair cells. A dual AAV vector strategy has been reported in the prior art. In this strategy, dual AAV achieves mRNA expression of long-fragment genes through mRNA trans-splicing after each mRNA is transcribed. This delivery scheme first divides the coding region of the target gene into two parts and constructs them into different AAV vector plasmids. Two different AAV vectors transcribe the N-terminal and C-terminal mRNA sequences of the target gene in the cell, and complete the splicing of the N-terminal and C-terminal mRNA of the target gene by splicing the donor sequence-splicing acceptor sequence (abbreviated as SD-SA sequence), thereby forming a complete target gene mRNA template to express the complete protein. In 2019, Sebastian Kuegler and Ellen Reisinger (EMBO Mol Med. 2019 Jan; 11(1): e9396.doi: 10.15252/emmm.201809396) and Omar Akil (Proc Natl Acad Sci U S A. 2019 Mar 5; 116(10): 4496-4501) published articles showing that the simultaneous injection of two AAVs containing the N and C segments of mouse otoferlin cDNA into the cochlea can efficiently transfect inner hair cells and restore some ABR thresholds in OTOF ^-/- mice. This groundbreaking research progress has laid the foundation for the dual AAV gene therapy strategy to enter clinical treatment. According to the University of Florida Gene Research Group, two cleavage sites, exon21/22 and exon23/24, have been screened in vitro through this method to express the full-length Myo7a protein.

Currently, there are no drugs or surgeries that can effectively treat hearing loss caused by Myo7a mutations, and this hearing loss is irreversible. Neither stimulating residual hearing through hearing aids during infancy nor cochlear implants can restore or even maintain residual hearing. The gene therapy strategy of dual AAV vectors has the potential to express MYO7A protein in hair cells, thereby restoring the morphology of hair cell stereocilia, maintaining hair cell survival, and protecting hearing. However, the efficiency of full-length protein produced by mRNA trans-splicing in existing technologies is relatively low, and may not be sufficient to maintain long-term survival of hair cells. It has not yet been applied to hearing treatment, and its reliability is still unknown.

The dual-vector system developed by the present invention and the MYO7A protein produced by intein splicing can replenish the MYO7A protein of hair cells in the early stage, repair the morphology and function of hair cell stereocilia, prevent hair cell death, and thus prevent further deterioration of hearing loss.

SUMMARY OF THE INVENTION

In the existing technology, there are no effective drugs or surgical treatments for USH1B, autosomal recessive hearing loss DFNB2, and autosomal dominant hearing loss DFNA11 caused by Myo7a gene mutations. The dual-vector system provided by the present invention can effectively express sufficient MYO7A protein in cochlear hair cells, thereby repairing the morphology and function of hair cell stereocilia, maintaining hair cell survival and protecting hearing. It can be used as a candidate drug for the future clinical treatment of hearing loss.

The AAV dual-vector system of the present invention provides a MYO7A cleavage site, a MYO7A protein cleavage site sequence, and a screening method thereof that can be used to treat USH1B, autosomal recessive hereditary deafness DFNB2, and autosomal dominant hereditary deafness DFNA11. This dual-vector system can be used in the clinical treatment of USH1B.

In a first aspect, the present invention provides a dual vector system for expressing MYO7A protein, comprising a first nucleic acid vector and a second nucleic acid vector, wherein

The first nucleic acid vector comprises a first nucleotide sequence; and the second nucleic acid vector comprises a second nucleotide sequence;

The first nucleotide sequence comprises an expression cassette inserted between two first ITR sequences;

The second nucleotide sequence comprises an expression cassette inserted between two second ITR sequences;

The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A, an N-terminal coding sequence of an intein, and polyA;

The expression cassette of the second nucleotide sequence comprises a promoter, a C-terminal coding sequence of an intein, a C-terminal coding sequence of MYO7A and polyA; and

A MYO7A cleavage site is provided in the MYO7A amino acid sequence, for example, the MYO7A amino acid sequence is as shown in SEQ ID NO: 2 or a functional fragment thereof, for example, the functional fragment is an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2;

The N-terminal coding sequence of MYO7A is a nucleotide coding sequence from the N-terminus of the MYO7A amino acid sequence to the MYO7A cleavage site; the C-terminal coding sequence of MYO7A is a nucleotide coding sequence from the amino acid after the MYO7A cleavage site to the C-terminus of the MYO7A amino acid sequence.

In some embodiments, the MYO7A cleavage site is located at the amino acid preceding serine, threonine, or cysteine in the MYO7A amino acid sequence.

In some embodiments, in the binary vector system for expressing MYO7A protein of the present invention, the promoter of the expression cassette of the first nucleotide sequence or the second nucleotide sequence is selected from CAG promoter, CMV promoter, CBA promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or promoters of genes encoding Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, OTOF and STRC;

In some embodiments, in the dual-vector system for expressing MYO7A protein of the present invention, the polyA of the expression cassette of the first nucleotide sequence or the second nucleotide sequence comprises AATAAA (SEQ ID NO: 33) and variants of AATAAA; the variants of AATAAA comprise ATTAAA (SEQ ID NO: 34), AGTAAA (SEQ ID NO: 35), CATAAA (SEQ ID NO: 36), TATAAA (SEQ ID NO: 37), GATAAA (SEQ ID NO: 38), ACTAAA (SEQ ID NO: 39), AATATA (SEQ ID NO: 40), AAGAAA (SEQ ID NO: 41), AATAAT (SEQ ID NO: 42), AAAAAA (SEQ ID NO: 43), AATGAA (SEQ ID NO: 44), NO: 44), AATCAA (SEQ ID NO: 45), AACAAA (SEQ ID NO: 46), AATCAA (SEQ ID NO: 47), AATAAC (SEQ ID NO: 48), AATAGA (SEQ ID NO: 49), AATTAA (SEQ ID NO: 50) or AATAAG (SEQ ID NO: 51); for example, the polyA is a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the poly A signal sequence shown in SEQ ID NO: 29 or SEQ ID NO: 32; and each of the two first ITR sequences and the two second ITR sequences is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9.

In some embodiments, in the dual-vector system for expressing MYO7A protein of the present invention, the expression cassette of the first nucleotide sequence or the second nucleotide sequence further comprises an expression regulatory element and/or a tag element, for example, the expression regulatory element is a woodchuck hepatitis posttranscriptional regulatory element (WPRE) or a variant thereof, preferably a WPRE truncated variant, for example, a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence shown in SEQ ID NO: 28, for example, the nucleotide sequence shown in SEQ ID NO: 31; for example, the tag element is HA.

In some embodiments, in the dual-vector system for expressing MYO7A protein of the present invention, the intein is derived from MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, NpuDnaE, AvaDnaE, CraDnaE, CspDnaE, CwaDnaE, MchtDnaE, OliDnaE, TerDnaE, gp41-1, gp41-8, IMPDH-1 or RmaDnaB, for example, the intein is derived from RmaDnaB, for example, the N-terminus of the intein is the N-terminus of the RmaDnaB intein as shown in SEQ ID NO:23, and the C-terminus of the intein is the C-terminus of the RmaDnaB intein as shown in SEQ ID NO:24. In some embodiments, the intein is derived from NpuDnaE, for example, the N-terminus of the intein is the N-terminus of the NpuDnaE intein as shown in SEQ ID NO:52, and the C-terminus of the intein is the C-terminus of the NpuDnaE intein as shown in SEQ ID NO:54.

In some embodiments, in the dual-vector system for expressing MYO7A protein of the present invention, the first nucleotide sequence is inserted into a plasmid comprising two first ITR sequences, and the second nucleotide sequence is inserted into a plasmid comprising two second ITR sequences, for example, the plasmid comprising two first ITR sequences and the plasmid comprising two second ITR sequences are the same or different, for example, the plasmid is pAAV, pAAV-CMV, pX601, pX551 or pAAV-MCS plasmid.

In some embodiments, in the dual-vector system for expressing MYO7A protein of the present invention, the MYO7A cleavage site is as shown in Table 1.

In some specific embodiments, in the dual-vector system for expressing the MYO7A protein of the present invention, amino acid 1043 of the MYO7A amino acid sequence shown in SEQ ID NO: 2 is used as the MYO7A cleavage site, and the RmaDnaB intein is used; the N-terminal coding sequence of MYO7A is connected and fused to the N-terminal coding sequence of the RmaDnaB intein, and then a first nucleotide sequence is constructed, using the pAAV-CMV plasmid as a vector; the C-terminal coding sequence of the RmaDnaB intein is connected and fused to the C-terminal coding sequence of MYO7A, and then a second nucleotide sequence is constructed, using the pAAV-CMV plasmid as a vector;

The 1058th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector;

The 1061st amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector;

The 1064th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector;

The 1071st amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector;

The 1076th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector;

The 1081st amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector;

The 1104th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector;

The 1105th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector;

The 1114th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector;

The 1119th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector;

Using amino acid 1122 of the MYO7A amino acid sequence shown in SEQ ID NO: 2 as the MYO7A cleavage site and using the RmaDnaB intein; connecting and fusing the N-terminal coding sequence of MYO7A with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, using the pAAV-CMV plasmid as a vector; connecting and fusing the C-terminal coding sequence of the RmaDnaB intein with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, using the pAAV-CMV plasmid as a vector; or

The 1126th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector;

For example, the N-terminal coding sequence of the RmaDnaB intein encodes the N-terminal portion of RmaDnaB shown in SEQ ID NO:23, and the C-terminal coding sequence of the RmaDnaB intein encodes the C-terminal portion of RmaDnaB shown in SEQ ID NO:24.

In some embodiments, in the dual-vector system for expressing MYO7A protein of the present invention, the expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A as shown in SEQ ID No: 4, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, a C-terminal coding sequence of an intein, a C-terminal coding sequence of MYO7A as shown in SEQ ID No: 6, and polyA;

The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A as shown in SEQ ID No: 8, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, an C-terminal coding sequence of an intein, a C-terminal coding sequence of MYO7A as shown in SEQ ID No: 10, and polyA;

The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A as shown in SEQ ID No: 12, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, an C-terminal coding sequence of an intein, a C-terminal coding sequence of MYO7A as shown in SEQ ID No: 14, and polyA;

The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A as shown in SEQ ID No: 16, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, an C-terminal coding sequence of an intein, an C-terminal coding sequence of MYO7A as shown in SEQ ID No: 18, and polyA; or

The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A as shown in SEQ ID No: 20, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, an C-terminal coding sequence of an intein, an C-terminal coding sequence of MYO7A as shown in SEQ ID No: 22, and polyA.

In some embodiments, in the dual-vector system for expressing MYO7A protein of the present invention, the expression cassette of the first nucleotide sequence and the expression cassette of the second nucleotide sequence each contain a combination of a WPRE nucleotide sequence and an SV40 polyadenylation sequence at the N-terminus of the 3’ITR sequence, for example, a nucleotide sequence shown in SEQ ID NO: 27 or a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 27; or a combination of a WPRE3 nucleotide sequence and an SV40 late polyadenylation sequence, for example, a nucleotide sequence shown in SEQ ID NO: 30 or a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 30.

In a second aspect, the present invention provides an adeno-associated virus packaging vector system, which comprises the dual-vector system for expressing MYO7A protein described in the first aspect of the present invention, a vector carrying AAV rep and cap genes, and a helper virus vector, which are packaged into an AAV vector. Preferably, the amino acid sequence of the MYO7A protein is as shown in SEQ ID NO: 2.

In some embodiments, in the adeno-associated virus packaging vector system of the present invention, the vector carrying the AAV rep and cap genes is selected from AAV1, AAV2, AAV5, AAV8, AAV9, Anc80, PHP.eB, AAV-DJ and AAVrh.10 vectors; and the helper virus vector is a pHelper plasmid.

In a third aspect, the present invention provides a method for packaging an adeno-associated virus, wherein the adeno-associated virus packaging vector system described in the second aspect of the present invention is transferred into a host cell for packaging.

In some embodiments, the host cell is selected from Hela-S3 cells, HEK-293 cells, HEK-293T cells, HEK-293FT cells, A549 cells, and Sf9 cells.

In a fourth aspect, the present invention provides a dual adeno-associated virus vector, which is obtained by the packaging method according to the second aspect of the present invention.

In a fifth aspect, the present invention provides the use of the dual vector system for expressing MYO7A protein described in the first aspect of the present invention or the dual adeno-associated virus vector described in the fourth aspect of the present invention for preparing a drug or preparation for treating deafness, hearing loss or hearing dysfunction.

In a sixth aspect, the present invention provides a medicine or preparation for treating deafness, hearing loss, or hearing dysfunction, which is prepared by the dual-vector system for expressing MYO7A protein described in the first aspect of the present invention or the adeno-associated virus described in the fourth aspect of the present invention, wherein the adeno-associated virus is obtained by transferring the adeno-associated virus packaging vector system into a host cell for packaging, and the adeno-associated virus packaging vector system includes a dual-vector system for expressing MYO7A protein, a vector carrying AAVrep and cap genes, and a helper virus vector.

In some embodiments, the drug or formulation of the present invention further comprises a neutral salt buffer, an acidic salt buffer, an alkaline salt buffer, glucose, mannose, mannitol, proteins, polypeptides, amino acids, antibiotics, chelating agents, adjuvants, preservatives, nanoparticles, liposomes and positive lipid particles.

In some embodiments, the drug or preparation of the present invention is administered by injection through the round window, oval window, semicircular canal, or common canal of the cochlea; and is administered once or multiple times throughout life, with a total dose of 1×10 ⁹ -1×10 ¹³ viral genomes.

In a seventh aspect, the present invention provides a method for screening MYO7A protein cleavage sites for intein splicing, comprising:

a) constructing the first nucleic acid vector and the second nucleic acid vector according to the first aspect of the present invention, wherein, optionally, the first residue at the N-terminus of the intein is Cys; the 3' terminal sequence at the C-terminus of the intein contains His and Asn, and the first amino acid residue at the 5' terminal of the C-terminus of MYO7A is Cys, Ser, or Thr;

b) co-transfecting the constructed first nucleic acid vector and the second nucleic acid vector into mammalian cells;

c) When the MYO7A protein can be highly expressed, the 3' terminal residue of the N-terminal portion of the MYO7A protein is used as the MYO7A protein cleavage site for intein splicing.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic diagram of intein-mediated full-length extein expression. In the figure, "N-extein" represents the N-terminal extein, "C-extein" represents the C-terminal extein, "intein ^N " represents the N-terminal intein, and "intein ^C " represents the C-terminal intein.

Figure 2 shows a schematic diagram of the screening process for Myo7a cleavage sites via intein splicing. The intein splicing method involves protein trans-splicing, where the N-terminal and C-terminal coding sequences (CDS) of Myo7a are constructed and expressed separately in two separate plasmids, and the full-length Myo7a protein is assembled via intein splicing. The N-terminal plasmid contains an intein N-terminal fragment sequence (e.g., the RmaDnaB intein N-terminal fragment (Rmintein ^N ) sequence, also referred to herein as the "Rm-N-intein" sequence, where the first residue of the Rm-N-intein sequence is Cys) at the 3' end of the Myo7a N-terminal CDS sequence. The C-terminal plasmid adds an intein C-terminal fragment (Rmintein ^C ) sequence before the 5' start of the C-terminal CDS of Myo7a (for example, the RmaDnaB intein C-terminal fragment sequence, also referred to as the "Rm-C-intein" sequence in this article, the 3' end sequence of the Rm-C-intein sequence contains His and Asn). The first amino acid residue 5' of the C-terminal CDS of Myo7a is Cys, Ser, and Thr for intein splicing.

Figure 3 illustrates a schematic diagram of the AAV binary vector plasmid elements. The pAAV-CMV-EGFP-WPRE-SV40 plasmid backbone sequence can be as shown in SEQ ID NO: 26, wherein the 5' ITR sequence is located between 1 bp and 141 bp, the CMV promoter sequence is located between 169 bp and 752 bp, the Kozak sequence is located between 792 bp and 797 bp, the EGFP sequence is located between 801 bp and 1517 bp, the WPRE sequence is located between 1536 bp and 2124 bp, the SV40 PolyA signal sequence is located between 2131 bp and 2252 bp, the 3' ITR sequence is located between 2290 bp and 2430 bp, the f1 Ori sequence is located between 2505 bp and 2960 bp, the kana resistance sequence is located between 3242 bp and 4156 bp, and the Ori sequence is located between 4327 bp and 4515 bp.

Figure 4 shows the N-terminal protein map of the MYO7A cleavage site S3.

Figure 5 shows the C-terminal protein map of the MYO7A cleavage site S3.

Figure 6 shows the N-terminal protein map of the MYO7A cleavage site S4.

Figure 7 shows the C-terminal protein map of the MYO7A cleavage site S4.

Figure 8 shows the N-terminal protein map of the MYO7A cleavage site S8.

Figure 9 shows the C-terminal protein plasmid map of the MYO7A cleavage site S8.

Figure 10 shows the N-terminal protein map of the MYO7A cleavage site S9.

Figure 11 shows the C-terminal protein map of the MYO7A cleavage site S9.

FIG12 shows the N-terminal protein map of the MYO7A cleavage site S11.

FIG13 shows a protein map of the C-terminal region of the MYO7A cleavage site S11.

Figure 14 shows the full-length protein expression levels of each MYO7A cleavage site plasmid pair. As shown in Figure 14, MYO7A cleavage sites S3, S4, S8, S9, and S11 produce significantly higher full-length protein expression levels.

Figure 15 shows the results of Western blot analysis of MYO7A protein expression in 293T cells for the MYO7A cleavage site S3, using vector plasmids containing the WPRE-SV40 element (the WPRE -SV40 element is the name for the combination of the WPR E element and the SV40 poly(A) signal sequence, and its sequence is shown in SEQ ID NO:27) or the W3SL element (the W3SL element is the name for the combination of the WPR E element and the SV40 late poly(A) signal sequence, and its sequence is shown in SEQ ID NO:30), as well as plasmids that do not contain the WPRE-SV40 element or the W3SL element. In the figure, "HA" indicates the results of detecting HA-tagged MYO7A protein expression using an antibody against the HA tag.

Figure 16 shows the immunofluorescence results of MYO7A protein expression in mouse cochlea. The left panel of Figure 16 shows the results of parvalbumin staining; the middle panel shows the results of Myo7a staining; and the right panel is an overlay of parvalbumin and Myo7a staining.

FIG17 shows the test results of treating hearing function in Myo7a knockout mice using Myo7a genes corresponding to various candidate cleavage sites of MYO7A.

Detailed Description of the Invention

Unless otherwise defined hereinafter, all technical and scientific terms used in this specification have the same meaning as those of ordinary skill in the art to which the present invention pertains. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In addition, the materials, methods and examples described herein are merely illustrative and are not intended to be restrictive. Other features, objects and advantages of the present invention will become apparent from this specification and the accompanying drawings and from the appended claims.

I. Definition

As used herein, the term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value. The term is also intended to encompass values within ±1%, ±0.5%, or ±0.1% of the specified number.

As used herein, the term "comprising" or "including" means including the recited elements, integers, steps, or groups of elements, integers, or steps, but does not exclude any other elements, integers, or steps, or other groups of elements, integers, or steps. As used herein, unless otherwise indicated, the term "comprising" or "including" also encompasses the situation consisting of the recited elements, integers, or steps. For example, when referring to a polynucleotide "comprising" a particular sequence, it is intended to encompass a polynucleotide consisting of that particular sequence.

Herein, the expression "and/or," when used in conjunction with two or more items, is intended to mean any one of the associated listed items, or any multiple or all possible combinations of the associated listed items.

The intein or protein intein (also known as intein) described herein is a polypeptide chain within an immature precursor protein. Through a series of self-catalytic reactions, such as rearrangement, transesterification, and cyclization, it can be excised from the precursor protein and its two end polypeptide segments (exteins) connected by a natural peptide bond. This is to say, protein self-splicing achieves a rearrangement of the protein structure. A split intein is a structural type of intein. Structurally, its N-terminal and C-terminal regions are separated from each other. When the two fragments containing the N-terminal and C-terminal regions of the intein are connected, the exteins at both ends can be spliced together according to the standard intein splicing pathway.

Most inteins consist of terminal splicing regions and a central endonuclease domain or linker domain. Inteins can be divided into three types: canonical inteins, miniinteins, and split inteins. Both canonical and miniinteins contain terminal splicing domains and a central region. The difference between them is that the central region of a canonical intein is an endonuclease domain, while that of a miniintein is a linker domain. The lengths of the linker domains vary between miniinteins. The central region of a split intein is disconnected at a specific site, forming an N-terminal fragment and a C-terminal fragment, respectively, located on two genes distant from each other in the genome. During the translation and maturation of the precursor protein, these two intein fragments recognize each other and restore endonuclease activity, mediating protein trans-splicing. Herein, a dual AAV vector system can be used to deliver a nucleic acid containing a nucleic acid encoding a split intein.

Typically, inteins are composed of 10 modules, starting from the N-terminus: A, N2, B, N4, C, D, E, H, F, and G. A, N2, B, and N4 are the N-terminal splicing regions, F and G are the C-terminal splicing regions, and C, D, E, and H are the homing endonuclease active regions or linker domains. The motifs involved in intein splicing within the A, B, F, and G modules contain highly conserved amino acid residues at the splice sites, essential for the affinity displacement reaction during intein splicing. The motifs within the A module of the intein typically contain amino acids with hydroxyl or sulfhydryl groups, such as Ser and Cys. The motif within the B module contains the highly conserved amino acid sequence Thr-X-X-His, which is also found in serine proteases. The conserved amino acid residues within the motif involved in the splicing reaction within the G module are Asn, Ser, Cys, Thr, and His. In addition, the conserved sites in the motif of the A module (such as Ser, Cys) can be replaced by Ala, Gln or Pro in some inteins, and the same is true for the motif of the G module.

In this article, "hearing loss" refers to hearing below the normal hearing threshold level determined by audiometry, including mild, moderate, severe and profound hearing loss, and deafness. Hearing loss can be described by a percentage of hearing loss, for example, 30%, 60%, 80% or even 100% hearing loss, or by a grading description of hearing loss. The hearing loss can be hearing loss caused by or associated with a gene defect, such as congenital deafness and pre-lingual deafness caused by genetic factors, or hearing loss associated with genetic factors, induced by environmental factors (for example, aging, noise, drugs or infection-induced). Hearing loss can be asymptomatic (that is, there is no associated visible outer ear or other organ abnormality) or symptomatic. In some embodiments, hearing loss is sensorineural hearing loss.

As used herein, a "hearing loss-associated gene" refers to a gene whose variation can cause hearing loss or susceptibility to hearing loss by altering the ability of the inner ear to function normally. In this article, such genes are also referred to as "hearing loss genes." More than 100 genes have been identified as being associated with hearing loss (see Hereditary Hearing Loss Homepage, https://hereditaryhearingloss.org/, which lists the gene locations and identification data for currently known single-gene asymptomatic hearing loss). In the case of causing susceptibility to hearing loss, individuals carrying the hearing loss gene variation may show a greater susceptibility to hearing loss due to environmental factors, such as aging, noise, drugs, or infections, relative to healthy individuals.

As used herein, "cochlear inner hair cells" refer to isolated or in vitro cochlear inner hair cells, cell lines, or cell populations derived from a mammal, or inner hair cells in the cochlea of a mammal.

As used herein, "cochlear outer hair cells" refer to cochlear outer hair cells, cell lines, or cell populations isolated from or in vitro of a mammal, or outer hair cells in the cochlea of a mammal.

As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been artificially synthesized or separated from at least some components of its natural environment. For example, an isolated nucleic acid can be part of a larger nucleic acid, or part of a vector or composition of matter, or can be contained within a cell and still be "isolated" provided that the larger nucleic acid, vector, composition of matter, or specific cell is not the natural environment of the nucleic acid.

As used herein, the term "operably linked," also referred to as "effectively linked" or "functionally linked," means that two or more polynucleotide (e.g., DNA) segments are in a relationship that allows them to function in the intended manner. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in a suitable host cell or other expression system. Generally, promoters that are operably linked to a transcribable sequence are contiguous with the transcribable sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences (e.g., enhancers) do not need to be physically adjacent to or in close proximity to the coding sequence whose transcription they enhance.

The term "full-length MYO7A protein" refers to a MYO7A protein produced by operatively linking the N-terminal portion of the MYO7A protein and the C-terminal portion of the MYO7A protein expressed in the dual vector system of the present invention. In some embodiments, the full-length MYO7A protein is a wild-type or functional human MYO7A protein. The amino acid sequences of wild-type and functional hMYO7A proteins and the polynucleotide sequences encoding them are known in the art (see, for example, GenBank accession numbers NP_000251 and U39226.1). In some specific embodiments, the full-length MYO7A protein is the full-length MYO7A protein shown in SEQ ID NO. 2, or a functional derivative or functional fragment thereof.

As used herein, the term "adeno-associated virus (AAV)" is named after its discovery in adenovirus products. AAV is a member of the Parvovirus family, which includes multiple serotypes and has a single-stranded DNA genome.

AAV is a dependent virus that requires other viruses such as adenovirus, herpes simplex virus, human papillomavirus, or auxiliary factors to provide auxiliary functional proteins for replication.

The first AAV virus isolated was serotype 2 (AAV2). The AAV2 genome is approximately 4.7 kb long, flanked by 145-bp inverted terminal repeats (ITRs) at each end, forming a palindromic hairpin structure. The genome contains two large open reading frames (ORFs), encoding the rep and cap genes, respectively.

ITRs are cis-acting elements of the AAV vector genome, playing a crucial role in AAV integration, rescue, replication, and genome packaging. The ITR sequence contains the Rep binding site (RBS) and the terminal resolution site (TRs), which are recognized by the Rep protein and produce a cleavage at the TRs. The ITR sequence also forms a unique "T"-shaped secondary structure, which plays a crucial role in the AAV life cycle.

The rest of the AAV2 genome can be divided into two functional regions, the rep gene region and the cap gene region.

The rep gene region encodes four Rep proteins: Rep78, Rep68, Rep52, and Rep40. Rep proteins play an important role in the replication, integration, rescue, and packaging of AAV viruses. Rep78 and Rep68 specifically bind to the terminal melting sites trs and GAGY repeat motifs in the ITR, initiating the replication of the AAV genome from single-stranded to double-stranded. The trs and GAGC repeat motifs and/or GAGY repeat motifs in the ITR are the center of AAV genome replication. Therefore, although the ITR sequences are different in various serotypes of AAV viruses, they can all form a hairpin structure and contain Rep binding sites. There is a p19 promoter at position 19 on the AAV2 genome map, which initiates the expression of Rep52 and Rep40, respectively. Rep52 and Rep40 have ATP-dependent DNA helicase activity but do not have the function of binding to DNA.

The cap gene encodes the AAV capsid proteins VP1, VP2, and VP3. VP3 has the smallest molecular weight but is the most abundant. In mature AAV particles, the ratio of VP1, VP2, and VP3 is approximately 1:1:10. VP1 is essential for the formation of infectious AAV; VP2 facilitates VP3 entry into the cell nucleus; and VP3 is the primary protein in AAV particles.

As used herein, the term "AAV vector" refers to an efficient exogenous gene transfer tool, i.e., an AAV vector, that has been transformed from wild-type AAV virus as people gain a better understanding of the AAV virus life cycle and its related molecular biological mechanisms. The modified AAV vector genome only contains the ITR sequence of the AAV virus and the exogenous sequence to be transferred. The Rep and Cap proteins required for AAV virus packaging are provided in trans by other exogenous plasmids, thereby reducing the possible harm caused by packaging the rep and cap genes into the AAV vector. Furthermore, the AAV virus itself is not pathogenic, which makes the AAV vector recognized as one of the safest viral vectors.

There are many AAV virus serotypes, and different serotypes have different tissue infection tropisms. Therefore, the use of AAV vectors can transport exogenous genes to specific organs and tissues.

The existing technology has a relatively mature packaging system for AAV vectors, which facilitates the large-scale production of AAV vectors.

The term "vector genome (vg)" refers to the nucleic acid sequence that is packaged within the rAAV capsid to form the rAAV vector.

As used herein, "individual" and "subject" are used interchangeably to refer to mammals. Examples of mammals include, but are not limited to, humans, non-human primates (e.g., cynomolgus monkeys, rhesus monkeys), rodents, and other mammals, such as cattle, pigs, horses, and dogs. As used herein, mammals include individuals at all stages of development, including embryonic and fetal stages.

As used herein, the term "treatment" refers to clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. The term "treatment" also encompasses modification or improvement of at least one physical parameter, including physical parameters that may not be discernible by the patient.

As used herein, the term "prevention" refers to preventing or delaying the onset or development or progression of a disease or condition. As used herein, "prevention" generally refers to hospital intervention performed before at least one symptom of a disease occurs.

Various aspects of the present invention are described below.

II. Dual Vector System

The present invention utilizes protein trans-splicing, that is, the N-terminal and C-terminal CDS of MYO7A are constructed into two different plasmids for expression, and the complete full-length protein is assembled through intein splicing. The N-terminal plasmid adds the N-terminal coding sequence of the intein at the 3' end of the MYO7A N-terminal CDS sequence, such as the nucleic acid sequence encoding the Rm-N-intein sequence (SEQ ID NO: 23), and the first amino acid residue of the Rm-N-intein sequence contains Cys. The C-terminal plasmid adds the C-terminal coding sequence of the intein at the 5' end of the MYO7A C-terminal CDS sequence, such as the nucleic acid sequence encoding the Rm-C-intein (SEQ ID NO: 24), and the terminal sequence of the Rm-C-intein contains His and Asn, and the first amino acid residue of the MYO7A C-terminal CDS sequence is Cys, Ser, and Thr for intein splicing.

The present invention provides a dual vector system for expressing MYO7A protein, which comprises a first nucleic acid vector and a second nucleic acid vector, wherein

The first nucleic acid vector comprises a first nucleotide sequence; and the second nucleic acid vector comprises a second nucleotide sequence;

The first nucleotide sequence comprises an expression cassette inserted between two first ITR sequences;

The second nucleotide sequence comprises an expression cassette inserted between two second ITR sequences;

The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A, an N-terminal coding sequence of an intein, and polyA;

The expression cassette of the second nucleotide sequence comprises a promoter, a C-terminal coding sequence of an intein, a C-terminal coding sequence of MYO7A and polyA.

Inteins

Inteins can splice proteins and exert their effects by covalently linking two different proteins after or during protein translation. The earliest inteins were discovered in fungi. Comparison and analysis of intein sequences predicts that there are over 600 intein genes present in viruses, bacteria, archaea, and eukaryotic microorganisms. Most inteins are complete proteins, but a small number of inteins have separate N- and C-termini. Inteins are linked to a portion of a protein at each end, and then reassemble after translation to produce the complete protein through nucleophilic chemical reactions and conformational changes.

In the present invention, preferably, the intein is separated from the N-terminus and the C-terminus. The intein can be derived from MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, NpuDnaE, AvaDnaE, CraDnaE, CspDnaE, CwaDnaE, MchtDnaE, OliDnaE, TerDnaE, gp41-1, gp41-8, IMPDH-1 or RmaDnaB.

In some embodiments, the intein is an RmaDnaB intein, e.g., having an N-terminal portion of the RmaDnaB intein set forth in SEQ ID NO: 23 and a C-terminal portion of the RmaDnaB intein set forth in SEQ ID NO: 24. In some embodiments, the intein is an NpuDnaE intein, e.g., having an N-terminal portion of the NpuDnaE intein set forth in SEQ ID NO: 52 and a C-terminal portion of the NpuDnaE intein set forth in SEQ ID NO: 54.

MYO7A protein

In some embodiments, the MYO7A protein comprises or consists of the amino acid sequence of SEQ ID No 2.

In some embodiments, a cleavage site is provided in the amino acid sequence of the MYO7A protein, dividing the MYO7A protein into the N-terminal portion of the MYO7A protein (also referred to herein as the "N-terminus of MYO7A") and the C-terminal portion of the MYO7A protein (also referred to herein as the "C-terminus of MYO7A"). The N-terminus of MYO7A is the sequence from the N-terminus of the MYO7A amino acid sequence to the cleavage site, and the C-terminus of MYO7A is the sequence from the amino acid residue immediately adjacent to the cleavage site to the C-terminus of the MYO7A amino acid sequence. The N-terminus of MYO7A is linked and fused to the N-terminus of the intein, and the C-terminus of the intein is linked and fused to the C-terminus of MYO7A. There are various options for the cleavage site of MYO7A. Table 1 below lists the locations of some of these cleavage sites on the MYO7A protein and the corresponding N-terminal and C-terminal portions of MYO7A.

Table 1. Location of cleavage sites on the MYO7A protein and the corresponding N-terminal and C-terminal portions of MYO7A

Table 1 continued

Table 1 continued

In some embodiments, the MYO7A protein is cleaved at one or more of the following groups of amino acid residues to form the N-terminal portion of the MYO7A protein and the C-terminal portion of the MYO7A protein: 1043; 1058; 1061; 1064; 1071; 1076; 1081; 1104; 1105; 1114; 1119; 1122; or 1126, wherein the amino acid position is relative to the position of SEQ ID No: 2.

In some embodiments, the MYO7A protein is cleaved into an N-terminal portion of the MYO7A protein and a C-terminal portion of the MYO7A protein selected from any one of the following groups:

(1)1-1043aa+1044-2215aa;

(2)1-1058aa+1059-2215aa;

(3)1-1061aa+1062-2215aa;

(4)1-1064aa+1065-2215aa;

(5)1-1071aa+1072-2215aa;

(6)1-1076aa+1077-2215aa;

(7)1-1081aa+1082-2215aa;

(8)1-1104aa+1105-2215aa;

(9)1-1105aa+1106-2215aa;

(10)1-1114aa+1115-2215aa;

(11)1-1119aa+1120-2215aa;

(12)1-1122aa+11123-2215aa; or

(13)1-1126aa+1127-2215aa, optionally wherein the amino acid position is relative to the position of SEQ ID No:2.

In some embodiments, the MYO7A protein is cleaved into an N-terminal portion of the MYO7A protein of 1-1061 aa and a C-terminal portion of the MYO7A protein of 1062-2215 aa. In some embodiments, the N-terminal portion of the MYO7A protein is represented by SEQ ID No: 4 and the C-terminal portion of the MYO7A protein is represented by SEQ ID No: 6.

In some embodiments, the MYO7A protein is cleaved into an N-terminal portion of the MYO7A protein ranging from 1 to 1064 aa and a C-terminal portion of the MYO7A protein ranging from 1065 to 2215 aa. In some embodiments, the N-terminal portion of the MYO7A protein is represented by SEQ ID No: 8 and the C-terminal portion of the MYO7A protein is represented by SEQ ID No: 10.

In some embodiments, the MYO7A protein is cleaved into an N-terminal portion of the MYO7A protein of 1-1104 aa and a C-terminal portion of the MYO7A protein of 1105-2215 aa. In some embodiments, the N-terminal portion of the MYO7A protein is represented by SEQ ID No: 12 and the C-terminal portion of the MYO7A protein is represented by SEQ ID No: 14.

In some embodiments, the MYO7A protein is cleaved into an N-terminal portion of the MYO7A protein of 1-1105 aa and a C-terminal portion of the MYO7A protein of 1115-2215 aa. In some embodiments, the N-terminal portion of the MYO7A protein is SEQ ID No: 16 and the C-terminal portion of the MYO7A protein is SEQ ID No: 18.

In some embodiments, the MYO7A protein is cleaved into an N-terminal portion of the MYO7A protein of 1-1119 aa and a C-terminal portion of the MYO7A protein of 1120-2215 aa. In some embodiments, the N-terminal portion of the MYO7A protein is represented by SEQ ID No: 20 and the C-terminal portion of the MYO7A protein is represented by SEQ ID No: 22.

vector plasmid

The vector plasmid of the present invention can be any plasmid that can replicate in a host cell and express a corresponding polypeptide.

In some embodiments, the vector plasmid comprises two ITR sequences, namely a 5' inverted terminal repeat (5' ITR) sequence and a 3' inverted terminal repeat (3' ITR) sequence.

In some embodiments, in the dual-vector system for expressing MYO7A protein of the present invention, the first nucleotide sequence is inserted into a plasmid comprising two first ITR sequences, and the second nucleotide sequence is inserted into a plasmid comprising two second ITR sequences, for example, the plasmid comprising two first ITR sequences and the plasmid comprising two second ITR sequences are the same or different, for example, the plasmid is pAAV, pAAV-CMV, pX601, pX551 or pAAV-MCS plasmid.

Dual vector system

The present invention provides a dual vector system comprising a first nucleic acid vector and a second nucleic acid vector, wherein:

The first nucleic acid vector comprises, in 5'-3' direction: a 5' inverted terminal repeat (5'ITR) sequence, a nucleic acid sequence encoding the N-terminal portion of the MYO7A protein, a nucleic acid sequence encoding the N-terminal portion of the intein, and a 3' inverted terminal repeat (3'ITR) sequence;

The second nucleic acid vector comprises, in the 5'-3' direction: a 5'ITR sequence, a nucleic acid sequence encoding the C-terminal portion of an intein, a nucleic acid sequence encoding the C-terminal portion of a MYO7A protein, and a 3'ITR sequence, and

Optionally, after the first nucleic acid vector and the second nucleic acid vector are introduced into a host cell, the N-terminal portion of the MYO7A protein and the C-terminal portion of the MYO7A protein are operably linked to produce the MYO7A protein.

In some embodiments, the present invention provides a two-vector system comprising a first nucleic acid vector and a second nucleic acid vector, wherein:

The first nucleic acid vector comprises, in the 5'-3' direction: a 5' inverted terminal repeat (5'ITR) sequence, a nucleic acid sequence encoding the N-terminal portion of the MYO7A protein, a nucleic acid sequence encoding the N-terminal portion of the intein, and a 3' inverted terminal repeat (3'ITR) sequence;

The second nucleic acid vector comprises, in 5'-3' direction: a 5' ITR sequence, a nucleic acid sequence encoding the C-terminal portion of an intein, a nucleic acid sequence encoding the C-terminal portion of a MYO7A protein, and a 3' ITR sequence, and

The MYO7A protein has a MYO7A cleavage site in its amino acid sequence, for example, the MYO7A protein has an amino acid sequence as shown in SEQ ID NO: 2 or a functional fragment thereof, for example, an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2;

The N-terminal portion of the MYO7A protein is the sequence from the N-terminus of the MYO7A amino acid sequence to the MYO7A cleavage site;

The C-terminal portion of the MYO7A protein is a sequence from the amino acid after the MYO7A cleavage site to the C-terminus of the MYO7A amino acid sequence;

Optionally, after the first nucleic acid vector and the second nucleic acid vector are introduced into cells, the N-terminal portion of the MYO7A protein and the C-terminal portion of the MYO7A protein are operably linked to produce a full-length MYO7A protein.

In some embodiments, the nucleotide sequences of the ITRs in the dual vector system are derived from the same AAV serotype or different AAV serotypes, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotypes. In some embodiments, the 5'-ITR and 3'-ITR of the first nucleic acid vector and the 5'-ITR and 3'-ITR of the second nucleic acid vector are derived from the same AAV serotype. In some embodiments, the 5'-ITR and 3'-ITR of the first nucleic acid vector and the 5'-ITR and 3'-ITR of the second nucleic acid vector are derived from different AAV serotypes.

In some embodiments, a tissue-specific promoter is used in a two-vector system, for example, a promoter that mediates expression in the ear, such as the synapsin promoter or the GFAP promoter.

In some embodiments, any one of the following promoters is used in the binary vector system: cytomegalovirus (CMV) promoter, SV40 promoter, Rous sarcoma virus (RSV) promoter, CAG promoter, chimeric CMV/chicken beta actin (CBA) promoter, truncated CBA (smCBA) promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or promoters of Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, OTOF and STRC encoding genes. In some embodiments, the promoter is a CMV promoter.

The dual vector system of the present invention may also include one or more additional regulatory sequences that can function before or after transcription. The regulatory sequences may be part of the native transgenic locus or may be heterologous regulatory sequences. A portion of the 5'UTR or 3'UTR of the native transgenic transcript may be included in the dual vector system of the present invention.

The regulatory sequence may be any sequence that promotes transgene expression, i.e., serves to increase transcript expression, improve nuclear export of mRNA, or enhance its stability. Such regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sequences.

Enhancers are cis-regulatory elements that affect the transcription of genes on the same molecule of DNA. Enhancers can be located upstream, downstream, within introns, or even relatively far from the genes they regulate.

The preferred post-transcriptional regulatory element used in the dual vector system of the present invention is the woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof. Compared with an AAV vector without WPRE or a variant thereof, an AAV vector containing WPRE or a variant thereof increases the expression of MYO7A protein.

In one embodiment, the dual vector system of the present invention comprises a WPRE nucleotide sequence as set forth in SEQ ID NO:28. In another embodiment, the dual vector system of the present invention comprises a post-transcriptional regulatory element having a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the WPRE nucleotide sequence as set forth in SEQ ID NO:28, wherein the nucleotide sequence substantially retains the functional activity of the post-transcriptional regulatory element as set forth in SEQ ID NO:28, for example, a truncated variant of WPRE. Reducing the size of the AAV genome enables increased flexibility in introducing other regulatory elements into the vector in addition to transgenes. In one embodiment, the truncated variant of WPRE has the WPRE3 nucleotide sequence as set forth in SEQ ID NO:31.

In one embodiment, the dual vector system of the present invention comprises a polyadenylation sequence, for example, a bovine growth hormone polyadenylation sequence, an SV40 polyadenylation sequence, and/or an SV40 late polyadenylation sequence. In one embodiment, the dual vector system of the present invention comprises an SV40 polyadenylation sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 29. In one embodiment, the dual vector system of the present invention comprises an SV40 late polyadenylation sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 32.

In some embodiments, the dual vector system of the present invention comprises a combination of a WPRE nucleotide sequence and an SV40 polyadenylation sequence. For example, the dual vector system of the present invention has the nucleotide sequence set forth in SEQ ID NO: 27, or a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27. The combination of the WPRE nucleotide sequence and the SV40 polyadenylation sequence allows for high-level expression of a transgene.

In some embodiments, the dual-vector system of the present invention comprises a combination of a WPRE3 nucleotide sequence and an SV40 late polyadenylation sequence. For example, the dual-vector system of the present invention has the nucleotide sequence set forth in SEQ ID NO: 30, or a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30. The combination of the WPRE3 nucleotide sequence and the SV40 late polyadenylation sequence (also referred to as "W3SL") can efficiently express larger exogenous genes while occupying less AAV packaging capacity.

The present invention uses the dual-vector system to deliver the MYO7A protein gene in two parts to inner ear cells, inner hair cells, or outer hair cells, where the N-terminal and C-terminal parts of the MYO7A protein expressed undergo trans-splicing to form the full-length MYO7A protein. The present invention demonstrates that the dual-vector system for expressing the MYO7A protein can effectively transduce the targeted inner ear cells, inner hair cells, or outer hair cells, producing the MYO7A protein in these cells and durably restoring hearing loss caused by MYO7A gene knockout.

In a preferred embodiment, the dual vector system of the present invention allows for the expression of homologous polypeptides having an amino acid sequence that is at least 70% identical and/or similar to SEQ ID NO: 2. More preferably, the homologous sequence is at least 75%, even more preferably at least 80%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 99%, or at least 99% identical and/or similar to SEQ ID NO: 2. When the homologous polypeptide is much shorter than SEQ ID NO: 2, a local alignment may be considered.

In another embodiment, the dual vector system of the present invention can allow for the expression of functional fragments of the MYO7A protein polypeptide. The term "functional fragment" herein refers to any fragment that retains at least one biological function of the target MYO7A protein polypeptide.

The full-length MYO7A protein can be obtained by transforming host cells using the dual vector system of the present invention. In some embodiments, the host cells are selected from Hela-S3 cells, HEK-293 cells, HEK-293T cells, HEK-293FT cells, A549 cells, and Sf9 cells.

III. Uses of the Dual Vector System

The dual-vector system of the present invention is used to administer to patients suffering from Myo7a mutation-induced hearing loss. "Patient suffering from Myo7a mutation-induced hearing loss" refers to a patient, particularly a human patient, who is believed to have (or has been diagnosed with) a mutation in the constitutive Myo7a gene that triggers abnormal expression, abnormal function, or both of the MYO7A protein. In some embodiments, the Myo7a mutation-induced hearing loss is USH1B, autosomal recessive hearing loss DFNB2, or autosomal dominant hearing loss DFNA11.

In some embodiments, the dual vector system of the present invention is a dual AAV vector system. In some embodiments, the first AAV vector and the second AAV vector in the dual AAV vector system are vectors each having a capsid of the same or different AAV origin, for example, the first AAV vector and the second AAV vector in the dual AAV vector system are vectors each having an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Anc80 capsid or an AAV vector with a chimeric capsid, in particular an AAV vector with an AAV-Anc80 capsid. Preferably, the synthetic adeno-associated virus vector Anc80L65 is used, which has been shown to have the highest transduction efficiency of inner ear hair cells reported to date (Suzuki et al., Sci. Rep. 7: 45524 (2017)).

After administration, the dual-vector system of the present invention can trigger the expression of the full-length MYO7A protein polypeptide, or a functional fragment thereof, in inner ear cells, inner hair cells, or outer hair cells.

The patients to whom the dual vector system of the present invention is administered are preferably newborn human infants, usually less than 6 months old, or even less than 3 months old (if they were diagnosed with DFNB16 deafness in childhood). These human infants are more preferably between 3 months and 1 year old.

The two-vector system of the present invention can also be administered to, for example, infants (2-6 years), children (6-12 years), adolescents (12-18 years), or adults (18 years and older).

As used herein, the term "treating" is intended to mean administering a therapeutically effective amount of the dual-vector system of the present invention to a patient suffering from DFNB16 deafness to partially or completely restore the patient's hearing. Restoration can be assessed by testing auditory brainstem responses (ABRs) using electrophysiological equipment. "Treatment of Myo7a mutation-induced hearing loss" specifically refers to complete restoration of hearing function. The term "preventing" refers to reducing or delaying hearing loss within the auditory frequency range.

Example

Example 1: Selecting intein cleavage sites in the amino acid sequence of MYO7A protein

Figure 1 shows a schematic diagram of intein-mediated full-length extein expression. An intein cleavage site is set within the amino acid sequence of the MYO7A protein. The N-terminal coding sequence of MYO7A is the nucleotide coding sequence from the N-terminus of the MYO7A amino acid sequence to the cleavage site, and the C-terminal coding sequence of MYO7A is the nucleotide coding sequence from the amino acid immediately following the cleavage site to the C-terminus of the MYO7A amino acid sequence. The N-terminal sequence of MYO7A is fused to the N-terminal sequence of the intein, and the C-terminal sequence of the intein is fused to the C-terminal sequence of MYO7A. Numerous cleavage sites can be selected within the amino acid sequence of the MYO7A protein. Table 2 lists 13 cleavage site schemes and the corresponding MYO7A N-terminal and C-terminal amino acid sequences. The goal is to identify cleavage sites with improved efficacy for Myo7A gene therapy. Figure 2 shows a schematic diagram of the screening process for Myo7A cleavage sites through intein splicing.

Table 2 Exemplary cleavage sites of the MYO7A protein shown in SEQ ID NO: 2

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, the 1043rd amino acid residue is used as the cleavage site (S1).

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, the 1058th amino acid residue is used as the cleavage site (S2).

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, amino acid residue 1061 is used as the cleavage site (S3). At the cleavage site S3, SEQ ID NO:3 and SEQ ID NO:4 are the nucleotide sequence and amino acid sequence of the N-terminus of MYO7A, respectively, and SEQ ID NO:5 and SEQ ID NO:6 are the nucleotide sequence and amino acid sequence of the C-terminus of MYO7A, respectively.

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, amino acid residue 1064 is used as the cleavage site (S4). At the cleavage site S4, SEQ ID NO:7 and SEQ ID NO:8 are the nucleotide sequence and amino acid sequence of the N-terminus of MYO7A, respectively, and SEQ ID NO:9 and SEQ ID NO:10 are the nucleotide sequence and amino acid sequence of the C-terminus of MYO7A, respectively.

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, the 1071st amino acid residue is used as the cleavage site (S5).

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, the 1076th amino acid residue is used as the cleavage site (S6).

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, the 1081st amino acid residue is used as the cleavage site (S7).

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, amino acid residue 1104 is used as the cleavage site (S8). At the cleavage site S8, SEQ ID NO:11 and SEQ ID NO:12 are the nucleotide sequence and amino acid sequence of the N-terminus of MYO7A, respectively, and SEQ ID NO:13 and SEQ ID NO:14 are the nucleotide sequence and amino acid sequence of the C-terminus of MYO7A, respectively.

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, amino acid residue 1105 is used as the cleavage site (S9). At the cleavage site S9, SEQ ID NO:15 and SEQ ID NO:16 are the nucleotide sequence and amino acid sequence of the N-terminus of MYO7A, respectively, and SEQ ID NO:17 and SEQ ID NO:18 are the nucleotide sequence and amino acid sequence of the C-terminus of MYO7A, respectively.

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, the 1114th amino acid residue is used as the cleavage site (S10).

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, amino acid residue 1119 is used as the cleavage site (S11). At the cleavage site S11, SEQ ID NO:19 and SEQ ID NO:20 are the nucleotide sequence and amino acid sequence of the N-terminus of MYO7A, respectively, and SEQ ID NO:21 and SEQ ID NO:22 are the nucleotide sequence and amino acid sequence of the C-terminus of MYO7A, respectively.

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, the 1122nd amino acid residue is used as the cleavage site (S12).

In the amino acid sequence of the MYO7A protein shown in SEQ ID NO:2, the 1126th amino acid residue is used as the cleavage site (S13).

Example 2. Construction of a dual-vector system for expressing MYO7A protein using a plasmid containing ITR sequences

Using the pAAV-CMV-EGFP-WPRE-SV40 plasmid (synthesized by Nanjing GenScript, Figure 3) as the plasmid backbone, a first vector plasmid expressing the N-terminus of the MYO7A protein and a second vector plasmid expressing the C-terminus of the MYO7A protein were constructed. The constructed plasmid elements are shown in the upper and lower panels of Figure 2, respectively. The second vector plasmid was connected to the end of the coding sequence (CDS) expressing the C-terminus of the MYO7A protein (HA tag sequence YPYDVPDYA (SEQ ID NO. 25) for verifying in vitro expression, thereby obtaining a dual-vector system.

Specifically, the first vector plasmid and the second vector plasmid in the dual-vector system are obtained by modifying the pAAV-CMV-EGFP-WPRE-SV40 plasmid backbone shown in Figure 3. After expression, the first vector plasmid and the second vector plasmid achieve expression of the MYO7A full-length protein by protein trans-splicing. The first vector plasmid is connected to the sequence encoding the N-terminal fragment of the intein (Rm-N, SEQ ID NO: 23) at the 3' end of the sequence encoding the Myo7a-N sequence (i.e., the sequence of the N-terminal part of Myo7a), and the second vector plasmid is connected to the sequence encoding the Myo7a-C sequence (i.e., the sequence of the C-terminal part of Myo7a) at the 3' end of the sequence encoding the C-terminal fragment of the intein (Rm-C sequence, SEQ ID NO: 24), and the end of the Myo7a-C sequence is connected to an HA tag.

The EGFP reporter gene sequence and other sequences in the pAAV-CMV-EGFP-WPRE-SV40 plasmid were replaced with a sequence encoding the Myo7a-N sequence and the N-terminal fragment of the intein (Rm-N, SEQ ID NO: 23) using EcoRI and EcoRV enzymes to obtain the first vector plasmid. The EGFP reporter gene sequence in the pAAV-CMV-EGFP-WPRE-SV40 plasmid was replaced with a sequence encoding the C-terminal fragment of the intein (Rm-C sequence, SEQ ID NO: 24) and the Myo7a-C sequence using EcoRI and EcoRV enzymes to obtain the second vector plasmid. The synthesis of these sequences and the construction of these vectors were commissioned to Nanjing GenScript Biotechnology Co., Ltd.

Thirteen pairs of plasmids corresponding to the cleavage sites in Table 2 were obtained for subsequent examples and were named

(1) pAAV-CMV-MYO7A-N-S1-Rm-N-intein plasmid; and pAAV-CMV-Rm-C-intein-MYO7A-C-S1 plasmid;

(2) pAAV-CMV-MYO7A-N-S2-Rm-N-intein plasmid; and pAAV-CMV-Rm-C-intein-MYO7A-C-S2 plasmid;

(3) pAAV-CMV-MYO7A-N-S3-Rm-N-intein plasmid (plasmid map is shown in FIG4 ); and pAAV-CMV-Rm-C-intein-MYO7A-C-S3 plasmid (plasmid map is shown in FIG5 );

(4) pAAV-CMV-MYO7A-N-S4-Rm-N-intein plasmid (plasmid map is shown in FIG6 ); and pAAV-CMV-Rm-C-intein-MYO7A-C-S4 plasmid (plasmid map is shown in FIG7 );

(5) pAAV-CMV-MYO7A-N-S5-Rm-N-intein plasmid; and pAAV-CMV-Rm-C-intein-MYO7A-C-S5 plasmid;

(6) pAAV-CMV-MYO7A-N-S6-Rm-N-intein plasmid; and pAAV-CMV-Rm-C-intein-MYO7A-C-S6 plasmid;

(7) pAAV-CMV-MYO7A-N-S7-Rm-N-intein plasmid; and pAAV-CMV-Rm-C-intein-MYO7A-C-S7 plasmid;

(8) pAAV-CMV-MYO7A-N-S8-Rm-N-intein plasmid (plasmid map is shown in FIG8 ); and pAAV-CMV-Rm-C-intein-MYO7A-C-S8 plasmid (plasmid map is shown in FIG9 );

(9) pAAV-CMV-MYO7A-N-S9-Rm-N-intein plasmid (plasmid map is shown in FIG10 ); and pAAV-CMV-Rm-C-intein-MYO7A-C-S9 plasmid (plasmid map is shown in FIG11 );

(10) pAAV-CMV-MYO7A-N-S10-Rm-N-intein plasmid; and pAAV-CMV-Rm-C-intein-MYO7A-C-S10 plasmid;

(11) pAAV-CMV-MYO7A-N-S11-Rm-N-intein plasmid (plasmid map is shown in FIG12 ); and pAAV-CMV-Rm-C-intein-MYO7A-C-S11 plasmid (plasmid map is shown in FIG13 );

(12) pAAV-CMV-MYO7A-N-S12-Rm-N-intein plasmid; and pAAV-CMV-Rm-C-intein-MYO7A-C-S12 plasmid;

(13) pAAV-CMV-MYO7A-N-S13-Rm-N-intein plasmid; and pAAV-CMV-Rm-C-intein-MYO7A-C-S13 plasmid.

The plasmid transcripts all contain a covalently linked MYO7A portion and an Rm intein portion. The plasmid names and transcripts are shown in Table 3 below.

Table 3. Exemplary plasmid names and transcription and translation products of each plasmid

Example 3. Recombination of the constructed plasmid in cells

3.1 Recombination of the binary vector system containing the full-length WPRE in cells

The 13 pairs of plasmids shown in Table 3 obtained in Example 2 were transfected into HEK-293T cells (cells purchased from ATCC) in pairs, and the expression of full-length MYO7A protein was analyzed by Western blotting 48 hours after transfection. The specific experimental method is as follows.

Cell transfection: Inoculate HEK-293T cells (Human Embryonic Kidney 293T cells, hereinafter referred to as "293T cells") to a density of 70-90% and prepare for transfection. Prepare Tube A: 125 μL serum-free DMEM medium + 8 μL Lipofectamine 3000 reagent (Invitrogen, catalog number: L3000015) and mix thoroughly. Prepare Tube B: 125 μL serum-free DMEM medium + 2 μg first vector plasmid + 2 μg second vector plasmid + 8 μL P3000 reagent (Invitrogen, catalog number: L3000015) and mix thoroughly. Add the mixture in Tube B to Tube A, mix gently and thoroughly, and let it stand at room temperature for 10-15 minutes. Tube A contains a mixture of culture medium and Lipofectamine 3000 transfection reagent, while Tube B contains a mixture of culture medium, vector plasmid DNA, and transfection enhancer P3000. The obtained DNA-liposome complex was added to 293T cells for transfection, and the cells were incubated at 37°C in 95% air and 5% _CO2 . After 48 hours of transfection, the cells were harvested by centrifugation.

After cell transfection, the cell pellet harvested by centrifugation was fully resuspended in an appropriate amount of RIPA lysis buffer (Thermo Fisher Scientific, catalog number: 89900) (supplemented with 1% protease inhibitor cocktail (Thermo Fisher Scientific, catalog number: 87786), 1% PMSF), and lysed on ice for 30 minutes. Vortex every 10 minutes during this period to fully resuspend the cell pellet in the lysis buffer. Centrifuge at 12000rpm for 15 minutes to collect the supernatant (do not aspirate the precipitate), use 5X loading buffer (Loading Buffer) to add the sample in proportion, boil at 75℃ for 15 minutes, cool on ice, and after centrifugation, take the supernatant for Western blotting analysis.

Western blotting to detect MYO7A protein expression: Wash the glass plates and secure them flat on a rack, clamping them with the concave surface facing inward. Position the plates symmetrically, front and back. Prepare separating gel and seal with isopropanol. After 0.5 hours, discard the isopropanol and place on its side with a pump to dry. Prepare stacking gel, adding until overflowing, and insert a comb. After 45 minutes, remove the gel plate and attach it to the clamps in the electrophoresis tank. Add running buffer from the center of the tank until it overflows to 1/2 of the tank volume. Carefully remove the comb. Load the sample and perform electrophoresis at a constant voltage of 100 V for 1 hour. Transfer the membrane at a constant current of 300 mA on ice for 90 minutes.

After transfer, remove the PVDF membrane and incubate it with blocking solution (5% skim milk powder, prepared in TBST buffer) at room temperature for 1 hour. Add the primary antibody (HA-Tag Mouse mAb, Cell Signaling Technology, catalog number: 6E2; β-Actin Mouse mAb, Cell Signaling Technology, catalog number: 8H10D10) to the blocked PVDF membrane and incubate it at 4°C overnight. Use HA antibody as the primary antibody to detect the expression of the full-length MYO7A protein, and use β-actin antibody as the primary antibody to detect the level of β-actin, an internal control protein in Western blotting. The protein level of β-actin does not usually change, so it can be used to check whether the sample amount is consistent during Western blotting.

The next day, the incubated PVDF membrane was removed and rinsed three times with 1X TBST for 5 minutes each time. The membrane was incubated with the secondary antibody (HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L), Proteintech, catalog number: SA00001-1) at room temperature for 1 hour and rinsed three times with 1X TBST for 5 minutes each time. The chemiluminescent reagent (ECL) was then added for development in a dark room.

The expression results of target proteins after 13 pairs of plasmids shown in Table 3 were transfected into 293T cells are shown in Figure 14. In Figure 14, lane "Ctrl" represents the protein control without transfection plasmid; lane "FL" represents the full-length protein MYO7A,

Lane “1” indicates the protein expression level of MYO7A after co-transfection of 293T cells with pAAV-CMV-MYO7A-N-S1-Rm-N-intein plasmid and pAAV-CMV-Rm-C-intein-MYO7A-C-S1 plasmid;

Lane "2" represents the protein expression level of MYO7A after co-transfection of 293T cells with pAAV-CMV-MYO7A-N-S2-Rm-N-intein plasmid and pAAV-CMV-Rm-C-intein-MYO7A-C-S2 plasmid; ...

Lane “13” indicates the protein expression level of MYO7A after co-transfection of pAAV-CMV-MYO7A-N-S13-Rm-N-intein plasmid and pAAV-CMV-Rm-C-intein-MYO7A-C-S13 plasmid into 293T cells.

As shown in Figure 14, co-transfection of 293T cells with paired plasmids corresponding to the MYO7A protein intein cleavage sites S3, S4, S8, S9, and S11 resulted in significantly higher expression of full-length MYO7A protein. The MYO7A protein intein cleavage sites S3, S4, S8, S9, and S11 enable efficient recombination of the corresponding paired plasmids in cells, demonstrating promising application prospects and serving as candidate sites for dual AAV vector therapy.

3.2 Recombination of the binary vector system containing truncated WPRE in cells

The intein cleavage site S3 of the MYO7A protein was selected, and a dual-vector system containing a truncated WPRE was constructed, wherein the WPRE+SV40 poly(A) (717bp) nucleotide sequence shown in SEQ ID NO:27 in the pAAV-CMV-EGFP-WPRE-SV40 plasmid backbone shown in Figure 3 was replaced with the WPRE3-SV40 late poly(A) (432bp) nucleotide sequence shown in SEQ ID NO:30, and a first vector plasmid and a second vector plasmid containing a truncated WPRE were constructed.

The first vector plasmid and the second vector plasmid containing the truncated WPRE were co-transfected into HEK-293T cells, and the expression of the full-length MYO7A protein was analyzed by Western blotting 48 hours after transfection. The results were compared with the Western blotting results of HEK-293T cells co-transfected with the first vector plasmid and the second vector plasmid (containing the full-length WPRE) using the intein cleavage site S3 of the MYO7A protein in Example 3.1, and the Western blotting results of HEK-293T cells co-transfected with the first vector plasmid and the second vector plasmid that did not contain the full-length WPRE or its truncated variants or the poly A sequence.

The results are shown in FIG15 . Compared with the control (“Ctrl”) containing neither the full-length WPRE nor its truncated variants nor the poly A sequence, the combination of the full-length WPRE and the truncated WPRE with the poly A sequence increased the expression of the full-length MYO7A protein.

Example 4. Preparation of adeno-associated virus

The 10 plasmids corresponding to the intein cleavage sites S3, S4, S8, S9, and S11 of the MYO7A protein constructed in Example 2 were mixed with pHelper plasmid (synthesized by Nanjing GenScript) and pRC plasmid (containing the PHP.B VP1 gene, partial CDS sequence ID: KU056473.1) at a molar ratio of 1:1:1, and co-transfected into HEK-293T cells using PEI transfection reagent (a total of 1 μg of the three plasmids was added per approximately one million cells). The cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% carbon dioxide incubator at 37°C for 3 days, washed once with PBS buffer, collected, and repeatedly frozen and thawed five times. NaCl was added to a final concentration of 500 mM, and 10,0 The cells were centrifuged at 00g for half an hour, and the supernatant was filtered with a 0.45μm filter membrane. The filtrate was purified and concentrated to obtain an adeno-associated virus with a virus titer of 4.19E+13GC/ml. The adeno-associated virus was named according to the name of the plasmid. For example, the adenovirus packaged with pAAV-CMV-MYO7A-N-S3-Rm-N-intein, pHelper plasmid and pRC plasmid was named pAAV-CMV-MYO7A-N-S3-Rm-N-intein adeno-associated virus (AAV); the adenovirus packaged with pAAV-CMV-Rm-C-intein-MYO7A-C-S3, pHelper plasmid and pRC plasmid was named pAAV-CMV-Rm-C-intein-MYO7A-C-S3 AAV.

Example 5. Expression of MYO7A protein at candidate intein cleavage sites in mice

To verify whether the candidate intein cleavage site can express MYO7A protein in the inner ear hair cells, the following paired adeno-associated viruses constructed in Example 4 were respectively injected into the ears of three P3 (i.e., 3 days after birth) Myo7a (p.Q720X) point mutation KO mice (the mice were commissioned by Saiye Biotechnology Co., Ltd. to construct using the CRISPR/Cas9 method. The Myo7a (p.Q720X) point mutation will lead to the loss of Myo7A protein expression in the inner ear hair cells, thereby leading to hair cell death and hearing loss) through the round window administration for in vivo expression verification.

(i) pAAV-CMV-MYO7A-N-S3-Rm-N-intein AAV; and

pAAV-CMV-Rm-C-intein-MYO7A-C-S3 AAV;

(ii) pAAV-CMV-MYO7A-N-S4-Rm-N-intein AAV; and

pAAV-CMV-Rm-C-intein-MYO7A-C-S4 AAV;

(iii) pAAV-CMV-MYO7A-N-S8-Rm-N-intein AAV; and

pAAV-CMV-Rm-C-intein-MYO7A-C-S8 AAV;

(iv) pAAV-CMV-MYO7A-N-S9-Rm-N-intein AAV; and

pAAV-CMV-Rm-C-intein-MYO7A-C-S9 AAV;

(v) pAAV-CMV-MYO7A-N-S11-Rm-N-intein AAV; and

pAAV-CMV-Rm-C-intein-MYO7A-C-S11 AAV.

First, the virus was injected. Mice were anesthetized hypothermically on ice for 1-2 minutes. After anesthesia, a postauricular incision was made to expose the round window to the visual field. A glass micropipette was used to inject a paired adeno-associated virus from (i) to (v) totaling 2×10 ¹⁰ viral genomes (i.e., 1×10 ¹⁰ viral genomes for each AAV). After injection, the skin wound was sealed with 3M Vethod tissue glue. Two weeks later, the mice were sacrificed, and the cochlea was removed and fixed with 4% paraformaldehyde (PFA). Then, the cochlea was decalcified with EDTA solution until ready for use. Finally, immunofluorescence staining was performed. The cochlea was dissected and the tectorial membrane was removed. The tissue was then blocked with blocking solution for 1 hour and incubated with the primary antibody at 4°C overnight (Proteus BioSciences, 25-6790, anti-Myo7a, 1:1000; Sigma, sab4200545, anti-parvalbumin, 1:1000); washed three times with PBS, and incubated with the secondary antibody at room temperature for 1 hour (Invitrogen, Donkey anti-rabbit Alexa Fluor 488, 1:500; Invitrogen, Goat anti-mouse IgG1 Alexa Fluor 647, 1:500). After incubation, the sections were washed three times with PBS and mounted. The stained cochlear sections were photographed using a Zeiss laser confocal microscope.

The immunofluorescence results in Figure 16 show that the dual AAV system achieves the expression of MYO7A protein in the mouse cochlea. In the figure, "WT" is a wild-type mouse, blue is parvalbumin-labeled hair cells, and green is Myo7a protein. As shown in Figure 16, Myo7a (p.Q720X) mice do not express Myo7a protein when they are not injected with paired AAV viruses. "Myo7a (p.Q720X) + AAV" is Myo7a (p.Q720X) mice injected with paired AAV viruses. After virus injection, Myo7a (p.Q720X) mice expressed Myo7a protein in the inner ear hair cells, and the expression level was similar to that of WT mice.

Example 6 Detection and Analysis of Candidate Intein Cleavage Sites in MYO7A Proteins for the Treatment of Hearing Function in Myo7a Knockout Mice

Myo7a knockout mice are Myo7a (p.Q720X) point mutation KO mice, which were commissioned to Saiye Biotechnology Co., Ltd. to construct. To verify whether the candidate intein cleavage site can effectively protect hearing by expressing MYO7A protein, the paired adeno-associated viruses of Example 5 were injected into the inner ear of P3 mice through the round window for in vivo expression verification. First, the virus was injected. The mice were anesthetized in ice for 1-2 minutes. After anesthesia, a post-auricular incision was made to expose the round window to the field of view, and the virus was injected with a glass micropipette. After the injection, the skin wound was sealed with 3M Vethod tissue glue.

Three weeks later, the auditory brainstem response (ABR) was used to detect the overall auditory function differences of Myo7a knockout mice after treatment with our AAV virus preparation (intein cleavage sites S3, S4, S8, S9, S11). Myo7a knockout mice and wild-type mice of the same litter served as control mice for auditory function analysis.

Using ABR testing, auditory response thresholds, latency, and inter-wave duration were measured. Clicks, particularly those at different frequencies (4 kHz, 8 kHz, 12 kHz, 16 kHz, 24 kHz, and 32 kHz), were used as stimuli to measure the mice's hearing thresholds. This analysis of the mice's hearing sensitivity allowed them to assess overall hearing function, from hair cells to the cerebral cortex. Higher ABR thresholds indicate more severe hearing loss in Myo7a knockout mice. Conversely, lower ABR thresholds indicate greater efficacy of gene therapy.

The audiometry results showed (Figure 17) that adeno-associated virus preparations carrying intein cleavage site S3, intein cleavage site S4, intein cleavage site S8, intein cleavage site S9, and intein cleavage site S11 could effectively protect hearing after injection. Compared with the ABR threshold of control mice, the hearing protection effect after administration of pAAV-CMV-MYO7A-N-S4-Rm-N-intein AAV and pAAV-CMV-Rm-C-intein-MYO7A-C-S4 AAV was that the ABR threshold increased by about 20-40dB; the hearing protection effect after administration of pAAV-CMV-MYO7A-N-S11-Rm-N-intein AAV and pAAV-CMV-Rm-C-intein-MYO7A-C-S11 AAV was the best, and the ABR threshold increased by about 5-20dB compared with the control mice.

While the exemplary embodiments of the present invention have been described above, it should be understood by those skilled in the art that these disclosures are merely exemplary and that various other substitutions, adaptations, and modifications may be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

Exemplary sequences

Nucleotide sequence encoding human myosin VIIa polypeptide SEQ ID NO: 1

Amino acid sequence encoding human myosin VIIa polypeptide SEQ ID NO: 2

Nucleotide sequence encoding amino acid sequence 1-1061 of human myosin VIIa polypeptide SEQ ID NO: 3

Amino acid sequence of human myosin VIIa polypeptide SEQ ID NO: 4

Nucleotide sequence encoding amino acid sequence 1062-2215 of human myosin VIIa polypeptide SEQ ID NO: 5

Amino acid sequence of human myosin VIIa polypeptide at positions 1062-2215 SEQ ID NO: 6

Nucleotide sequence encoding amino acid sequence 1-1064 of human myosin VIIa polypeptide SEQ ID NO: 7

Amino acid sequence of human myosin VIIA polypeptide SEQ ID NO: 8

Nucleotide sequence encoding amino acid sequence 1065-2215 of human myosin VIIa polypeptide SEQ ID NO: 9

Amino acid sequence of human myosin VIIA polypeptide at positions 1065-2215 SEQ ID NO: 10

Nucleotide sequence encoding amino acid sequence 1-1104 of human myosin VIIa polypeptide SEQ ID NO: 11

Amino acid sequence of human myosin VIIA polypeptide SEQ ID NO: 12

Nucleotide sequence encoding amino acid sequence 1105-2215 of human myosin VIIa polypeptide SEQ ID NO: 13

Amino acid sequence of human myosin VIIA polypeptide at positions 1105-2215 SEQ ID NO: 14

Nucleotide sequence encoding amino acid sequence 1-1105 of human myosin VIIa polypeptide SEQ ID NO: 15

Amino acid sequence of human myosin VIIA polypeptide SEQ ID NO: 16

Nucleotide sequence encoding amino acid sequence 1106-2215 of human myosin VIIa polypeptide SEQ ID NO: 17

Amino acid sequence of human myosin VIIA polypeptide at positions 1106-2215 SEQ ID NO: 18

Nucleotide sequence encoding amino acids 1-1119 of human myosin VIIa polypeptide SEQ ID NO: 19

Amino acid sequence of human myosin VIIA polypeptide SEQ ID NO: 20

Nucleotide sequence encoding amino acid sequence 1120-2215 of human myosin VIIa polypeptide SEQ ID NO: 21

Amino acid sequence of human myosin VIIA polypeptide SEQ ID NO: 22

The amino acid sequence of the N-terminal portion of the RmaDnaB intein is SEQ ID NO: 23

The amino acid sequence of the C-terminal portion of the RmaDnaB intein is SEQ ID NO: 24

Amino acid sequence of HA tag SEQ ID NO: 25

The nucleotide sequence of the vector pAAV-CMV-EGFP-WPRE-SV40 plasmid shown in Figure 3 is shown in SEQ ID NO:26.

Nucleotide sequence of WPRE+SV40 poly(A) signal (717 bp): SEQ ID NO: 27

Nucleotide sequence of WPRE (589 bp): SEQ ID NO: 28

Nucleotide sequence of SV40 poly(A) signal (122 bp): SEQ ID NO: 29

Nucleotide sequence of WPRE3-SV40 late poly(A) (432 bp): SEQ ID NO: 30

Nucleotide sequence of WPRE3: SEQ ID NO: 31

Nucleotide sequence of SV40 late poly(A) signal: SEQ ID NO: 32

Sequences in polyA

AATAAA (SEQ ID NO: 33), ATTAAA (SEQ ID NO: 34), AGTAAA (SEQ ID NO: 35), CATAAA (SEQ ID NO: 36), TATAAA (SEQ ID N O: 37), GATAAA (SEQ ID NO: 38), ACTAAA (SEQ ID NO: 39), AATATA (SEQ ID NO: 40), AAGAAA (SEQ ID NO: 41), AATAAT (SEQ ID NO: 42), AAAAAA (SEQ ID NO: 43), AATGAA (SEQ ID NO: 44), AATCAA (SEQ ID NO: 45), AACAAA (SEQ ID NO: 46), AATC AA(SEQ ID NO:47), AATAAC(SEQ ID NO:48), AATAGA(SEQ ID NO:49), AATTAA(SEQ ID NO:50) or AATAAG(SEQ ID NO:51)

The amino acid sequence of the N-terminal portion of the NpuDnaE intein is SEQ ID NO: 52

The nucleotide sequence of the N-terminal portion of the NpuDnaE intein is SEQ ID NO: 53

The amino acid sequence of the C-terminal portion of the NpuDnaE intein is SEQ ID NO: 54

The nucleotide sequence of the C-terminal portion of the NpuDnaE intein is SEQ ID NO: 55

Claims (18)

A dual vector system for expressing MYO7A protein, comprising a first nucleic acid vector and a second nucleic acid vector, wherein The first nucleic acid vector comprises a first nucleotide sequence; and the second nucleic acid vector comprises a second nucleotide sequence; The first nucleotide sequence comprises an expression cassette inserted between two first ITR sequences; The second nucleotide sequence comprises an expression cassette inserted between two second ITR sequences; The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A, an N-terminal coding sequence of an intein, and polyA; The expression cassette of the second nucleotide sequence comprises a promoter, a C-terminal coding sequence of an intein, a C-terminal coding sequence of MYO7A and polyA; and A MYO7A cleavage site is provided in the MYO7A amino acid sequence, for example, the MYO7A amino acid sequence as set forth in SEQ ID NO: 2 or a functional fragment thereof, for example, an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2; The N-terminal coding sequence of MYO7A is a nucleotide coding sequence from the N-terminus of the MYO7A amino acid sequence to the MYO7A cleavage site; the C-terminal coding sequence of MYO7A is a nucleotide coding sequence from the amino acid after the MYO7A cleavage site to the C-terminus of the MYO7A amino acid sequence. The dual-vector system for expressing MYO7A protein according to claim 1, wherein the MYO7A cleavage site is located at the amino acid preceding serine, threonine or cysteine in the MYO7A amino acid sequence. The dual-vector system for expressing MYO7A protein according to claim 1, wherein the promoter of the expression cassette of the first nucleotide sequence or the second nucleotide sequence is selected from CAG promoter, CMV promoter, CBA promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or promoters of genes encoding Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, OTOF and STRC; The polyA of the expression cassette of the first nucleotide sequence or the second nucleotide sequence comprises AATAAA (SEQ ID NO: 33) and variants of AATAAA; the variants of AATAAA comprise ATTAAA (SEQ ID NO: 34), AGTAAA (SEQ ID NO: 35), CATAAA (SEQ ID NO: 36), TATAAA (SEQ ID NO: 37), GATAAA (SEQ ID NO: 38), ACTAAA (SEQ ID NO: 39), AATATA (SEQ ID NO: 40), AAGAAA (SEQ ID NO: 41), AATAAT (SEQ ID NO: 42), AAAAAA (SEQ ID NO: 43), and AAAAAA (SEQ ID NO: 44). NO: 43), AATGAA (SEQ ID NO: 44), AATCAA (SEQ ID NO: 45), AACAAA (SEQ ID NO: 46), AATCAA (SEQ ID NO: 47), AATAAC (SEQ ID NO: 48), AATAGA (SEQ ID NO: 49), AATTAA (SEQ ID NO: 50) or AATAAG (SEQ ID NO: 51); for example, the polyA is a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the poly A signal sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 32; and Each of the two first ITR sequences and the two second ITR sequences is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9. The dual-vector system for expressing MYO7A protein according to claim 1, wherein the expression cassette of the first nucleotide sequence or the second nucleotide sequence further comprises an expression regulatory element and/or a tag element, for example, the expression regulatory element is a woodchuck hepatitis posttranscriptional regulatory element (WPRE) or a variant thereof, preferably a WPRE truncated variant, for example, a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence shown in SEQ ID NO: 28, for example, the nucleotide sequence shown in SEQ ID NO: 31; for example, the tag element is HA. The dual vector system for expressing MYO7A protein according to claim 1, wherein the intein is derived from MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, NpuDnaE, AvaDnaE, CraDnaE, CspDnaE, CwaDnaE, MchtDnaE, OliDnaE, TerDnaE, gp41-1, gp41-8, IMPDH-1 or RmaDnaB, for example For example, the intein is derived from RmaDnaB, for example, the N-terminus of the intein is the N-terminus of the RmaDnaB intein as shown in SEQ ID NO:23, and the C-terminus of the intein is the C-terminus of the RmaDnaB intein as shown in SEQ ID NO:24; or, the intein is derived from NpuDnaE, for example, the N-terminus of the intein is the N-terminus of the NpuDnaE intein as shown in SEQ ID NO:52, and the C-terminus of the intein is the C-terminus of the NpuDnaE intein as shown in SEQ ID NO:54. The dual-vector system for expressing MYO7A protein according to claim 1, wherein the first nucleotide sequence is inserted into a plasmid comprising two first ITR sequences, and the second nucleotide sequence is inserted into a plasmid comprising two second ITR sequences, for example, the plasmid comprising two first ITR sequences and the plasmid comprising two second ITR sequences are the same or different, for example, the plasmid is pAAV, pAAV-CMV, pX601, pX551 or pAAV-MCS plasmid. The dual-vector system for expressing the MYO7A protein according to any one of claims 1 to 6, wherein the MYO7A cleavage site is as shown in Table 1; preferably, amino acid 1043 of the MYO7A amino acid sequence shown in SEQ ID NO: 2 is used as the MYO7A cleavage site, and the RmaDnaB intein is used; the N-terminal coding sequence of MYO7A is connected and fused to the N-terminal coding sequence of the RmaDnaB intein, to construct a first nucleotide sequence, and the pAAV-CMV plasmid is used as a vector; the C-terminal coding sequence of the RmaDnaB intein is connected and fused to the C-terminal coding sequence of MYO7A, to construct a second nucleotide sequence, and the pAAV-CMV plasmid is used as a vector; The 1058th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; The 1061st amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; The 1064th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; The 1071st amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; The 1076th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; The 1081st amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; The 1104th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; The 1105th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; The 1114th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; The 1119th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; Using amino acid 1122 of the MYO7A amino acid sequence shown in SEQ ID NO: 2 as the MYO7A cleavage site and using the RmaDnaB intein; connecting and fusing the N-terminal coding sequence of MYO7A with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, using the pAAV-CMV plasmid as a vector; connecting and fusing the C-terminal coding sequence of the RmaDnaB intein with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, using the pAAV-CMV plasmid as a vector; or The 1126th amino acid of the MYO7A amino acid sequence shown in SEQ ID NO: 2 was used as the MYO7A cleavage site, and the RmaDnaB intein was used; the N-terminal coding sequence of MYO7A was connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; the C-terminal coding sequence of the RmaDnaB intein was connected and fused with the C-terminal coding sequence of MYO7A to construct a second nucleotide sequence, and the pAAV-CMV plasmid was used as a vector; For example, the N-terminal coding sequence of the RmaDnaB intein encodes the N-terminal portion of RmaDnaB shown in SEQ ID NO:23, and the C-terminal coding sequence of the RmaDnaB intein encodes the C-terminal portion of RmaDnaB shown in SEQ ID NO:24. The dual vector system for expressing MYO7A protein according to any one of claims 1 to 7, wherein The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A as shown in SEQ ID No: 4, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, an C-terminal coding sequence of an intein, a C-terminal coding sequence of MYO7A as shown in SEQ ID No: 6, and polyA; The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A as shown in SEQ ID No: 8, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, an C-terminal coding sequence of an intein, a C-terminal coding sequence of MYO7A as shown in SEQ ID No: 10, and polyA; The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A as shown in SEQ ID No: 12, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, a C-terminal coding sequence of an intein, a C-terminal coding sequence of MYO7A as shown in SEQ ID No: 14, and polyA; The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A as shown in SEQ ID No: 16, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, an C-terminal coding sequence of an intein, an C-terminal coding sequence of MYO7A as shown in SEQ ID No: 18, and polyA; or The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of MYO7A as shown in SEQ ID No: 20, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, an C-terminal coding sequence of an intein, an C-terminal coding sequence of MYO7A as shown in SEQ ID No: 22, and polyA. A dual vector system for expressing MYO7A protein according to any one of claims 1 to 8, wherein the expression cassette of the first nucleotide sequence and the expression cassette of the second nucleotide sequence each comprise a combination of a WPRE nucleotide sequence and an SV40 polyadenylation sequence at the N-terminus of the 3’ITR sequence, for example, a nucleotide sequence shown in SEQ ID NO: 27 or a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 27; or a combination of a WPRE3 nucleotide sequence and an SV40 late polyadenylation sequence, for example, a nucleotide sequence shown in SEQ ID NO: 30 or a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 30. An adeno-associated virus packaging vector system, wherein the packaging vector system comprises a dual-vector system for expressing MYO7A protein according to any one of claims 1-9, a vector carrying AAV rep and cap genes, and a helper virus vector, which is packaged into an AAV vector. Preferably, the amino acid sequence of the MYO7A protein is as shown in SEQ ID NO: 2. The adeno-associated virus packaging vector system according to claim 10, wherein the vector carrying the AAV rep and cap genes is selected from AAV1, AAV2, AAV5, AAV8, AAV9, Anc80, PHP.eB, AAV-DJ and AAVrh.10 vectors; and the helper virus vector is a pHelper plasmid. A method for packaging an adeno-associated virus, wherein the adeno-associated virus packaging vector system according to claim 10 or 11 is transferred into a host cell for packaging. The method for packaging adeno-associated virus according to claim 12, wherein the host cell is selected from Hela-S3 cells, HEK-293 cells, HEK-293T cells, HEK-293FT cells, A549 cells and Sf9 cells. An adeno-associated virus obtained by the packaging method according to claim 12 or 13. Use of the dual vector system for expressing MYO7A protein according to any one of claims 1 to 9 or the adeno-associated virus according to claim 14 for preparing a medicament or preparation for treating deafness, hearing loss or hearing dysfunction. A medicine or preparation for treating deafness, hearing loss or hearing dysfunction, which is prepared by the dual-vector system for expressing MYO7A protein according to any one of claims 1 to 9 or the adeno-associated virus according to claim 14, wherein the adeno-associated virus is obtained by transferring the adeno-associated virus packaging vector system into a host cell for packaging, and the adeno-associated virus packaging vector system includes a dual-vector system for expressing MYO7A protein, a vector carrying AAV rep and cap genes, and a helper virus vector. The drug or preparation according to claim 16, wherein the drug or preparation further comprises a neutral salt buffer, an acidic salt buffer, an alkaline salt buffer, glucose, mannose, mannitol, proteins, polypeptides, amino acids, antibiotics, chelating agents, adjuvants, preservatives, nanoparticles, liposomes and positive lipid particles. The drug or preparation according to claim 16 or 17, wherein the drug is administered by injection through the round window, oval window, semicircular canal, or common canal of the cochlea; and the drug is administered single or multiple times throughout life, with a total dose of 1×10 ⁹ -1×10 ¹³ viral genomes.