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WO2025153705A1 - Utilisation d'intéine divisée pour le traitement d'une maladie associée à myo7a - Google Patents

Utilisation d'intéine divisée pour le traitement d'une maladie associée à myo7a

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Publication number
WO2025153705A1
WO2025153705A1 PCT/EP2025/051193 EP2025051193W WO2025153705A1 WO 2025153705 A1 WO2025153705 A1 WO 2025153705A1 EP 2025051193 W EP2025051193 W EP 2025051193W WO 2025153705 A1 WO2025153705 A1 WO 2025153705A1
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WIPO (PCT)
Prior art keywords
seq
protein
myosin
vila
residue
Prior art date
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Pending
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PCT/EP2025/051193
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English (en)
Inventor
Silvia FRUTOS
Gerard CAELLES
Miquel VILA-PERELLO
Sandra MOTAS
Aikaterina PAPAGIANNI
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Splicebio SL
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Splicebio SL
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Publication of WO2025153705A1 publication Critical patent/WO2025153705A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/92—Fusion polypeptide containing a motif for post-translational modification containing an intein ("protein splicing")domain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Usher IB the most prevalent form of Usher type 1, is caused by mutations in the gene encoding the unconventional molecular motor, MY07A.
  • lentiviral-mediated MYO7Agene delivery in USH1B model is the lentiviral-mediated MYO7Agene delivery in USH1B model. This approach was shown to correct the melanosome mislocalization and opsin accumulation at the photoreceptor connecting cilium in the Myo7a-deficient shakerl mouse model of USH1B (Hashimoto T, et al.. Gene Then 2007;14:584-594).
  • An equine infectious anemia virus-based lentiviral vector equine infectious anemia virus-CMV-MYO7A, UshStat
  • Adeno-associated viruses have been widely used for viral delivery in gene therapy; however, the size of a gene that can be encapsulated into AAV has been reported to be limited to ⁇ 5 kb (Grieger and Samulski 2005, J Virol 79: 9933-9944). Due to its large size, MY07A gene of 100 Mb and a cDNA of nearly 7 kb cannot be encapsulated into a single AAV, and there remains a need to develop new strategies to deliver MY07A gene inside target cells.
  • the first and second fusion proteins according to the present disclosure comprises amino acid sequences selected from any one of the following pairs: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, and, SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 30-43.
  • each polynucleotide is comprised within an expression vector, preferably a viral vector, preferably an adeno associated viral (AAV) vector, preferably said AAV vector comprises capsid protein of AAV selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or RhlO, preferably AAV2, AAV8 or AAV5.
  • AAV vector comprises capsid protein of AAV selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or RhlO, preferably AAV2, AAV8 or AAV5.
  • FIG. 2 Splicing efficiency of Myosin-VIIa protein at position 1172, 1197, 1200 monitored by Western blot: Myosin-VIIa protein was first split at position 1172, 1197 or 1200 and the N-terminal fragment (residues 1-1172 or 1-1197 or 1-1200) recombinantly fused to N- inteins, and the C-terminal fragment (residues 1173-2215, 1198-2215 or 1201-2215) to the C- inteins.
  • Cultured HEK293T cells were co-transfected with equimolar amounts of plasmids encoding for the N- and C-terminal fragments and splicing efficiency monitored by Western blotting.
  • HEK293T cells transfected with a full-length Myosin-VIIa protein serve as control. Split position is numbered according to Myosin-VIIa human amino acid sequence (SEQ ID NO: 15).
  • FIG. 3 Splicing efficiency of Myosin-VIIa protein at position 1226 or 1283 monitored by Western blot: Myosin-VIIa protein was first split at position 1172, 1197, 1200, 1226 or 1283 and the N-terminal fragment (residues 1-1172 or 1-1197 or 1-1200 or 1-1226 or 1-1283) recombinantly fused to N-inteins, and the C-terminal fragment (residues 1173-2215, 1198-2215 1201-2215, 1227-2215, or 1284-2215) to the C-inteins.
  • HEK293T cells were cotransfected with equimolar amounts of plasmids encoding for the N- and C-terminal fragments and splicing efficiency monitored by Western blotting.
  • HEK293T cells transfected with a full- length Myosin-VIIa protein serve as control. Split position is numbered according to Myosin- VIIa human amino acid sequence (SEQ ID NO: 15).
  • FIG. 4 Comparison of splicing yields of MYO7A at split sites MYO7A-1172, MYO7A- 1197, MY07A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, MYO7A-1325 with full- length protein in HEK293T cell line.
  • Cells were transfected with a full-length MY07A plasmid, or co-transfected with both MYO7A N -IntN and IntC-MYO7A c plasmids for each split site. Cells were lysed 48 hours after transfection and then analyzed by western blot.
  • FIG. 1 Comparison of splicing yields of MYO7A at split sites MYO7A-1172, MYO7A- 1197, MY07A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, MYO7A-1325 with full- length protein in HeLa cell line.
  • Cells were transfected with a full-length MY07A plasmid, or co-transfected with both MY07A N -IntN and IntC-MY07A c plasmids for each split site. Cells were lysed 48 hours after transfection and then analyzed by western blot.
  • FIG. 8 Maintenance of ATPase activity in reconstituted MYO7A protein via PTS in all different split sites.
  • HEK293T cells were transfected with full-length (FL) MY07A plasmid, only C-fragment, or co-transfected with both MY07AN-IntN and IntC-MYO7AC plasmids for each split site. Cells were lysed 48 hours after transfection and then analyzed by ATPase activity assay. Results equal to or greater than 1.2 a.u. were considered positive, as a cutoff value of 1.2 a.u.
  • the present disclosure relates to a combination of polynucleotides comprising: i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’ : a N-terminal fragment of Myosin- Vila protein and N-split intein, fused directly or indirectly via a linker, ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in a cell generates full length Myosin- Vila protein by protein splicing.
  • first and second polynucleotides encoding said first and second fusion proteins in a cell generates Myosin- Vila protein by protein splicing.
  • N- and/or C- split inteins according to the present disclosure comprised in the first and second fusion proteins respectively can be engineered N- and/or C- split inteins.
  • Said engineered N- and/or C- split inteins can be engineered by introducing mutations in natural N- and/or C- split intein sequences, in particular to enhance protein splicing activity.
  • the N-split intein and/or C-split intein sequences comprised in the first and second fusion proteins respectively comprise or consist of amino acid sequences selected from any of N- and C-split inteins listed in Table 1 below:
  • Table 1 Examples of pairs of N- and C-split inteins that can be used according to the present disclosure.
  • the N- and C-split inteins comprised in the first and second fusion proteins respectively according to the present disclosure are N- and C-split inteins comprising or consisting of amino acid sequences of SEQ ID No: 1 (Cfa-N split intein) and SEQ ID No: 2 (Cfa-C-split intein:), SEQ ID No: 3 (Npu-N) and 4 (Npu-C), SEQ ID No: 5 (Cat-N) and 6 (Cat- C), SEQ ID No: 7 (Gp41-N) and 8 (Gp41-C), SEQ ID No: 9 (ConN) and 10 (ConC) or SEQ ID No: 11 (Nrdj 1-N) and 12 (Nrdj 1-C) or any functional variant(s) thereof.
  • variant refers to a polypeptide sequence that is derived from N- and/or C-split inteins as described above and comprises an alteration, i.e., a substitution, insertion, and/or deletion, at one or more positions, but retain the capacity when bound to form a functional enzyme to catalyze a protein splicing reaction that excises the N and C-intein sequences and joins flanking sequences (N- and C-exteins) with a peptide bond.
  • the variant may be obtained by various techniques well known in the art. Examples of techniques for altering the nucleotide sequence encoding the native protein, include, but are not limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction.
  • the protein splicing efficiency of N- and/or C-split intein functional variants may be assessed for instance by measuring the protein reconstitution efficiency in a cell.
  • the protein reconstitution efficiency can be measured by expressing in a cell a combination of polynucleotides, said first polynucleotide encodes a N-terminal fragment of a reporter protein (e.g., GFP) fused to N-split intein and a second polynucleotide encodes a C-terminal fragment of said gene reporter fused to the C-split intein.
  • the reconstitution efficiency of the reporter protein can then be monitored by determining the level of expression of reconstituted protein.
  • the functional variant of Cfa N- and/or Cfa C-Split intein retain the functional splicing activity of native Cfa split intein, more preferably have a protein splicing efficiency higher than Npu split-intein.
  • N-terminal Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29.
  • the combination of polynucleotides according to the present disclosure comprises polynucleotides encoding a first fusion protein and a second fusion protein comprising amino acid sequences selected from the pairs disclosed in Table 4, preferably selected from the pairs consisting of: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, and SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 30-43.
  • Table 4 Preferred first and second protein fusions according to the present disclosure.
  • a degron can be fused directly or indirectly via a linker to the first and/or second fusion proteins to mediate degradation of the excised intein.
  • said degron is fused, directly or indirectly via a linker, to the N- split intein and C-split intein comprised in the first and second fusion proteins, respectively, preferably said degron is located at the 3’-end of the N-Split intein and at the 5’end of the C- split intein.
  • the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ a Cfa-c-Split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fuse
  • the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:
  • N-terminal Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29 and wherein said first or second fusion protein further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split
  • N-terminal Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively; wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29 and wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split
  • the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-c-Split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila 1 protein
  • N-terminal Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively; wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29 and wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split
  • the combination of polynucleotides according to the present disclosure encode a first fusion protein and a second fusion protein comprising amino acid sequences selected from the pairs consisting of: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41; and SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 30-43 and wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein or at the 5 ’end of the C-spli
  • the combination of polynucleotides according to the present disclosure encode a first fusion protein and a second fusion protein comprising amino acid sequences selected from the pairs consisting of: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41; and SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 30-43 and wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and at the 5’end of the C-split
  • said degron can be selected as non-limiting examples in the degrons listed in Table 5 below.
  • Table 5 Examples of degrons and corresponding sequences.
  • the degron comprised in the first and/or second fusion protein according to the present disclosure can be selected from the group consisting of: CL1 (SEQ ID NO: 44), Degl (SEQ ID NO: 45), PEST (SEQ ID NO: 46), DD1 (SEQ ID NO: 47), DD2 (SEQ ID NO: 48), VI 5 (SEQ ID NO: 49), Ml (SEQ ID NO: 50), M2 (SEQ ID NO: 51), SopE (SEQ ID NO: 52), SopEl-78 (SEQ ID NO: 53), SopE 15-78 (SEQ ID NO: 54), SopE-15-50 (SEQ ID NO: 55), L2 (SEQ ID NO: 56), L6 (SEQ ID NO: 57), L9 (SEQ ID NO: 58), LIO (SEQ ID NO: 59), Lil (SEQ ID NO: 60), L12 (SEQ ID NO: 61), L15 (SEQ ID NO: 62), L16 (SEQ ID NO: 59), Lil (
  • the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and/or at the 5’end of the C
  • the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:
  • N-terminal Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29 and wherein said first and/or second fusion protein further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-
  • the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-c-Split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila 1 protein
  • N-terminal Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively; wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29 and wherein said first and/or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-
  • the polynucleotides encoding said first and second protein is transfected in a cell, and the amount of starting material and excised intein is determined for example by Western blot.
  • the degron induces degradation of starting material (i.e., the fusion protein comprising the protein of interest, the intein and the degron) and excised intein(s) when the amount of the starting material and excised intein is lower than the starting material and excised intein amount in a cell transfected with fusion proteins without degrons, preferably when the amount of intein is at least 1.2, 1.3, 1.4, 1.5, 1.8 or 2.0 fold lower than the starting material and intein amount in a cell transfected with fusion proteins without degrons.
  • the expression level of reconstituted protein may be determined by any suitable methods known by skilled persons.
  • the quantity of the reconstituted protein may be measured, for example, by semi-quantitative Western blots, enzyme-labelled and mediated immunoassays, such as ELISAs, biotin/avidin type assays, radioimmunoassay, immunoelectrophoresis, mass spectrometry, or immunoprecipitation or by protein or antibody arrays.
  • the present disclosure relates to i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N-terminal fragment of a protein of interest and N-split intein, fused directly or indirectly via a linker, ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of said protein of interest, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in a cell generates full length protein of interest by protein splicing, wherein said first and/or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end
  • Said nucleic acid construct comprises one or more control sequence required for expression of said coding sequence.
  • the nucleic acid construct comprises a coding sequence and regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence that are required for expression of the selected gene product.
  • a nucleic acid construct typically comprises a promoter sequence, a coding sequence and a 3' untranslated region that usually contains a polyadenylation site and/or transcription terminator.
  • said nucleic acid construct may comprise a SV40 intron.
  • promoter refers to a regulatory element that directs the transcription of a nucleic acid to which it is operably linked.
  • a promoter can regulate both rate and efficiency of transcription of an operably linked nucleic acid.
  • a promoter may also be operably linked to other regulatory elements which enhance (“enhancers”) or repress (“repressors”) promoterdependent transcription of a nucleic acid.
  • enhance enhance
  • repressors repress
  • These regulatory elements include, without limitation, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter, including e.g., attenuators, enhancers, and silencers.
  • operably linked refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically but not necessarily contiguous; where it is necessary to join two protein encoding regions, they are contiguous and in reading frame.
  • each polynucleotide or nucleic acid construct according to the present disclosure may be comprised in an expression vector.
  • expression vector refers to a nucleic acid molecule used as a vehicle to transfer genetic material, and in particular to deliver a nucleic acid into a host cell, either in vitro or in vivo.
  • Expression vector also refers to a nucleic acid molecule capable of effecting expression of a gene (transgene) in host cells or host organisms compatible with such sequences.
  • Expression vectors typically include at least suitable transcription regulatory sequences and optionally 3 ’-transcription termination signals.
  • Vectors include, but are not limited to, plasmids, phasmids, cosmids, transposable elements, viruses, and artificial chromosomes (e.g., YACs).
  • HIV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • FV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • EIAV equine infectious anemia virus
  • adenoviral (Ad) vectors adeno-associated viral (AAV) vectors
  • AAV adeno-associated viral vectors
  • SV-40 simian virus 40 vectors
  • bovine papilloma virus vectors Epstein-Barr virus
  • herpes virus vectors vaccinia virus vectors
  • Harvey murine sarcoma virus vectors murine mammary tumor virus vectors
  • suitable sequences should be introduced in the vector of the disclosure for obtaining a functional viral vector, such as AAV ITRs for an AAV vector, or LTRs for lentiviral vectors.
  • said vector is an AAV vector.
  • AAV has arisen considerable interest as a potential vector for human gene therapy.
  • favorable properties of the virus are its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and the wide range of cell lines derived from different tissues that can be infected.
  • the AAV genome is composed of a linear, single-stranded DNA molecule which contains 4681 bases (Berns and Bohenzky, 1987, Advances in Virus Research (Academic Press, Inc.) 32:243-307).
  • the genome includes inverted terminal repeats (ITRs) at each end, which function in cis as origins of DNA replication and as packaging signals for the virus.
  • the ITRs are approximately 145 bp in length.
  • the internal non-repeated portion of the genome includes two large open reading frames, known as the AAV rep and cap genes, respectively. These genes code for the viral proteins involved in replication and packaging of the virion. In particular, at least four viral proteins are synthesized from the AAV rep gene, Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight.
  • the AAV cap gene encodes at least three proteins, VP1, VP2 and VP3.
  • the polynucleotides, nucleic acid constructs or expression vectors according to the present disclosure thereof further comprises a 5’ITR and a 3TTR sequences, preferably a 5’ITR and a 3’ ITR sequences of an adeno-associated virus.
  • inverted terminal repeat refers to a nucleotide sequence located at the 5’-end (5’ITR) and a nucleotide sequence located at the 3’-end (3’ITR) of a virus, that contain palindromic sequences and that can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication. They are also needed for viral genome integration into the host genome; for the rescue from the host genome; and for the encapsidation of viral nucleic acid into mature virions. The ITRs are required in cis for the vector genome replication and its packaging into the viral particles.
  • the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure further comprises a 5’ITR and a 3TTR of an AAV, preferably of a serotype AAV2.
  • the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins as described above may be packaged into a virus capsid to generate a "viral particle”, also named “viral vector particle”.
  • the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure is packaged into an AAV-derived capsids to generate an "adeno- associated viral particles" or "AAV particles”.
  • the present disclosure relates to viral particles comprising the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure and preferably comprising capsid proteins of adeno-associated virus.
  • AAV viral particles The construction of recombinant AAV viral particles is generally known in the art and has been described for instance in US 5,173,414 and US5,139,941; WO 92/01070, WO 93/03769, Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533- 539; Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.
  • the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins as described above including ITR(s) of a given AAV serotype can be packaged, for example, into: a) a viral particle constituted of capsid proteins derived from the same or different AAV serotype [e.g.
  • AAV2 ITRs and AAV5 capsid proteins AAV2 ITRs and AAV8 capsid proteins; AAV2 ITRs and Anc80 capsid proteins; AAV2 ITRs and AAV9 capsid proteins];
  • a mosaic viral particle constituted of a mixture of capsid proteins from different AAV serotypes or mutants [e.g. AAV2 ITRs with AAV1 and AAV5 capsid proteins];
  • a chimeric viral particle constituted of capsid proteins that have been truncated by domain swapping between different AAV serotypes or variants e.g. AAV2 ITRs with AAV5 capsid proteins with AAV3 domains.
  • the present disclosure relates to a viral particle comprising the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins as described above and preferably comprising capsid proteins of adeno-associated virus such as capsid proteins from AAV9 and AAV-PHP.B, AAV2, AAV8 and AAV5.
  • adeno-associated virus such as capsid proteins from AAV9 and AAV-PHP.B, AAV2, AAV8 and AAV5.
  • subject refers to mammals.
  • Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, nonhuman primates such as apes, chimpanzees, monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like.
  • said subject is a human patient.
  • the disclosure also provides a method for treating a MYO7A-associated disease as described above in a patient in need thereof comprising administering to said patient a therapeutically effective amount of the polynucleotides, nucleic acid constructs, expression vectors or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins as described above or pharmaceutical composition thereof.
  • the combination of polynucleotides, nucleic acid constructs, expression vectors or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure for its therapeutic use is administered to the subject or patient by a parenteral route, in particularly by intravenous, intraarterial, intramuscular, intranasal, intraocular, intravitreal, suprachoroidal or subretinal route.
  • the amount of product of the disclosure that is administered to the subject or patient may vary depending on the particular circumstances of the individual subject or patient including, age, sex, and weight of the individual; the nature and stage of the disease, the aggressiveness of the disease; the route of administration; and/or concomitant medication that has been prescribed to the subject or patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For any particular subject, specific dosage regimens may be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions.
  • the dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.
  • the disclosure further relates to a kit, preferably for use in the treatment of MYO7A-associated disease as described above, preferably Usher syndrome type IB comprising a combination of polynucleotides as described above, comprising: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, optionally wherein said first and/or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second
  • the kit comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : Cfa C-split intein of SEQ ID NO: 2 or Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, and optionally wherein said
  • the kit comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:
  • the kit comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa C-split intein of SEQ ID NO: 2 or Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and
  • the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vila fragment respectively consists of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NOs: 16 to 29 and optionally wherein said first and/or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C- split
  • the kit comprises a combination of polynucleotides encoding a first fusion protein and a second fusion protein comprising amino acid sequences selected from the groups of pairs consisting of: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, and, SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% identity to any one of sequences SEQ ID NO: 30-43, and optionally wherein said first and/or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and/or second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and
  • the present disclosure relates to a kit comprising a combination of polynucleotides comprising a first polynucleotide encoding a first fusion protein comprising from 5 ’ to 3 ’ : a N-terminal fragment of a protein of interest and N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of said protein of interest, fused directly or indirectly via a linker, wherein said first and/or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and/or at the 5’
  • the present disclosure relates to a kit comprising a combination of polynucleotides comprising: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of protein of interest and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-C-split intein of SEQ ID NO: 2 or Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of the protein of interest, fused directly
  • the present disclosure relates to a kit comprising a nucleic acid construct comprising a degron comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 75-119, or any combination thereof or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 75-119.
  • the kit may include instructions or packaging materials that describe how to administer the polynucleotides contained within the kit to a patient.
  • Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration.
  • the kits may include one or more ampoules or syringes that contain the products of the invention in a suitable liquid or solution form.
  • Oligonucleotides were purchased from Eurofins genomics. Synthetic genes were purchased from GENEWIZ. Pfu Ultra fusion polymerase for cloning and all restriction enzymes were purchased from Thermofisher Scientific. High-competency cells used for cloning were generated from XLIO-Gold chemically competent E. coli. HEK293T and HeLa cells were purchased from ATCC. DNA purification kits were purchased from Thermofisher Scientific. All plasmids were sequenced by Macrogen. Luria Bertani (LB) media, and all buffering salts were purchased from Thermofisher Scientific.
  • LB Luria Bertani
  • Coomassie brilliant blue, NH4HCO3, DTT, formic acid, fetal bovine serum and asolectin from soybean were purchased from Sigma- Aldrich.
  • Acetonitrile (ACN) was purchased from Carlo-Erba.
  • EDTA-free complete protease inhibitors were purchased from Roche.
  • Lipofectamine 2000 transfection reagent, DMEM high glucose GlutaMAX supplement, RPMI 1640 medium GlutaMAX supplement, RIPA lysis and extraction buffer, BCA protein assay kit, MES-SDS running buffer, pre-stained protein ladder and SDS-PAGE (Bis-tris and Tris-acetate gels) were purchased from Thermofisher Scientific.
  • HRP-conjugated secondary antibody was purchased from Vitro. Dodecyl maltoside (D310) and cholesteryl hemisuccinate (CH210) solution were purchased from Anatrace. Trypsin was purchased from Promega. ADP-GloTM Kinase Assay was purchased from Promega. SuperSignal ELISA Femto Maximum Sensitivity Substrate was purchased from Thermo Fisher Scientific.Equipment:
  • Synthetic genes to prepare constructs MY07A-l-1200-CfaN-3FT (SEQ ID NO: 124), MY07A- 1201-2215-CfaCmut-3FT (SEQ ID NO: 125) were purchased and introduced into plasmid expression vectors using Kpnl and Notl restriction enzymes.
  • HEK293T cells (ATCC, CRL-3216) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S) at 37°C in a 5% CO2 atmosphere.
  • DMEM Dulbecco’s Modified Eagle’s Medium
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • mice Four weeks after vector administration via SRI in mice, animals were euthanized and eyes were enucleated. Retinas were individually dissected and snap-frozen in liquid nitrogen.
  • inteins have been engineered based on consensus design (Stevens et al., 2016; Stevens, Sekar, Gramespacher, Cowburn, & Muir, 2018) and shown to have superior properties than naturally occurring inteins.
  • One of these inteins termed Cfa
  • Cfa has faster kinetics, higher expression levels and high tolerance to extreme conditions such as high temperature and concentration of denaturing agents.
  • Cfa variants with degrees of homology from 90% or higher display similar properties.
  • a major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site.
  • Deviation from this preferred sequence context leads to a marked reduction in splicing activity, limiting the applicability of protein trans-splicing (PTS)-based methods.
  • PTS protein trans-splicing
  • Recently engineered versions of naturally split inteins that possess greatly improved extein tolerance have been developed (Stevens et al., 2017). This intein is termed Cfa mu t.
  • MY07A is a large protein, which is mutated in USHER1B syndrome.
  • Usher syndrome is an inherited disease that causes severe hearing loss and retinitis pigmentosa, an eye disorder that causes vision to deteriorate over time. It is the most common condition that affects both hearing and vision.
  • Reconstitution of MY07A has been proposed as a viable strategy to treat the disease.
  • Several approaches based on AAV gene therapy are currently being explored to reconstitute it.
  • MYO7A(l-1172)-CfaN, CfaCmut- MY07A (1173-2215) constructs were used to evaluate splicing MY07A at position 1172 (SEQ ID NO: 120 and SEQ ID NO: 121), MY07A (1-1197)-CfaN and CfaCmut- MY07A(l 198-2215) to evaluate splitting MY07A at position 1197 (SEQ ID NO: 122 and SEQ ID NO: 123), and MY07A (1-1200)- CfaN, CfaCmut-MYO7A(1201-2215) constructs were used to evaluate splicing MY07A at position 1200 (SEQ ID NO: 124 and SEQ ID NO: 125).
  • Sites were selected based on the topological structure of Myosin- Vila and taking into consideration the presence of folded domains. Sites were selected outside of well-defined folded domains. Briefly, constructs were co-transfected into HEK293T cells as well as full-length Myo7A (SEQ ID NO: 134) was transfected to serve as control. Cells were lysed and protein reconstitution yields determined by Western Blot to compare the yields of reconstituted protein versus the full-length protein. Results showed that the yield for these three sites are comparable to the protein obtained only with the full-length protein (Figure 2).
  • MYO7A(l-1226)-CfaN and CfaCmut- MYO7A(1227-2215) constructs SEQ ID NO: 126 and SEQ ID NO: 127) and MY07A(l- 1283)-CfaN, CfaCmut-MYO7A(1284-2215) constructs (SEQ ID NO: 128 and SEQ ID NO: 129) were co-transfected into HEK293T cells as well as full-length MYO7A(SEQ ID NO: 134) was transfected to serve as control.
  • Cells were lysed and protein reconstitution yields determined by Western Blot to compare the yields of reconstituted protein versus the full-length protein. Results showed that the yield for these two sites are comparable to the protein obtained only with the full-length protein (Figure 3).
  • MYO7A(l-823)-CfaN and CfaCmut-MYO7A(824-2215) constructs SEQ ID NO: 130 and SEQ ID NO: 131) and MYO7A(l-1325)-CfaN, CfaCmut-MYO7A(1326-2215) constructs (SEQ ID NO: 132 and SEQ ID NO: 133) were co-transfected into HEK293T cells as well as full-length MY07A (SEQ ID NO: 89) was transfected to serve as control as well as all previous different split sites evaluated to compare all of them. Cells were lysed and protein reconstitution yields determined by Western Blot to compare the yields of reconstituted protein versus the full-length protein. Results demonstrated that the yield for all seven different sites are comparable to the protein obtained only with the full-length protein ( Figure 4).
  • constructs were also characterized in HeLa cell lines.
  • cells were transfected either with full-length MY07A plasmid or co-transfected with both N-fragment and C-fragment of MY07A plasmids.
  • Cells were lysed using a non-cationic detergent mixture as lysis buffer.
  • Expression levels were analyzed by western blot. For detection of protein anti-flag tag and anti-tubulin antibodies were used.
  • results showed excellent in vitro reconstitution of MY07A by protein trans-splicing reaching equivalent levels of expression as full-length MY07A construct in HeLa cell lines ( Figure 5).
  • CoIP coimmunoprecipitation
  • pairs of interest were evaluated in vitro in HEK293T cells.
  • cells were transfected either with full-length MY07A plasmid or co-transfected with both N- fragment and C-fragment of MY07 A plasmids for all seven different split sites MY07A-1172, MYO7A-1197, MY07A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, MYO7A-1325.
  • Cells were lysed using a non-cationic detergent mixture as lysis buffer.
  • a specific ATPase activity assay was developed. This assay was based on an ELISA-like system to enrich MY07A sample followed by the use of a commercial kit to detect ATPase activity and using only C- fragment sample as negative control of the assay due to its lack of ATPase activity as well as its capacity to inhibit it (Umeki N, et. al., Proc. Natl. Acad. Set. U.S.A., 2009. 106:8483-8488.
  • degron-containing candidates included degrons of different size, different families as well as different mechanism of action (e.g. proteosome mediated or chaperone-mediated autophagy) (Table 7).
  • degron-containing pair candidates were evaluated at functionality level in vitro.
  • candidates were tested by co-immunoprecipitation assay based on MY07A as bait protein, and all candidates assessed showed positive interaction with SANS ( Figure 10 and Table 7).
  • candidates were evaluated by ATPase activity assay, and again all candidates tested showed positive ATPase activity ( Figure 11 and Table 7).

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Abstract

La présente invention concerne l'utilisation d'intéines divisées pour exprimer la protéine myosine-VIIa codée par le gène MYO7A chez un sujet en ayant besoin pour une thérapie génique, en particulier pour le traitement d'une maladie associée à MYO7A , de préférence le syndrome d'Usher de type 1B.
PCT/EP2025/051193 2024-01-18 2025-01-17 Utilisation d'intéine divisée pour le traitement d'une maladie associée à myo7a Pending WO2025153705A1 (fr)

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