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WO2025162267A1 - Système à double vecteur pour exprimer une protéine myo7a, et son utilisation - Google Patents

Système à double vecteur pour exprimer une protéine myo7a, et son utilisation

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Publication number
WO2025162267A1
WO2025162267A1 PCT/CN2025/074724 CN2025074724W WO2025162267A1 WO 2025162267 A1 WO2025162267 A1 WO 2025162267A1 CN 2025074724 W CN2025074724 W CN 2025074724W WO 2025162267 A1 WO2025162267 A1 WO 2025162267A1
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Prior art keywords
myo7a
intein
seq
coding sequence
terminal coding
Prior art date
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Pending
Application number
PCT/CN2025/074724
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English (en)
Chinese (zh)
Inventor
柴人杰
谈方志
齐洁玉
余朝荣
张李燕
陈�田
张善中
谭畅
陆玲
丁泽阳
崔志平
宋怀恩
孙思睫
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Otovia Therapeutics
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Otovia Therapeutics
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Publication of WO2025162267A1 publication Critical patent/WO2025162267A1/fr
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Definitions

  • 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.
  • 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.
  • Rapidly evolving gene therapy strategies offer a potential treatment for hearing loss caused by Myo7a mutations.
  • 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.
  • 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).
  • 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.
  • 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.
  • AAV 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.
  • SD-SA sequence donor sequence-splicing acceptor sequence
  • 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.
  • 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.
  • 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
  • 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;
  • 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.
  • the MYO7A cleavage site is located at the amino acid preceding serine, threonine, or cysteine in the MYO7A amino acid sequence.
  • 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, EF1 ⁇ promoter, PGK promoter, or promoters of genes encoding Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, ⁇ 9AchR, ⁇ 10AchR, 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), 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), AATAAA (SEQ ID NO: 33) and variants of AATAAA; the variants of AATAAA comprise
  • the expression cassette of the first nucleotide sequence or the second nucleotide sequence further comprises an expression regulatory element and/or a tag element
  • 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.
  • WPRE woodchuck hepatitis posttranscriptional regulatory element
  • the intein in the dual-vector system for expressing MYO7A protein of the present invention, 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.
  • 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 first nucleotide sequence is inserted into a plasmid comprising two first ITR sequences
  • the second nucleotide sequence is inserted into a plasmid comprising two second ITR sequences
  • 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 MYO7A cleavage site is as shown in Table 1.
  • 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 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;
  • 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;
  • the N-terminal coding sequence of the RmaDnaB intein encodes the N-terminal portion of RmaDnaB shown in SEQ ID NO:23
  • the C-terminal coding sequence of the RmaDnaB intein encodes the C-terminal portion of RmaDnaB shown in SEQ ID NO:24.
  • 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.
  • 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.
  • 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.
  • 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.
  • the amino acid sequence of the MYO7A protein is as shown in SEQ ID NO: 2.
  • 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.
  • 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.
  • the host cell is selected from Hela-S3 cells, HEK-293 cells, HEK-293T cells, HEK-293FT cells, A549 cells, and Sf9 cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides a method for screening MYO7A protein cleavage sites for intein splicing, comprising:
  • 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;
  • the 3' terminal residue of the N-terminal portion of the MYO7A protein is used as the MYO7A protein cleavage site for intein splicing.
  • Figure 1 shows a schematic diagram of intein-mediated full-length extein expression.
  • N-extein represents the N-terminal extein
  • C-extein represents the C-terminal extein
  • intein N represents the N-terminal intein
  • 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.
  • CDS C-terminal coding sequences
  • 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.
  • 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
  • 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.
  • FIG. 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
  • 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.
  • "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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a split intein 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.
  • a dual AAV vector system can be used to deliver a nucleic acid containing a nucleic acid encoding a split intein.
  • 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
  • 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.
  • 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.
  • 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.
  • hearing loss is sensorineural hearing loss.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • promoters that are operably linked to a transcribable sequence are contiguous with the transcribable sequence, i.e., they are cis-acting.
  • some transcriptional regulatory sequences e.g., enhancers
  • 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.
  • 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).
  • 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.
  • AAV adeno-associated virus
  • the first AAV virus isolated was serotype 2 (AAV2).
  • AAV2 serotype 2
  • 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.
  • 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.
  • 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.
  • the AAV virus itself is not pathogenic, which makes the AAV vector recognized as one of the safest viral vectors.
  • AAV virus serotypes 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.
  • vector genome refers to the nucleic acid sequence that is packaged within the rAAV capsid to form the rAAV vector.
  • 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.
  • mammals include individuals at all stages of development, including embryonic and fetal stages.
  • 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.
  • treatment also encompasses modification or improvement of at least one physical parameter, including physical parameters that may not be discernible by the patient.
  • prevention refers to preventing or delaying the onset or development or progression of a disease or condition.
  • prevention generally refers to hospital intervention performed before at least one symptom of a disease occurs.
  • 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
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the MYO7A protein comprises or consists of the amino acid sequence of SEQ ID No 2.
  • 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
  • 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
  • the C-terminus of the intein is linked and fused to the C-terminus of MYO7A.
  • Table 1 lists the locations of some of these cleavage sites on the MYO7A protein and the corresponding N-terminal and C-terminal portions of MYO7A.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the vector plasmid of the present invention can be any plasmid that can replicate in a host cell and express a corresponding polypeptide.
  • 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.
  • the first nucleotide sequence is inserted into a plasmid comprising two first ITR sequences
  • the second nucleotide sequence is inserted into a plasmid comprising two second ITR sequences
  • 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 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;
  • 5'ITR 5' inverted terminal repeat
  • 3'ITR 3' inverted terminal repeat
  • 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
  • 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.
  • 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;
  • 5'ITR 5' inverted terminal repeat
  • 3'ITR 3' inverted terminal repeat
  • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, EF1 ⁇ promoter, PGK promoter, or promoters of Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, ⁇ 9AchR, ⁇ 10AchR, OTOF and STRC encoding genes.
  • 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.
  • WPRE woodchuck hepatitis post-transcriptional regulatory element
  • an AAV vector containing WPRE or a variant thereof increases the expression of MYO7A protein.
  • the dual vector system of the present invention comprises a WPRE nucleotide sequence as set forth in SEQ ID NO:28.
  • 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.
  • the truncated variant of WPRE has the WPRE3 nucleotide sequence as set forth in SEQ ID NO:31.
  • 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.
  • 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.
  • 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.
  • the dual vector system of the present invention comprises a combination of a WPRE nucleotide sequence and an SV40 polyadenylation sequence.
  • 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.
  • the dual-vector system of the present invention comprises a combination of a WPRE3 nucleotide sequence and an SV40 late polyadenylation sequence.
  • 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 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.
  • 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.
  • the dual vector system of the present invention can allow for the expression of functional fragments of the MYO7A protein polypeptide.
  • 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.
  • the host cells are selected from Hela-S3 cells, HEK-293 cells, HEK-293T cells, HEK-293FT cells, A549 cells, and Sf9 cells.
  • 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.
  • the Myo7a mutation-induced hearing loss is USH1B, autosomal recessive hearing loss DFNB2, or autosomal dominant hearing loss DFNA11.
  • the dual vector system of the present invention is a dual AAV vector system.
  • 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.
  • 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)).
  • 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).
  • 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 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
  • 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.
  • the 1043rd amino acid residue is used as the cleavage site (S1).
  • the 1058th amino acid residue is used as the cleavage site (S2).
  • amino acid residue 1061 is used as the cleavage site (S3).
  • S3 and SEQ ID NO:4 are the nucleotide sequence and amino acid sequence of the N-terminus of MYO7A, respectively
  • SEQ ID NO:5 and SEQ ID NO:6 are the nucleotide sequence and amino acid sequence of the C-terminus of MYO7A, respectively.
  • amino acid residue 1064 is used as 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
  • SEQ ID NO:9 and SEQ ID NO:10 are the nucleotide sequence and amino acid sequence of the C-terminus of MYO7A, respectively.
  • the 1071st amino acid residue is used as the cleavage site (S5).
  • the 1076th amino acid residue is used as the cleavage site (S6).
  • the 1081st amino acid residue is used as the cleavage site (S7).
  • amino acid residue 1104 is used as 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
  • SEQ ID NO:13 and SEQ ID NO:14 are the nucleotide sequence and amino acid sequence of the C-terminus of MYO7A, respectively.
  • amino acid residue 1105 is used as 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
  • SEQ ID NO:17 and SEQ ID NO:18 are the nucleotide sequence and amino acid sequence of the C-terminus of MYO7A, respectively.
  • the 1114th amino acid residue is used as the cleavage site (S10).
  • amino acid residue 1119 is used as 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
  • SEQ ID NO:21 and SEQ ID NO:22 are the nucleotide sequence and amino acid sequence of the C-terminus of MYO7A, respectively.
  • the 1122nd amino acid residue is used as the cleavage site (S12).
  • 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
  • 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.
  • 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.
  • 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.
  • Example 2 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.
  • HEK-293T cells Human Embryonic Kidney 293T cells, hereinafter referred to as "293T cells"
  • 293T cells Human Embryonic Kidney 293T cells
  • Prepare Tube A 125 ⁇ L serum-free DMEM medium + 8 ⁇ L Lipofectamine 3000 reagent (Invitrogen, catalog number: L3000015) and mix thoroughly.
  • Prepare Tube B 125 ⁇ L serum-free DMEM medium + 2 ⁇ g first vector plasmid + 2 ⁇ g second vector plasmid + 8 ⁇ L P3000 reagent (Invitrogen, catalog number: L3000015) and mix thoroughly.
  • Tube B contains a mixture of culture medium and Lipofectamine 3000 transfection reagent
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 ⁇ g 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 0.45 ⁇ m 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.
  • 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 4 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.
  • 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.
  • PFA paraformaldehyde
  • the immunofluorescence results in Figure 16 show that the dual AAV system achieves the expression of MYO7A protein in the mouse cochlea.
  • WT is a wild-type mouse
  • blue is parvalbumin-labeled hair cells
  • green is Myo7a protein.
  • 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.
  • Myo7a knockout mice are Myo7a (p.Q720X) point mutation KO mice, which were commissioned to Saiye Biotechnology Co., Ltd. to construct.
  • 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.
  • ABR auditory brainstem response
  • mice's hearing thresholds 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.
  • amino acid sequence of the N-terminal portion of the RmaDnaB intein is SEQ ID NO: 23
  • amino acid sequence of the C-terminal portion of the RmaDnaB intein is SEQ ID NO: 24
  • 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 SV40 poly(A) signal (122 bp): SEQ ID NO: 29
  • Nucleotide sequence of SV40 late poly(A) signal SEQ ID NO: 32
  • amino acid sequence of the N-terminal portion of the NpuDnaE intein is SEQ ID NO: 52
  • nucleotide sequence of the N-terminal portion of the NpuDnaE intein is SEQ ID NO: 53
  • amino acid sequence of the C-terminal portion of the NpuDnaE intein is SEQ ID NO: 54
  • nucleotide sequence of the C-terminal portion of the NpuDnaE intein is SEQ ID NO: 55

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Abstract

La présente invention concerne un système à double vecteur pour exprimer une protéine MYO7A. Le système à double vecteur comporte un premier vecteur d'acide nucléique et un deuxième vecteur d'acide nucléique, le premier vecteur d'acide nucléique comportant une première séquence nucléotidique et le deuxième vecteur d'acide nucléique comportant une deuxième séquence nucléotidique. La première séquence nucléotidique comporte une cassette d'expression insérée entre deux premières séquences ITR, et la deuxième séquence nucléotidique comporte une cassette d'expression insérée entre deux deux deuxièmes séquences ITR. La cassette d'expression de la première séquence nucléotidique comporte un promoteur, une séquence codante N-terminale de MYO7A, une séquence codante N-terminale d'intéine et polyA ; et la cassette d'expression de la deuxième séquence nucléotidique comporte un promoteur, une séquence codante C-terminale d'intéine, une séquence codante C-terminale de MYO7A et polyA. La présente invention concerne également un système de vecteur d'encapsulation d'un virus adéno-associé, un procédé d'encapsulation d'un virus adéno-associé, et un virus adéno-associé ainsi obtenu. Le système à deux vecteurs pour l'expression de la protéine MYO7A ou le virus adéno-associé peut être utilisé pour la thérapie génique, en particulier pour le traitement de la perte auditive, par exemple pour le traitement de l'USH1B liée à une mutation MYO7A, de la surdité héréditaire autosomique récessive DFNB2 et de la surdité héréditaire autosomique dominante DFNA11.
PCT/CN2025/074724 2024-02-01 2025-01-24 Système à double vecteur pour exprimer une protéine myo7a, et son utilisation Pending WO2025162267A1 (fr)

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CN202410142670.0A CN120400250A (zh) 2024-02-01 2024-02-01 表达myo7a蛋白的双载体系统及其用途

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140256802A1 (en) * 2011-11-16 2014-09-11 University Of Florida Research Foundation, Inc. Dual-AAV Vector-Based Systems and Methods for Delivering Oversized Genes to Mammalian Cells
WO2021209574A1 (fr) * 2020-04-15 2021-10-21 Fondazione Telethon Constructions comprenant des intéines
CN115074369A (zh) * 2022-06-24 2022-09-20 成都金唯科生物科技有限公司 表达全长abca4基因的腺相关病毒载体及应用
CN115666658A (zh) * 2020-04-01 2023-01-31 佛罗里达州大学研究基金会 具有提高的治疗ush1b的安全性的双aav-myo7a载体
US20230242942A1 (en) * 2022-05-17 2023-08-03 Eye & Ent Hospital Of Fudan University Dual-vector system for treating hearing impairment and use thereof
CN117106824A (zh) * 2022-05-17 2023-11-24 复旦大学附属眼耳鼻喉科医院 一种治疗听力损伤的双载体系统及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140256802A1 (en) * 2011-11-16 2014-09-11 University Of Florida Research Foundation, Inc. Dual-AAV Vector-Based Systems and Methods for Delivering Oversized Genes to Mammalian Cells
CN115666658A (zh) * 2020-04-01 2023-01-31 佛罗里达州大学研究基金会 具有提高的治疗ush1b的安全性的双aav-myo7a载体
WO2021209574A1 (fr) * 2020-04-15 2021-10-21 Fondazione Telethon Constructions comprenant des intéines
US20230242942A1 (en) * 2022-05-17 2023-08-03 Eye & Ent Hospital Of Fudan University Dual-vector system for treating hearing impairment and use thereof
CN117106824A (zh) * 2022-05-17 2023-11-24 复旦大学附属眼耳鼻喉科医院 一种治疗听力损伤的双载体系统及其应用
CN115074369A (zh) * 2022-06-24 2022-09-20 成都金唯科生物科技有限公司 表达全长abca4基因的腺相关病毒载体及应用

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