Use of split-intein for treating myo7a-associated diseases

Abstract

The present disclosure relates to the use of split inteins for expressing a myosin VIIa protein encoded by a gene in a subject in need thereof, in particular for treating a related disease, preferably Usher Syndrome type 1B. MYO7A The present disclosure relates to the use of split inteins for expressing a myosin VIIa protein encoded by a gene in a subject in need thereof, in particular for treating a related disease, preferably Usher Syndrome type 1B. MYO7A The present disclosure relates to the use of split inteins for expressing a myosin VIIa protein encoded by a gene in a subject in need thereof, in particular for treating a related disease, preferably Usher Syndrome type 1

Description

Technical Field

[0001] This disclosure relates to the use of cleavage introns in subjects who require them to express the expression of [the substance / method / etc.]. MYO7A The uses of the gene-encoded myosin VIIa protein, particularly for the treatment of myosin VIIa. MYO7A For related diseases, Usher syndrome type 1B is preferred. Background Technology

[0002] Usher syndrome (USH) represents a group of genetically heterogeneous autosomal recessive disorders characterized by combined visual and sensorineural hearing loss, sometimes accompanied by vestibular dysfunction. It primarily affects light-sensitive photoreceptor cells in the retina and auditory hair cells in the cochlea. Visual loss in all cases is progressive, manifesting as retinitis pigmentosa (RP), characterized by the gradual degeneration of photoreceptor cells. Peripheral rod cell function is lost first, leading to night blindness and narrowed visual field, followed by cone cell death and severe visual impairment (Dinculescu et al. IntOphthalmol Clin. 2021 Fall; 61(4): 109–124).

[0003] Usher 1B is the most common form of Usher 1, caused by mutations in the gene encoding the unconventional molecular motor MYO7A.

[0004] Several different gene therapy methods have been tested for retinal gene therapy to prevent blindness in Usher 1B.

[0005] One example is lentivirus-mediated MYO7A gene delivery in the USH1B model. This method has been shown to correct melanosome misalignment and opsin accumulation at the photoreceptor junctions in the Myo7a-deficient shaker1 mouse model of USH1B (Hashimoto T, et al. Gene Ther. 2007;14:584–594). A lentiviral vector based on equine infectious anemia virus (equine infectious anemia virus-CMV-MYO7A, UshStat) has also been used for subretinal delivery to USH1B patients to prevent or slow the progression of retinitis pigmentosa (ClinicalTrials.gov identifier: NCT01505062).

[0006] However, these integrative vector systems have a major drawback: the potential risk of insertion mutations.

[0007] Adeno-associated virus (AAV) has been widely used for viral delivery in gene therapy; however, the reported size limit for encapsulating AAV genes is approximately 5 kb (Grieger and Samulski 2005, J Virol 79: 9933–9944). Due to its relatively large size, 100 Mb... MYO7A Genes and nearly 7 kb of cDNA cannot be encapsulated into a single AAV, therefore new strategies still need to be developed to encapsulate them. MYO7A Genes are delivered into target cells.

[0008] One strategy to overcome this problem is to insert genes or cDNA (such as...) MYO7A The expression of MYO7A is divided into two segments, each contained in a separate AAV vector. However, a major problem with dual AAV systems (such as hybridization dual vectors) is achieving high levels of MYO7A expression.

[0009] It has been proven that the use of MYO7A The overlapping sequence of the AAV dual vector produces a nonfunctional MYO7A protein in vivo (Lopes et al. 2013, Gene Therapy, 20(8):824-33). Furthermore, the individual first half of the heterozygous AAV dual vector has been shown to be toxic.

[0010] Therefore, new strategies still need to be developed to... MYO7A Genes are delivered efficiently into cells to prevent the formation of toxic truncated fragments.

[0011] Inteins are genetic elements that undergo trans-splicing, in which two protein fragments combine to form a catalytic enzyme that then catalyzes the excision of itself and the ligation of their flanking sequences. Splitting inteins is primarily used to fuse different functional protein domains in protein purification systems and labeling steps. In recent years, several novel inteins have been engineered based on shared design (Stevens et al., J Am Chem Soc. 2016 Feb 24;138(7):2162-5;Stevens, Sekar, Gramespacher, Cowburn,&Muir, J Am Chem Soc. 2018 Sep 19;140(37):11791-11799), demonstrating superior properties compared to naturally occurring inteins.

[0012] Recent studies have investigated the use of trans-splicing of proteins mediated by splitting inteptides to reconstruct therapeutic proteins in vivo (WO2021 / 191447 and WO2020 / 079034). Invention Overview In this application, the inventors demonstrate for the first time that cleavage-involved peptide-mediated protein trans-splicing can be used to effectively reconstitute proteins by splitting within the cell. MYO7A Genetically encoded myosin VIIa. Recombination of myosin VIIa using cleavage intron-mediated protein transsplicing represents an effective strategy for treating MYO7A-related diseases such as Usher syndrome type 1B. Using cleavage introns to recombinant myosin VIIa protein, particularly when combined with degraders, can prevent the in vivo toxicity of the half-fragment.

[0014] This disclosure relates to polynucleotide combinations for treating MYO7A-related diseases in subjects requiring treatment, preferably selected from the group consisting of: non-syndromic hearing loss DFNA11, non-syndromic hearing loss DFNB2, or Usher syndrome type 1B, wherein said combination comprises: i) A first polynucleotide encoding the first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and an N-cleavage inteptide, which are fused directly or indirectly through a linker. ii) A second polynucleotide encoding the second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. The expression of the first and second polynucleotides in the subjects generates myosin VIIa through protein splicing.

[0015] In a preferred embodiment, the N-split inteptide is the N-Cfa-inteptide of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity with SEQ ID NO: 1; and the C-split inteptide is the C-Cfa-inteptide of SEQ ID NO: 2 or any functional variant thereof having at least 90% identity with SEQ ID NO: 2, preferably wherein amino acid residues 20 to 22 of SEQ ID NO: 2 are GEP, more preferably the C-Cfa-inteptide of SEQ ID NO: 13. mut Intended peptides or any functional variant thereof having at least 90% identity with SEQ ID NO: 13.

[0016] In a specific embodiment, the N-fractionation inteptide is the N-Npu-inteptide of SEQ ID NO: 3 or any functional variant thereof having at least 90% identity with SEQ ID NO: 3; and the C-fractionation inteptide is the C-Npu-inteptide of SEQ ID NO: 4 or any functional variant thereof having at least 90% identity with SEQ ID NO: 4, preferably wherein amino acid residues 20 to 22 of SEQ ID NO: 4 are GEP, more preferably the C-Npu-inteptide of SEQ ID NO: 14.mut Intended peptides or any functional variant thereof having at least 90% identity with SEQ ID NO: 14.

[0017] In a preferred embodiment, the myosin VIIa protein is human myosin VIIa protein, preferably comprising or consisting of SEQ ID NO: 15 or any functional variant thereof having at least 90% identity with SEQ ID NO: 15.

[0018] In a more preferred embodiment, the N-terminal fragment of myosin VIIa protein terminates at residue 823, and the C-terminal fragment of myosin VIIa protein begins at residue 824; the N-terminal fragment of myosin VIIa protein terminates at residue 1172, and the C-terminal fragment of myosin VIIa protein begins at residue 1173; the N-terminal fragment of myosin VIIa protein terminates at residue 1197, and the C-terminal fragment of myosin VIIa protein begins at residue 1198; and the N-terminal fragment of myosin VIIa protein terminates at residue 1200. The C-terminal fragment of myosin VIIa protein begins at residue 1201; the N-terminal fragment of myosin VIIa protein terminates at residue 1226, and the C-terminal fragment of myosin VIIa protein begins at residue 1227; the N-terminal fragment of myosin VIIa protein terminates at residue 1283, and the C-terminal fragment of myosin VIIa protein begins at residue 1284; or the N-terminal fragment of myosin VIIa protein terminates at residue 1325, and the C-terminal fragment of myosin VIIa protein begins at residue 1326, wherein the residues are specified according to SEQ ID NO: 15. More preferably, the N-terminal fragment of myosin VIIa protein terminates at residue 1172, and the C-terminal fragment of myosin VIIa protein begins at residue 1173; the N-terminal fragment of myosin VIIa protein terminates at residue 1197, and the C-terminal fragment of myosin VIIa protein begins at residue 1198; the N-terminal fragment of myosin VIIa protein terminates at residue 1200, and the C-terminal fragment of myosin VIIa protein begins at residue 1201; the N-terminal fragment of myosin VIIa protein terminates at residue 1226, and the C-terminal fragment of myosin VIIa protein begins at residue 1227; or the N-terminal fragment of myosin VIIa protein terminates at residue 1283, and the C-terminal fragment of myosin VIIa protein begins at residue 1284, wherein the residues are specified according to SEQ ID. Number NO: 15.

[0019] In a more preferred embodiment, the first and second fusion proteins according to this disclosure comprise amino acid sequences selected from any 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 with any one of sequences SEQ ID NO: 30-43.

[0020] In a particular embodiment, the first or second fusion protein may further comprise a degrader, preferably selected from: SEQ ID NO: 44 to 119 or any combination thereof, or any functional variant thereof having at least 90% identity with any one of the sequences SEQ ID NO: 44 to 119 or any combination thereof, more preferably the first fusion protein further comprises a degrader located at the 3' end of the N-cleavage inteptide, and / or the second fusion protein further comprises a degrader located at the 5' end of the C-cleavage inteptide, which are fused directly or indirectly through a linker.

[0021] According to this disclosure, each polynucleotide of the combination further comprises several regulatory elements, such as promoters, transcription termination sequences, translation termination sequences, introns, enhancers, signal peptides, and polyadenylation elements, preferably promoters selected from the group consisting of: cytomegalovirus (CMV) promoters (GenBank accession number: AF 396260.1, bp 150-812, last updated August 13, 2001), and chimeric reduced versions of the CMV and chicken β-actin (CEBA) promoters (GenBank accession number: AF396260.1, bp 160-526, last updated August 13, 2001; GenBank accession number: X). 00182.1, bp268-571, last updated November 14, 2006), human phosphoglycerate kinase (hPGK) promoter (GenBank accession number: AH002938.2, bp2-516, last updated August 1, 2016), photoreceptor cell-specific human rhodopsin kinase (hGRK1) promoter, rod cell-specific IRBP promoter, VMD2 (vitiligo macular dystrophy / Best disease) promoter, and EF1α promoter.

[0022] Preferably, each polynucleotide is contained in an expression vector, preferably a viral vector, preferably an adeno-associated virus (AAV) vector, preferably the AAV vector contains an AAV capsid protein selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or Rh10, preferably AAV2, AAV8 or AAV5.

[0023] In a preferred embodiment, the combination is administered to the subject via a parenteral route, more preferably via an intravenous, intra-arterial, intramuscular, intranasal, intraocular, intravitreal, suprachoroidal, or subretinal route.

[0024] This disclosure also relates to a reagent kit containing: (i) The first polynucleotide encoding the first fusion protein, comprising from 5' to 3': - N-terminal fragment of myosin VIIa protein and - Any functional variant of the N-Cfa inteptide of SEQ ID NO: 1 that has at least 90% identity with SEQ ID NO: 1. It fuses directly or indirectly through a joint. (ii) A second polynucleotide encoding the second fusion protein, comprising from 5' to 3': -C-Cfa intima-peptide of SEQ ID NO: 2 or C-Cfa of SEQ ID NO: 13 mut Intended peptides or any functional variant thereof having at least 90% identity with SEQ ID NO: 2 or 13, and - The C-terminal fragment of myosin VIIa protein, It fuses directly or indirectly through a joint. The first and second fusion proteins comprise: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. - Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284; or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO: 15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are each composed of amino acid sequences selected from the following pairs: 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%, or 99% sequence identity with any one of sequences SEQ ID NO: 16 to 29, more preferably wherein the first and second fusion proteins comprise 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 with any one of the sequences SEQ ID NO: 30-43. Attached Figure Description

[0025] Figure 1 Comparison of myosin VIIa protein isotypes (isotype 1 (SEQ ID NO: 15), isotype 2 (SEQ ID NO: 135), isotype 3 (SEQ ID NO: 136), isotype 4 (SEQ ID NO: 137), isotype 5 (SEQ ID NO: 138), isotype 6 (SEQ ID NO: 139), isotype 7 (SEQ ID NO: 140), isotype 8 (SEQ ID NO: 141)). Cleavage positions are indicated by arrows.

[0026] Figure 2The splicing efficiency of myosin VIIa protein at positions 1172, 1197, and 1200 was monitored using Western blotting. Myosin VIIa protein first splits at positions 1172, 1197, or 1200, and the N-terminal fragment (residues 1-1172, 1-1197, or 1-1200) fuses with the N-intein, while the C-terminal fragment (residues 1173-2215, 1198-2215, or 1201-2215) fuses with the C-intein. HEK293T cells were co-transfected with plasmids encoding the N-terminal and C-terminal fragments in equimolar amounts, and splicing efficiency was monitored using Western blotting. HEK293T cells transfected with full-length myosin VIIa protein served as a control. Splicing positions were numbered according to the amino acid sequence of human myosin VIIa (SEQ ID NO: 15).

[0027] Figure 3 The splicing efficiency of myosin VIIa protein at positions 1226 or 1283 was monitored by Western blotting: Myosin VIIa protein first splits at positions 1172, 1197, 1200, 1226, or 1283, and the N-terminal fragment (residues 1-1172, 1-1197, 1-1200, 1-1226, or 1-1283) is recombinantly fused with the N-intein, and the C-terminal fragment (residues 1173-2215, 1198-2215, 1201-2215, 1227-2215, or 1284-2215) is fused with the C-intein. HEK293T cells were co-transfected with plasmids encoding the N-terminal and C-terminal fragments in equimolar amounts, and the splicing efficiency was monitored by Western blotting. HEK293T cells transfected with full-length myosin VIIa protein were used as a control. Division sites were numbered according to the amino acid sequence of human myosin VIIa (SEQ ID NO: 15).

[0028] Figure 4 In the HEK293T cell line, the splicing yields of MYO7A and full-length MYO7A at the mitotic sites MYO7A-1172, MYO7A-1197, MYO7A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, and MYO7A-1325 were compared. Transfection with the full-length MYO7A plasmid, or with MYO7A for each mitotic site... N -IntN and IntC-MYO7A CCells were co-transfected with plasmids. Cells were lysed 48 hours post-transfection and then analyzed by Western blotting. (A) Results of Western blotting with monoclonal antibodies against the flag tag and tubulin. (B) Quantitative analysis of Western blotting density. Full-length and PTS recombinant myosin VIIa protein levels were normalized with β-tubulin and fold changes relative to the mean under “FL” conditions were calculated and expressed as a percentage. Results are shown as mean ± SD, n = 3–17. At the cleavage site MYO7A-823, the smallest fragment is the N fragment, the opposite of all other sites. FL, full length. PTS, protein trans-splicing.

[0029] Figure 5 The splicing yields of MYO7A and full-length MYO7A at the mitotic sites MYO7A-1172, MYO7A-1197, MYO7A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, and MYO7A-1325 were compared in HeLa cell lines. Transfection with the full-length MYO7A plasmid, or with MYO7A for each mitotic site... N -IntN and IntC-MYO7A C Cells were co-transfected with plasmids. Cells were lysed 48 hours post-transfection and then analyzed by Western blotting. (A) Results of Western blotting with monoclonal antibodies against the flag tag and microtubules. (B) Densitometry quantification by Western blotting. Full-length and PTS recombinant myosin VIIa protein levels were normalized with β-tubulin and fold changes were calculated relative to “FL” conditions for each experiment, expressed as a percentage. Results are shown as mean ± SD, n = 4. At the cleavage site MYO7A-823, the smallest fragment is the N fragment, the opposite of all other sites. FL, full length. PTS, protein trans-splicing.

[0030] Figure 6 The maintenance of protein-protein interactions between SANS protein and MYO7A, which is recombined at all different cleavage sites via PTS (MYO7A acts as a bait protein). Transfection with a full-length (FL) MYO7A plasmid, transfecting only the N or C fragment, or transfecting with MYO7A for each cleavage site. N -IntN and IntC-MYO7A C HEK293T cells were co-transfected with the plasmid. Cells were lysed 48 hours post-transfection, and then immunoprecipitation analysis was performed using MYO7A as the bait protein. Results with an interaction greater than 50% were considered positive. Results are shown as mean ± SD, n = 3–4. The C segment of the split site MYO7A-823 contains the entire MF1 domain and is therefore expected to interact with SANS. -: Negative control, consisting of cells transfected with the SANS plasmid and supplemented with an empty pUC vector; FL, full length; N, N-fragment; C, C-fragment; PTS, protein trans-splicing.

[0031] Figure 7 The maintenance of protein-protein interactions between the SANS protein and MYO7A, which is recombined at all different cleavage sites via PTS (SANS acts as a bait protein). Transfection with a full-length (FL) MYO7A plasmid, or with MYO7A for each cleavage site. N -IntN and IntC-MYO7A C HEK293T cells were co-transfected with plasmids. Cells were lysed 48 hours post-transfection, and immunoprecipitation analysis was performed using SANS as the bait protein, followed by Western blotting. Anti-FLAG and anti-HA antibodies were used to detect myosin VIIa-3xFLAG and SANS-HA proteins, respectively. M, MYO7A; FL, full-length; PTS, trans-splicing; IP, input; E, elution.

[0032] Figure 8 Maintenance of ATPase activity in MYO7A protein recombined at all different cleavage sites via PTS. Transfection with full-length (FL) MYO7A plasmid, transfection of only the C fragment, or transfection with MYO7A for each cleavage site. N -IntN and IntC-MYO7A C HEK293T cells were co-transfected with the plasmid. Cells were lysed 48 hours post-transfection, followed by ATPase activity assays. Results equal to or greater than 1.2 au were considered positive, as a 1.2 au cutoff was established based on the mean of full-length positive controls. Results are shown as mean ± SD, n = 3–13. FL, full-length; PTS, trans-splicing; au, arbitrary units; C + Positive control; C - , negative control.

[0033] Figure 9 Comparison of the degradation effects of different degrader sequences mediated by different MYO7A cleavage sites in HEK293T cells. MYO7A cells fused with degraders selected at each cleavage site. N -IntN and IntC-MYO7A C Cells were co-transfected with plasmids. Cells were lysed 48 hours post-transfection and then analyzed by Western blotting. Western blotting was performed using monoclonal antibodies against the flag tag and microtubules. Representative images are provided. FL, full length.

[0034] Figure 10. Maintenance of protein-protein interactions between SANS protein and PTS-recombinant MYO7A protein containing degraders. Transfection with full-length (FL) MYO7A plasmid or with MYO7A containing degraders. N -IntN and IntC-MYO7A C HEK293T cells were co-transfected with plasmids. Cells were lysed 48 hours post-transfection, and then immunoprecipitation analysis was performed using MYO7A as the bait protein, followed by Western blotting. Anti-FLAG and anti-HA antibodies were used to detect myosin VIIa-3xFLAG and SANS-HA proteins, respectively. (A) Representative Western blotting images of some candidate proteins. (B) Quantitative analysis of fold change in immunoprecipitation assays. Results with an interaction greater than 50% were considered positive. Results are shown as mean ± SD, n = 3–4. C - : Negative control, consisting of cells transfected with the SANS plasmid and supplemented with an empty pUC vector; FL, full length; M, MYO7A; IP, input; E, elution.

[0035] Figure 11 Maintenance of ATPase activity in PTS-containing recombinant MYO7A protein with degraders. Transfection with full-length (FL) MYO7A plasmid, transfecting only the C fragment, or using MYO7A containing degraders. N -IntN and IntC-MYO7A C HEK293T cells were co-transfected with the plasmid. Cells were lysed 48 hours post-transfection, followed by ATPase activity assays. Results equal to or greater than 1.2 au were considered positive, as a 1.2 au cutoff was established based on the mean of full-length positive controls. Results are shown as mean ± SD, n = 3–13. FL, full-length; au, any unit; C+, positive control; C-, negative control.

[0036] Figure 12 Protein transsplicing for recombinant MYO7A in the retina of WT mice. An in vivo proof-of-concept study using the GRK1 promoter was conducted in adult WT mice after subretinal injection. The studies were performed at (A) VHIO (Barcelona, ​​Spain) and (B) EyeCRO (USA). Subretinal delivery of AAVs containing N and C fragments of MYO7A (cleavage site MYO7A-1172) conjugated with a splitting inteptide was performed at low doses (LD) of 5E+9 vg / eye and high doses (HD) of 1E+10 vg / eye. Samples were analyzed by Western blot 4 weeks post-injection. Unreacted N fragment starting material was rarely detected in only a few animals, while C fragment accumulation was slightly detected. Each lane corresponded to a different injected retina. LD, low dose; HD, high dose; NT, untreated; C +An in vitro positive control of HEK293T cells transfected with MYO7A N and C fragments and coupled with the dividing intron. Invention Details The polynucleotide combination encoding the first and second fusion proteins The limited carrying capacity of AAV vectors prevents them from being used to deliver large genes, such as in gene therapy. MYO7A Genes. For delivery to patients. MYO7A The inventors utilized the intrinsic ability of trans-splicing proteins mediated by splitting intini to achieve large-scale... MYO7A After the gene is fragmented into two laterally attached polypeptides containing peptides, it is reconstructed from a large... MYO7A Genetically encoded macromyosin VIIa protein.

[0038] This disclosure relates to combinations of polynucleotides, comprising: i) A first polynucleotide encoding the first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and an N-cleavage inteptide, which are fused directly or indirectly through a linker. ii) A second polynucleotide encoding the second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. The expression of the first and second polynucleotides in the subjects generates myosin VIIa protein through protein splicing.

[0039] According to this disclosure, the combination of polynucleotides comprises a first polynucleotide encoding a first fusion protein and a second polynucleotide encoding a second fusion protein.

[0040] According to this disclosure, the term "combination" means that the first and second polynucleotides according to this disclosure can be formulated in a single formulation or as separate formulations.

[0041] According to this disclosure, the terms "nucleic acid sequence," "nucleic acid molecule," "nucleotide sequence," or "polynucleotide" are used interchangeably and refer to any molecule composed of or containing monomeric nucleotides. Polynucleotides can be DNA or RNA.

[0042] In this document, the terms “peptide,” “oligopeptide,” “polypeptide,” and “protein” are used interchangeably and refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming the chain.

[0043] The term "amino acid" refers to both naturally occurring and non-natural amino acids (also referred to herein as "non-natural amino acids"), such as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds having the same basic chemical structure as naturally occurring amino acids, such as an α-carbon bound to hydrogen, carboxyl, amino, and R groups, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds having a structure different from the general chemical structure of amino acids, but functioning similarly to naturally occurring amino acids. The terms "amino acid" and "amino acid residue" are used interchangeably throughout the text.

[0044] As used herein, the term "fusion protein" refers to a recombinant protein comprising two or more protein domains, preferably covalently linked, from at least two different proteins. The fusion protein is obtained or is available through genetic fusion, for example, through the genetic fusion of at least two gene segments encoding separate domains of different proteins. In a preferred embodiment, the fusion protein is a single-chain polypeptide that may be entirely encoded by a nucleic acid sequence and comprises at least two protein domains directly covalently linked by peptide bonds or optionally covalently linked by peptide linkers.

[0045] According to this disclosure, the first fusion protein comprises an N-terminal fragment of myosin VIIa protein and an N-terminal inteptide fused directly or indirectly through a linker, and the second fusion protein comprises a C-terminal inteptide fused directly or indirectly through a linker and a C-terminal fragment of myosin VIIa protein.

[0046] As used herein, the term "connector" refers to a chemical group or molecule that links two adjacent molecules or portions. In some embodiments, the polynucleotide encodes a connector selected from the group consisting of (GGS)n (SEQ ID NO: 142), (GGGGS)n (SEQ ID NO: 143), (G)n (SEQ ID NO: 144), (EAAAK)n (SEQ ID NO: 145), XTEN-based connectors, or (XP)n motifs, or any combination thereof, wherein n is independently an integer from 1 to 50. In other embodiments, no connector is used. Instead, for example, the polynucleotide sequence comprises nucleic acids encoding first and second protein domains, and further comprises additional nucleic acids at at least one end thereof that function as a connector.

[0047] According to this disclosure, the expression of the first and second polynucleotides encoding the first and second fusion proteins in cells generates myosin VIIa protein through protein splicing.

[0048] The term “protein trans-splicing” or “protein splicing” refers to the process of cutting off the inteptide from a larger precursor polypeptide by cleaving two peptide bonds and simultaneously linking flanking protein fragments (also known as exopeptides) by forming new peptide bonds, thereby forming a mature protein and a free inteptide (Shah NH and Muir TW, Chem Sci. 2014; 5(1):446–461. 2013).

[0049] The term "intrapeptide" as used in this article refers to a protein that can attach flanking sequences (exopeptides) to a new protein.

[0050] As used herein, the terms “split inteptide” or “trans-split inteptide” refer to naturally occurring or engineered protein fragments (i.e., N-inteptides and C-inteptides) that combine to form a catalytically competent enzyme capable of catalyzing a protein splicing reaction that removes the N- and C-inteptide sequences and links flanking sequences (N- and C-inteptides) with peptide bonds.

[0051] As used herein, the term "peptide bond" refers to the covalent chemical bond -CO-NH- formed between two molecules when the carboxyl part (carboxyl component, C-component, or C-terminal component) of one molecule reacts with the amino part (amino component, N-component, or N-terminal component) of another molecule.

[0052] According to this disclosure, the terms "N-split inteptide," "N-inteptide," or "N-terminal split inteptide" refer to any N-terminal amino acid sequence of a split inteptide capable of binding to the C-terminal amino acid sequence of the split inteptide to form a functional split inteptide capable of catalyzing a protein splicing reaction that removes the N- and C-inteptide sequences and connects the flanking sequences (N- and C-inteptides) with peptide bonds.

[0053] According to this disclosure, the terms "C-inte," "C-split inteptide," or "C-terminal split inteptide" refer to any C-terminal amino acid sequence of a split inteptide capable of binding to the N-terminal amino acid sequence of the split inteptide to form a functional split inteptide capable of catalyzing a protein splicing reaction that removes the N- and C-inteptide sequences and links flanking sequences (N- and C-inteptides) with peptide bonds.

[0054] In one specific embodiment, the N- and C-cleavage inteptides according to this disclosure, respectively contained in the first and second fusion proteins, can be derived from DNA polymerase III from different organisms. DnaE The catalytic subunit of the gene, such as in cyanobacteria, including Nostoc punctata ( Nostoc punctiforme, Npu Synechocystis ( Synechocystis sp., Ssp ) PCC6803 strain, Fischneria ( Fischerella sp. PCC 9605, Monofibrillaria thunbergii ( Scytonema tolypothrichoides ), cyanobacteria ( Cyanobacteria bacterium SW_9_47-5, Foamy Cladosporium ( Nodularia spumigena ), Nostoc commune ( Nostoc flagelliforme ), *Cyclocarya var. vara* ( Crocosphaera watsonii WH 8502, Chlorella vulgaris ( Chroococcidiopsis cubana CCALA 043 or Red Sea Trichophyton ( Trichodesmium erythraeum ); preferably from Npu or Ssp.

[0055] In another specific embodiment, the N- and C-fractionation inteptides may be derived from cyanobacteria, including marine halophilic bacteria (Halophyta rubrum). R. marinus, Rma Synechocystis ( Synechocystis sp., Ssp PC6803, Porphyromonas chloroplasts (Ppu), or possibly derived from gp41-1, gp41-8, NrdJ-1 or IMPDH-1 (Carvajal-Vallejos P. et al. J Biol Chem. 2012 Aug 17; 287(34): 28686–28696).

[0056] In a preferred embodiment, the N- and / or C-cleaving inteins of this disclosure, respectively contained in the first and second fusion proteins, can be engineered N- and / or C-cleaving inteins. The engineered N- and / or C-cleaving inteins can be engineered by introducing mutations into the native N- and / or C-cleaving intein sequences, particularly to enhance protein splicing activity.

[0057] In a preferred embodiment, the N- and / or C- cleavage inteptide sequences contained in the first and second fusion proteins respectively comprise or consist of amino acid sequences selected from any N- and C-cleavage inteptides listed in Table 1 below:

[0058] Table 1: Examples of N- and C-split inteptide pairs that may be used according to this disclosure. Bold amino acids may be replaced with GEP amino acids to improve exopeptide tolerance.

[0059] In specific embodiments, the N- and C-split inteins according to this disclosure contained in the first and second fusion proteins respectively are N- and C-split inteins comprising or composed of SEQ ID NO: 1 (Cfa-N-split inteptide) and SEQ ID NO: 2 (Cfa-C-split inteptide), 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 (Nrdj1-N) and 12 (Nrdj1-C) or any functional variant thereof.

[0060] As used herein, the term "variant" or "functional variant" refers to a polypeptide sequence derived from the N- and / or C-cleaved inteptides described above, which contains alterations, i.e., substitutions, insertions, and / or deletions, at one or more positions, but retains the ability to form a functional enzyme when combined to catalyze a protein splicing reaction that cleaves the N- and C-inteptide sequences and links flanking sequences (N- and C-exo peptides) with peptide bonds.

[0061] Variants can be obtained using a variety of techniques well known in the art. Examples of techniques for altering the nucleotide sequence encoding a natural protein include, but are not limited to, site-directed mutagenesis, random mutagenesis, and the construction of synthetic oligonucleotides.

[0062] The protein splicing efficiency of N- and / or C-split intein functional variants can be assessed, for example, by measuring the protein reconstruction efficiency in cells. Specifically, protein reconstruction efficiency can be determined by expressing a combination of polynucleotides in cells, the first polynucleotide encoding an N-terminal fragment of a reporter protein (e.g., GFP) fused to an N-split intein, and the second polynucleotide encoding a C-terminal fragment of the reporter molecule fused to a C-split intein. The reporter protein reconstruction efficiency can then be monitored by measuring the expression level of the reconstituted protein.

[0063] The expression level of the reconstructed protein can be determined by any suitable method known to those skilled in the art. The amount of protein can be measured, for example, by semi-quantitative Western blotting, enzyme labeling and mediated immunoassays such as ELISA, biotin / antibiotin proteophoresis, radioimmunoassay, immunoelectrophoresis, mass spectrometry or immunoprecipitation, or by a protein or antibody array. In a particular embodiment, when the reporter protein is a fluorescent protein, the amount of protein can be measured by flow cytometry or fluorescence microscopy.

[0064] The expression levels are then compared to control values. According to a preferred embodiment, the term "control value" refers to the expression level of the protein reconstituted with native N- and C-cleaving introns in cells expressing a combination of polynucleotides, wherein the first polynucleotide encodes an N-terminal fragment of a reporter protein (e.g., GFP) fused with a native N-cleaving intron, and the second polynucleotide encodes a C-terminal fragment of the reporter molecule fused with a native C-cleaving intron.

[0065] When the expression level of the protein reconstructed in cells using a functional variant of the N- and / or C-cleavage inteptide is similar to that of the control (i.e., the expression level of the protein reconstructed using native N- and C-cleavage inteptides), especially when the change in expression level is less than 40%, 30%, 20%, or 10% of the control, the protein reconstruction efficiency of the functional variant is similar to that of the native cleavage inteptide in cells.

[0066] As used herein, the term “variant” or “functional variant” may refer to a polypeptide having an amino acid sequence that is at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity with any of the above-mentioned N- and / or C-split inpeptides (particularly Table 1), and preferably retains the protein splicing capability of the above-mentioned polypeptides.

[0067] In a particular embodiment, the N-fractional inteptide and C-fractional inteptide contained in the first and second fusion proteins according to the present disclosure comprise or consist of SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10 or SEQ ID NO: 11 and 12 or any functional variant thereof, preferably having 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity with any amino acid sequence selected from SEQ ID NO: 1 to 12.

[0068] As used in this paper, the percentage identity between two sequences is a function of the number of common positions shared by the sequences (i.e., % identity = number of common positions / total number of positions × 100), taking into account the number of gaps and the length of each gap; the gap length needs to be introduced for optimal alignment of the two sequences. Sequence comparison and the determination of the percentage identity between two sequences can be accomplished using mathematical algorithms, as described below. The Needleman and Wunsch algorithms (NEEDLEMAN and Wunsch) can be used to determine the percentage identity between two amino acid sequences.

[0069] The percentage identity between two nucleotide or amino acid sequences can also be determined using algorithms such as EMBOSSNeedle (pairwise alignment; available at www.ebi.ac.uk, Rice et al 2000 Trends Genet 16:276-277). For example, EMBOSSNeedle can be used with the BLOSUM62 matrix, with a "vacancy open penalty" of 10 and a "vacancy extension penalty" of 0.5, and a pseudo-"terminal vacancy penalty" of 10 and 0.5. Typically, "percentage identity" is a function of the number of matching positions divided by the number of comparison positions and multiplied by 100. For example, if 6 out of 10 sequence positions are identical between the two comparison sequences after alignment, then the identity is 60%. % identity is usually determined over the full length of the query sequence being analyzed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical, regardless of any chemical and / or biological modifications.

[0070] The term "variant" or "functional variant" can also refer to a polypeptide having an amino acid sequence that differs from the native sequence by less than 10, 9, 8, 7, 6, 5, 4, or 3 substituted, inserted, and / or deleted amino acids. In a preferred embodiment, the variant differs from the native sequence in one or more conserved substitutions, preferably less than 10, 9, 8, 7, 6, 5, 4, or 3 conserved substitutions. Examples of conserved substitutions include basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (methionine, leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, and threonine).

[0071] The inventors previously engineered N- and C-cleaving intekines with excellent protein trans-splicing properties. They demonstrated that Cfa-N and Cfa-C-cleaving intekines have higher protein splicing efficiency than Npu cleaving intekines (WO2017 / 132582 and WO2021 / 191447).

[0072] In a preferred embodiment, the N- and C-cleaving intekines according to this disclosure contained in the first and second fusion proteins, respectively, are Cfa-N- and Cfa-C-cleaving intekines, comprising or consisting of SEQ ID NO: 1 (Cfa-N-cleaving intekine) and SEQ ID NO: 2 (Cfa-C-cleaving intekine) or any functional variant thereof, preferably having 70, 75, 80, 85, 90, 95, 98, or 99% sequence identity with SEQ ID NO: 1 and / or 2.

[0073] In a preferred embodiment, functional variants of the Cfa N- and / or Cfa C- cleavage inteptides retain the functional splicing activity of the native Cfa cleavage inteptide, and more preferably have a higher protein splicing efficiency than the Npu cleavage inteptide.

[0074] The protein splicing efficiency of functional variants of Cfa-N- and / or Cfa-C-split inteins can be evaluated as described above. In a preferred embodiment, the expression levels of the reconstituted protein and functional variants of Cfa-N- and / or Cfa-C-split inteins are compared to a control, which refers to the expression level of the protein reconstituted with Npu inteins.

[0075] The protein reconstruction efficiency in cells is higher when the expression level of the protein reconstituted in cells with engineered Cfa-N- and / or Cfa-C- cleavage inteptides is at least 1.5 times higher, or 2, 3, 4, 5 times higher, or even higher than the control value (e.g., with Npu inteptides).

[0076] In a more preferred embodiment, functional variants of the Cfa N- and / or Cfa C- cleaving intepids can also splice faster than the Npu cleaving intepid, preferably at least 1.5 times, or 2, 3 times, or even more. Protein trans-splicing activity can be measured by incubating the N- and C-intepids separately in a splicing buffer containing 2 mM tris(2-carboxyethyl)phosphine (TCEP) (e.g., 100 mM sodium phosphate, 150 mM sodium chloride, 1 mM EDTA, pH 7.2) for 15 minutes. Splicing is initiated by mixing the N- and C-intepids and quenched by adding 8 M guanidine hydrochloride and 4% trifluoroacetic acid (TFA) (3:1 v / v). The splicing reaction progress can be monitored by RP-HPLC or SDS-PAGE. When using RP-HPLC, the area of ​​each individual peak is normalized relative to the total area of ​​all merged peaks, and a reaction curve is plotted (see detailed protocol, paragraphs

[00724] -

[00731] of application WO2017 / 132580). When using SDS-PAGE, β-tubulin is used as a loading control, and the splice products are quantified by density assay.

[0077] A key indication of the splicing-based approach is that all characterized introns exhibit sequence preference at exopeptide residues adjacent to the splice site. Engineered versions of naturally occurring split introns, particularly those containing GEP amino acids at positions 20, 21, and 22, demonstrate improved exopeptide tolerance, with the residues numbered according to SEQ ID NO: 2.

[0078] According to this disclosure, it is derived from DNA polymerase III ( DnaEThe C-cleavage inteptide of the catalytic subunit of the gene may contain GEP amino acids at positions 20, 21, and 22, wherein the residues are identified according to SEQ ID NO: 2 (see Stevens etal., J Am Chem Soc. 2016 Feb 24; 138(7): 2162–2165, or WO2017 / 132580). Figure 7 A and B, C-inpeptides of SEQ ID NO: 5-358, specifically replacing the amino acid positions shown in bold in Table 1.

[0079] In a preferred embodiment, the C-fractionation inteptide is selected from any C-fractionation inteptide disclosed in Table 2.

[0080] Table 2: Engineered C-cleavage inteptides with improved tolerance to exopeptides In a preferred embodiment, the C-split inteptide is a C-split inteptide or any functional variant thereof having a mutation of a GEP amino acid at positions 20, 21, and 22, wherein the residues, according to SEQ ID NO: 2, preferably retain the functional splicing activity of the aforementioned split inteptide, and more preferably have improved exopeptide tolerance.

[0081] In a particular embodiment, the C-split inteptide may be a functional variant of the C-split inteptide as described in Table 2, preferably comprising or consisting of the amino acid sequence SEQ ID NO: 13 or 14, or any functional variant thereof having 70, 75, 80, 85, 90, 95, 98, or 99% sequence identity with any one of the sequences SEQ ID NO: 13 or 14, preferably retaining the functional splicing activity of the above-described C-split inteptide, and more preferably having improved exopeptide tolerance.

[0082] Exopeptide tolerance can be assessed, for example, in a kanamycin resistance assay as described on page 60 of WO2017 / 132580, paragraphs

[00735] -

[00738] , wherein a fragmented aminoglycoside phosphotransferase encoding a cleavage inteptide fused to a C-exopeptide with F, G, R, or E present at position +2 is transformed into DH5α competent cells and cultured at different concentrations of kanamycin. Cell density at 650 nm is measured at the 24-hour endpoint, and IC50 values ​​are determined and compared with Cfa-cmut. A functional variant with improved exopeptide tolerance is a C-cleavage inteptide having a similar IC50 to a cleavage inteptide containing GEP amino acids at positions 20, 21, and 22, wherein the residues are numbered according to SEQ ID NO: 2.

[0083] In a preferred embodiment, the C-split inteptide is a Cfa-Cmut split inteptide comprising or composed of SEQ ID NO: 13 or any functional variant thereof having 70, 75, 80, 85, 90, 95, 98, or 99% sequence identity with SEQ ID NO: 13, preferably retaining the functional splicing activity of the C-split inteptide as described above, more preferably having improved exopeptide tolerance, and even more preferably having a similar IC50 to Cfa-Cmut comprising or composed of SEQ ID NO: 2. In particular, the IC50 variation is less than 40%, 30%, 20%, or 10% of the positive control value (i.e., the IC50 of Cfa-c-mut comprising or composed of SEQ ID NO: 13).

[0084] In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises a first polynucleotide encoding a first fusion protein, which from 5' to 3' comprises: an N-terminal fragment of myosin VIIa protein, and a Cfa N-cleavage inteptide of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 1, fused directly or indirectly via a linker; and a second polynucleotide encoding a second fusion protein, which from 5' to 3' comprises: a Cfa C-cleavage inteptide of SEQ ID NO: 2 or a Cfa Cmut-cleavage inteptide of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of myosin VIIa protein, fused directly or indirectly via a linker.

[0085] The inventors utilized the intrinsic ability of trans-splicing of proteins mediated by the aforementioned splitting intini peptides to reconstruct the macromyosin VIIa protein after fragmenting it into two polypeptides with splitting intini peptides attached to its sides.

[0086] Depend on MYO7A The gene encodes an unconventional myosin VIIa protein (also referred to herein as MYO7A protein), a member of the myosin gene family. Myosin is a mechanochemical protein characterized by the presence of a motor domain, an actin-binding domain, a neck domain for interacting with other proteins, and a tail domain that acts as an anchor. This gene encodes an unconventional myosin with a very short tail.

[0087] Specifically, myosin VIIa protein is human myosin VIIa protein (UniProtKB accession number: Q13402; updated September 13, 2023, by...). MYO7AGene coding (gene ID: 4647, updated September 7, 2023), also known as DFNB2; MYU7A; NSRD2; USH1B; DFNA11; MYOVIIA Alternative splicing produces multiple protein isotypes, such as myosin VIIa isotype 1 (NCBI reference sequence: NP_000251.3, updated June 4, 2023, SEQ ID NO: 15) or myosin VIIa isotype 2 (NCBI reference sequence: NP_001120652.1, updated June 4, 2023, SEQ ID NO: 90). According to this disclosure, the term "myosin VIIa protein" encompasses all known protein isotypes of myosin VIIa protein.

[0088] In a preferred embodiment, the myosin VIIa protein according to this disclosure may be the human myosin VIIa protein disclosed as described above or any functional variant thereof.

[0089] Preferably, the term "variant" or "functional variant" as used herein refers to a polypeptide having an amino acid sequence that is at least 70, 75, 80, 85, 90, 95, 98, or 99% sequence identity with the natural sequence and retaining the function of the polypeptide, in this context being myosin VIIa protein, preferably human myosin VIIa protein (SEQ ID NO: 15), particularly the actin-based motor activity of human myosin VIIa protein.

[0090] Myosin VIIa is a functional actin-based motor. Myosin VIIa binds to melanosomes, moves along actin filaments to the apical retinal pigment epithelium (RPE), and binds to RPE phagosomes, promoting its motility.

[0091] The activity of functional variant myosin VIIa can be assessed by correcting melanosome localization and ciliary opsin distribution in RPE in Myo7a mutant Shaker-1 mouse model (Self T et al. development, 1998, Feb;125(4):557-66).

[0092] In a preferred embodiment, the myosin VIIa protein is human myosin VIIa protein, comprising or consisting of any functional variant of SEQ ID NO: 15 or having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 15.

[0093] More preferably, the term "variant" or "functional variant" refers to a polypeptide having a sequence differing from the native sequence by less than 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, or 5 substituted, inserted, and / or deleted amino acid sequences. In a preferred embodiment, the functional variant differs from the native sequence in that it has one or more conserved substitutions, preferably less than 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, or 5 conserved substitutions. In some embodiments, the first methionine in the myosin VIIa protein sequence may be removed. Many different mammalian myosin VIIa are known, including but not limited to human, pig, chimpanzee, dog, cow, mouse, rabbit, or rat myosin, and can be readily found in sequence databases. The coding sequence can be readily determined by those skilled in the art based on the polypeptide sequence.

[0094] According to this disclosure, a polynucleotide encoding the myosin VIIa protein as described above is split into at least two nucleic acid sequences encoding N- and C-terminal myosin VIIa protein fragments, each fragment being fused with at least the N- and C-terminal introns as described above to form first and second fusion proteins, in such a manner that, after expression of the first and second fusion proteins, a protein splicing reaction can occur in the cell and induce the excision of the N- and C-terminal intron sequences and the linkage of flanking sequences of the myosin VIIa fragments (N- and C-terminal myosin VIIa fragments, also referred to as N- and C-terminal exopeptides) via peptide bonds to reconstruct the full-length myosin VIIa protein as described above in the cell.

[0095] According to this disclosure, the myosin VIIa protein or any functional variant thereof, as described above, can cleave into N-terminal and C-terminal myosin VIIa fragments at any location. However, the inventors have demonstrated that certain specific cleavage positions are particularly advantageous for improving the recombination efficiency of the myosin VIIa protein. Specifically, the myosin VIIa protein or any functional variant thereof, as described above, can cleave into N-terminal and C-terminal myosin VIIa fragments at cleavage positions between amino acids 823-824, 1172-1173, 1197-1198, 1200-1201, 1226-1227, 1283-1284, or 1325-1326, preferably between amino acids 1172-1173, 1197-1198, 1200-1201, 1226-1227, or 1283-1284, wherein said residues are numbered according to SEQ ID NO: 15. Those skilled in the art will readily determine the cleavage site in myosin VIIa isotypes or functional variants of myosin VIIa, particularly by means of, Figure 1 The sequence alignment shown.

[0096] In a preferred embodiment, the N-terminal myosin VIIa fragment terminates at residue 823 and the C-terminal myosin VIIa protein fragment begins at residue 824, wherein the residues are numbered according to SEQ ID NO: 15.

[0097] In another preferred embodiment, the N-terminal myosin VIIa fragment terminates at residue 1172 and the C-terminal myosin VIIa protein fragment begins at residue 1173, wherein the residues are numbered according to SEQ ID NO:15.

[0098] In another preferred embodiment, the N-terminal myosin VIIa fragment terminates at residue 1197 and the C-terminal myosin VIIa protein fragment begins at residue 1198, wherein the residues are numbered according to SEQ ID NO:15.

[0099] In another preferred embodiment, the N-terminal myosin VIIa fragment terminates at residue 1200 and the C-terminal myosin VIIa protein fragment begins at residue 1201, wherein the residues are numbered according to SEQ ID NO:15.

[0100] In another preferred embodiment, the N-terminal myosin VIIa fragment terminates at residue 1226 and the C-terminal myosin VIIa protein fragment begins at residue 1227, wherein the residues are numbered according to SEQ ID NO:15.

[0101] In another preferred embodiment, the N-terminal myosin VIIa fragment terminates at residue 1283 and the C-terminal myosin VIIa protein fragment begins at residue 1284, wherein the residues are numbered according to SEQ ID NO:15.

[0102] In another preferred embodiment, the N-terminal myosin VIIa fragment terminates at residue 1325 and the C-terminal myosin VIIa protein fragment begins at residue 1326, wherein the residues are numbered according to SEQ ID NO:15.

[0103] In a particular embodiment, the first and second fusion proteins according to this disclosure comprise an N-terminal myosin VIIa fragment and a C-terminal myosin VIIa fragment, respectively composed of amino acid sequences selected from Table 3, preferably selected from the following pairs: 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, more preferably SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, and SEQ ID NO: 26 and 27, or any functional variant thereof, preferably with SEQ ID NO: 16 to 29, preferably SEQ ID NO: Any one of 18 to 27 has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity, and preferably the reconstructed functional variant of myosin VIIa protein retains the function of said myosin VIIa, particularly the actin-based motor activity of human myosin VIIa protein. The function of the myosin VIIa functional variant can be assessed, for example, by the ability of said variant to correct the melanosome localization and ciliated opsin distribution of RPE in the Myo7a mutant Shaker-1 mouse model (Self T et al. development, 1998, Feb;125(4):557-66).

[0104] Table 3: Examples of N-terminal and C-terminal myosin VIIa fragments. Bold amino acids in full-length myosin VIIa indicate preferred cleavage positions. The sequences listed in Table 3 indicate the first methionine and N-terminal fragment sequences of myosin VII. In some embodiments, the first methionine may be retained or removed.

[0105] In a preferred embodiment, the polynucleotide combination according to this disclosure comprises: - The first polynucleotide encoding the first fusion protein, comprising from 5' to 3': the N-terminal fragment of myosin VIIa protein and an N-cleavage inteptide, which are fused directly or indirectly via a linker, and - A second polynucleotide encoding a second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. The first and second fusion proteins contain: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284. The residues thereon are numbered according to SEQ ID NO: 15, or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO: 15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are composed of amino acid sequences selected from the following pairs: 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%, or 99% sequence identity with any one of the sequences SEQ ID NO: 16 to 29.

[0106] In a more preferred embodiment, the polynucleotide combination according to this disclosure comprises: - A first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and the Cfa N-cleavage inteptide of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with 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': the Cfa-C cleavage inteptide of SEQ ID NO: 2 or the Cfa-Cmut cleavage inteptide of SEQ ID NO: 13, or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of myosin VIIa protein, which is fused directly or indirectly through a linker. The first and second fusion proteins contain: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284. The residues thereon are numbered according to SEQ ID NO: 15, or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO: 15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are composed of amino acid sequences selected from the following pairs: 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%, or 99% sequence identity with any one of the sequences SEQ ID NO: 16 to 29.

[0107] In a more preferred embodiment, the polynucleotide combination according to the present disclosure comprises a polynucleotide encoding a first fusion protein and a second fusion protein, which is selected from the amino acid sequences of the pairs disclosed in Table 4, preferably selected from 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 70, 75, 80, 85, 90, 95, 98 or 99% identity with any one of the sequences SEQ ID NO: 30-43.

[0108] Table 4: Preferred second protein fusions according to this disclosure. The sequences of the Cfa-N-cleavage inpeptide and the Cfa-C-mut-cleavage inpeptide are shown in bold. In some embodiments, the first methionine of the fusion protein is indicated and may be retained or removed.

[0109] To prevent the accumulation of unwanted starting material and to eliminate cleaved inte peptide fragments, degraders can be fused directly or indirectly to the first and / or second fusion proteins via linkers to mediate the degradation of the cleaved inte peptides.

[0110] A “protein degradation signal” or “degrader” refers to a peptide fragment that induces the degradation of a protein containing that fragment. Protein degradation can occur through any of many known protein degradation pathways, including but not limited to ubiquitination, lysosomal degradation, or autophagy. In a preferred embodiment, the degrader targets the protein to the ubiquitin-proteasome pathway. In a more preferred embodiment, the degrader according to this disclosure comprises an amino acid sequence having fewer than 200, 190, 180, 170, 150, 130, 125, 110, 100, 95, 90, 85, 80, or 75 amino acids.

[0111] In a particular embodiment, the degrader is fused directly or indirectly via a linker to an N-fractionation inteptide or a C-fractionation inteptide respectively contained in the first and second fusion proteins, preferably the degrader is located at the 3' end of the N-fractionation inteptide or the 5' end of the C-fractionation inteptide.

[0112] In a particular embodiment, the degrader is fused directly or indirectly via a linker to the N-fractionation inteptide and the C-fractionation inteptide contained in the first and second fusion proteins, respectively, preferably the degrader is located at the 3' end of the N-fractionation inteptide and the 5' end of the C-fractionation inteptide.

[0113] In a preferred embodiment, the polynucleotide combination according to this disclosure comprises: - A first polynucleotide encoding the first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein, an N-cleavage inteptide, which is fused directly or indirectly via a linker, and - A second polynucleotide encoding a second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. The first or second fusion protein further comprises a degrader, preferably fused directly or indirectly through a linker to an N-cleavage inteptide or C-cleavage inteptide contained in the first or second fusion protein, more preferably the degrader is located at the 3' end of the N-cleavage inteptide or the 5' end of the C-cleavage inteptide.

[0114] In a preferred embodiment, the polynucleotide combination according to this disclosure comprises: - A first polynucleotide encoding the first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein, an N-cleavage inteptide, which is fused directly or indirectly via a linker, and - A second polynucleotide encoding a second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. The first and second fusion proteins further comprise degraders, preferably fused directly or indirectly via a linker to the N-fractional inteptides and C-fractional inteptides contained in the first and second fusion proteins, more preferably the degraders are located at the 3' end of the N-fractional inteptide and the 5' end of the C-fractional inteptide.

[0115] In a more preferred embodiment, the polynucleotide combination according to the present disclosure comprises: - A first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and the Cfa N-cleavage inteptin of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with 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': the Cfa-c-cleavage inteptide of SEQ ID NO: 2 or the Cfa-Cmut-cleavage inteptide of SEQ ID NO: 13, or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of myosin VIIa protein, which is fused directly or indirectly through a linker. Furthermore, the first or second fusion protein further comprises a degrader, preferably fused directly or indirectly through a linker to an N-cleavage inteptide or C-cleavage inteptide contained in the first or second fusion protein, more preferably the degrader is located at the 3' end of the N-cleavage inteptide or the 5' end of the C-cleavage inteptide.

[0116] In a more preferred embodiment, the polynucleotide combination according to the present disclosure comprises: - A first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and the Cfa N-cleavage inteptin of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with 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': the Cfa-c-cleavage inteptide of SEQ ID NO: 2 or the Cfa-Cmut-cleavage inteptide of SEQ ID NO: 13, or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of myosin VIIa protein, which is fused directly or indirectly through a linker. Furthermore, the first and second fusion proteins further comprise degraders, preferably fused directly or indirectly via a linker to the N-fractional inteptides and C-fractional inteptides contained in the first and second fusion proteins, more preferably the degraders are located at the 3' end of the N-fractional inteptide and the 5' end of the C-fractional inteptide.

[0117] In a more preferred embodiment, the polynucleotide combination according to the present disclosure comprises: - A first polynucleotide encoding the first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein, an N-cleavage inteptide, which is fused directly or indirectly via a linker, and - A second polynucleotide encoding a second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. The first and second fusion proteins contain: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284. The residues thereon are numbered according to SEQ ID NO: 15, or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO: 15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are composed of amino acid sequences selected from the following pairs: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21 or 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%, or 99% sequence identity with any one of sequences SEQ ID NO: 16 to 29, and The first or second fusion protein further comprises a degrader, preferably fused directly or indirectly through a linker to an N-cleavage inteptide or C-cleavage inteptide contained in the first or second fusion protein, more preferably the degrader is located at the 3' end of the N-cleavage inteptide or the 5' end of the C-cleavage inteptide.

[0118] In a more preferred embodiment, the polynucleotide combination according to the present disclosure comprises: - A first polynucleotide encoding the first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein, an N-cleavage inteptide, which is fused directly or indirectly via a linker, and - A second polynucleotide encoding a second fusion protein, comprising from 5' to 3': a C-terminal inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. The first and second fusion proteins contain: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. - Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284; or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO: 15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are composed of amino acid sequences selected from the following pairs: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21 or 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%, or 99% sequence identity with any one of sequences SEQ ID NO: 16 to 29, and The first and second fusion proteins further comprise degraders, preferably fused directly or indirectly via a linker to the N-fractional inteptides and C-fractional inteptides contained in the first and second fusion proteins, more preferably the degraders are located at the 3' end of the N-fractional inteptide and the 5' end of the C-fractional inteptide.

[0119] In a more preferred embodiment, the polynucleotide combination according to the present disclosure comprises: - A first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and the Cfa N-cleavage inteptin of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with 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': the Cfa-c-cleavage inteptide of SEQ ID NO: 2 or the Cfa-Cmut-cleavage inteptide of SEQ ID NO: 13, or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of myosin VIIa protein, which is fused directly or indirectly through a linker. The first and second fusion proteins contain: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. - Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284; or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO: 15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are composed of amino acid sequences selected from the following pairs: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21 or 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%, or 99% sequence identity with any one of SEQ ID NO: 16 to 29, and The first or second fusion protein further comprises a degrader, preferably fused directly or indirectly through a linker to an N-cleavage inteptide or C-cleavage inteptide contained in the first or second fusion protein, more preferably the degrader is located at the 3' end of the N-cleavage inteptide or the 5' end of the C-cleavage inteptide.

[0120] In a more preferred embodiment, the polynucleotide combination according to the present disclosure comprises: - A first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and the Cfa N-cleavage inteptin of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with 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': the Cfa-c-cleavage inteptide of SEQ ID NO: 2 or the Cfa-Cmut-cleavage inteptide of SEQ ID NO: 13, or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of myosin VIIa protein, which is fused directly or indirectly through a linker. The first and second fusion proteins contain: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. - Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284; or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO: 15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are composed of amino acid sequences selected from the following pairs: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21 or 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%, or 99% sequence identity with any one of sequences SEQ ID NO: 16 to 29, and The first and second fusion proteins further comprise degraders, preferably fused directly or indirectly via a linker to the N-fractional inteptides and C-fractional inteptides contained in the first and second fusion proteins, more preferably the degraders are located at the 3' end of the N-fractional inteptide and the 5' end of the C-fractional inteptide.

[0121] In a more preferred embodiment, the polynucleotide combination according to the present disclosure encodes a first fusion protein and a second fusion protein, the first fusion protein and the second fusion protein comprising amino acid sequences selected from 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 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with any of the sequences SEQ ID NO: 30-43, and wherein the first or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage inteptide or a C-cleavage inteptide contained in the first or second fusion protein, more preferably the degrader being located at the 3' end of the N-cleavage inteptide or the 5' end of the C-cleavage inteptide.

[0122] In a more preferred embodiment, the polynucleotide combination according to the present disclosure encodes a first fusion protein and a second fusion protein, the first fusion protein and the second fusion protein comprising amino acid sequences selected from 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 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with any of the sequences SEQ ID NO: 30-43, and wherein the first and second fusion proteins further comprise degraders, preferably fused directly or indirectly via a linker to the N-cleavage inteptide and C-cleavage inteptide contained in the first and second fusion proteins, more preferably, the degraders being located at the 3' end of the N-cleavage inteptide and the 5' end of the C-cleavage inteptide.

[0123] According to this disclosure, the degradants may be selected from non-limiting examples of the degradants listed in Table 5 below.

[0124] Table 5: Examples of degradants and their corresponding sequences.

[0125] In a preferred embodiment, the degraders included in the first and / or second fusion protein according to this disclosure may be selected from the group consisting of: CL1 (SEQ ID NO: 44), Deg1 (SEQ ID NO: 45), PEST (SEQ ID NO: 46), DD1 (SEQ ID NO: 47), DD2 (SEQ ID NO: 48), V15 (SEQ ID NO: 49), M1 (SEQ ID NO: 50), M2 (SEQ ID NO: 51), SopE (SEQ ID NO: 52), SopE1-78 (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), L10 (SEQ ID NO: 59), L11 (SEQ ID NO: 60), L12 (SEQ ID NO: 59), and L12 (SEQ ID NO: 50). 61), L15 (SEQ ID NO: 62), L16 (SEQ ID NO: 63), M3 (SEQ ID NO: 64), M4 (SEQ ID NO: 65), M5 (SEQ ID NO: 66), V12 (SEQ ID NO: 67), DD4 (SEQ ID NO: 68), DD5 (SEQ ID NO: 69), DD6 (SEQ ID NO: 69) NO:70), DD7 (SEQ ID NO: 71), T1 (SEQ ID NO: 72), T2 (SEQ ID NO: 73), T3 (SEQ ID NO: 74), CL10+CL10 (SEQ ID NO: 75), CL10+CL1 (SEQ ID NO: 76), CL1+CL10 (SEQ ID NO: 77), DD8 (SEQ ID NO: 78), DD9 (SEQ ID NO: 79), DD10 (SEQ ID NO: 80), DD11 (SEQ ID NO: 81), DD12 (SEQ ID NO: 81) NO: 82), DD13 (SEQ ID NO: 83), CHA1 (SEQ ID NO: 84), CHA2 (SEQ ID NO: 85), CHA3 (SEQ ID NO: 86), CHA4 (SEQ ID NO: 87), CHA5 (SEQ ID NO: 88), AU2 (SEQ ID NO: 89), RB1 (SEQ ID NO: 90), RB2 (SEQ ID NO: 91), RB3 (SEQ ID NO:92), RB4 (SEQ ID NO: 93), RB5 (SEQ ID NO: 94), GID4-1 (SEQ ID NO: 95), GID4-2 (SEQ ID NO: 96), GID4-3 (SEQ ID NO: 97); GID4-4 (SEQ ID NO: 98), GID4-10-1 (SEQ ID NO: 99), GID4-10-3 (SEQ ID NO: 100), GID7 (SEQ ID NO: 101), GID8 (SEQ ID NO: 102), DegV1 (SEQ ID NO: 103), DegOOF (SEQ ID NO: 104), UbVR (SEQ ID NO: 105), UbR (SEQ ID NO: 106), UbAR (SEQ ID NO: 107), UbVV (SEQ ID NO: 108), Ndc10-2p (SEQ ID NO: 109), RpoS (SEQ ID NO: 110), FtsZ (SEQ ID NO: 111), SspB (SEQ ID NO: 112), Spx (SEQ ID NO: 113), Dps (SEQ ID NO: 114), ComK (SEQ ID NO: 115), SulA (SEQ ID NO: 116), HdiR (SEQ ID NO: 117), YopE (SEQ ID NO: 118), and HdiR-flip (SEQ ID NO: 119), preferably DD1, DD3, PEST, SopE, V12, M4, L2, L9, more preferably SopE, L2, L9, M4, or V12, or any combination thereof or any functional variant thereof, which induce the degradation of a fusion protein (i.e., starting material) comprising the target protein, integrins, and degraders, and also induce the degradation of the cleaved integrins fused with the degraders, preferably while maintaining the target protein (i.e., myosin VIIa protein, preferably with sequence SEQ ID NO: 119). NO: Reconstruction of any one of 44 to 119 with at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity, or any combination thereof.

[0126] In a preferred embodiment, the polynucleotide combination according to this disclosure comprises: - A first polynucleotide encoding the first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein, an N-cleavage inteptide, which is fused directly or indirectly via a linker, and - A second polynucleotide encoding a second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. The first or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to the N-cleavage inteptide and / or C-cleavage inteptide contained in the first and second fusion proteins, more preferably, the degrader is located at the 3' end of the N-cleavage inteptide and / or at the 5' end of the C-cleavage inteptide, and wherein the degrader contained in the first and / or second fusion protein according to this disclosure is selected from the group consisting of: SEQ ID NO: 44-119, preferably SEQ ID NO: 47, 49, 46, 52, 67, 65, 56 or 58, more preferably SEQ ID NO: 52, 56, 58, 65 or 67 or any combination thereof or any functional variant thereof (e.g., inducing degradation of the fusion protein (i.e., the starting material) containing the target protein, inteptide and degrader, and also inducing degradation of the cleaved inteptide fused with the degrader, preferably while maintaining the target protein (i.e., myosin VIIa protein, preferably with sequence SEQ ID NO: The reconstruction of any one of 44 to 119 having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity, or any combination thereof.

[0127] In a more preferred embodiment, the polynucleotide combination according to this disclosure comprises: - A first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and the Cfa N-cleavage inteptide of SEQ ID NO: 1 or any functional variant having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with 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': the Cfa-c-cleavage inteptide of SEQ ID NO: 2 or the Cfa-Cmut-cleavage inteptide of SEQ ID NO: 13 or any functional variant having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of myosin VIIa protein, which is fused directly or indirectly through a linker. The first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to the N-fractionation inteptide and / or C-fractionation inteptide contained in the first and second fusion proteins, respectively. More preferably, the degrader is located at the 3' end of the N-fractionation inteptide and / or at the 5' end of the C-fractionation inteptide. Furthermore, the degrader included in the first and / or second fusion protein according to this disclosure is selected from the group consisting of: SEQ ID NO: 44-119 or any combination thereof, preferably SEQ ID NO: 47, 49, 46, 52, 67, 65, 56 or 58, more preferably SEQ ID NO: 52, 56, 58, 65 or 67 or any functional variant thereof (e.g., inducing degradation of the fusion protein (i.e., the starting material) comprising the target protein, integrin and degrader, and also inducing degradation of the cleaved integrin fused with the degrader, preferably while maintaining the reconstruction of the target protein (i.e., myosin VIIa protein, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity with any one of the sequences SEQ ID NO: 44 to 119, or any combination thereof).

[0128] In a more preferred embodiment, the polynucleotide combination according to this disclosure comprises: - A first polynucleotide encoding the first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein, an N-cleavage inteptide, which is fused directly or indirectly via a linker, and - A second polynucleotide encoding a second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. The first and second fusion proteins comprise: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. The residues thereon are numbered according to SEQ ID NO: 15. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284. The residues thereon are numbered according to SEQ ID NO: 15, or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO: 15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are each composed of amino acid sequences selected from the following pairs: 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%, and 99% sequence identity with SEQ ID NO: 16 to 29, and wherein the first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to the N-cleavage inteptide and / or C-cleavage inteptide contained in the first and second fusion proteins, more preferably, the degrader is located at the 3' end of the N-cleavage inteptide and / or at the 5' end of the C-cleavage inteptide. Furthermore, the degrader included in the first and / or second fusion protein according to this disclosure is selected from the group consisting of: SEQ ID NO: 44-119 or any combination thereof, preferably SEQ ID NO: 47, 49, 46, 52, 67, 65, 56 or 58, more preferably SEQ ID NO: 52, 56, 58, 65 or 67 or any functional variant thereof (e.g., inducing degradation of the fusion protein (i.e., the starting material) comprising the target protein, integrin and degrader, and also inducing degradation of the cleaved integrin fused with the degrader, preferably while maintaining the reconstruction of the target protein (i.e., myosin VIIa protein, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity with any one of the sequences SEQ ID Nos: 44 to 119, or any combination thereof).

[0129] In a more preferred embodiment, the polynucleotide combination according to this disclosure comprises: - A first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and the Cfa N-cleavage inteptide of SEQ ID NO: 1 or any functional variant having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with 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': the Cfa-c-cleavage inteptide of SEQ ID NO: 2 or the Cfa-Cmut-cleavage inteptide of SEQ ID NO: 13 or any functional variant having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of myosin VIIa 1 protein, which is fused directly or indirectly through a linker. The first and second fusion proteins comprise: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. - Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284; or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO: 15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are each composed of amino acid sequences selected from the following pairs: 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%, or 99% sequence identity with any one of sequences SEQ ID NO: 16 to 29, and The first and / or second fusion protein further comprises a degrader, preferably fused directly or via a linker to the N-fractionation inteptide and / or C-fractionation inteptide contained in the first and second fusion proteins, respectively. More preferably, the degrader is located at the 3' end of the N-fractionation inteptide and / or at the 5' end of the C-fractionation inteptide. Furthermore, the degrader included in the first and / or second fusion protein according to this disclosure is selected from the group consisting of: SEQ ID NO: 44-119 or any combination thereof, preferably SEQ ID NO: 47, 49, 46, 52, 67, 65, 56 or 58, more preferably SEQ ID NO: 52, 56, 58, 65 or 67 or any functional variant thereof (e.g., inducing degradation of the fusion protein (i.e., the starting material) comprising the target protein, integrin and degrader, and also inducing degradation of the cleaved integrin fused with the degrader, preferably while maintaining the reconstruction of the target protein (i.e., myosin VIIa protein, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity with any one of the sequences SEQ ID NO: 44 to 119, or any combination thereof).

[0130] In a more preferred embodiment, the polynucleotide combination according to the present disclosure encodes a first fusion protein and a second fusion protein comprising amino acid sequences selected from 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 70, 75, 80, 85, 90, 95, 98, or 99% identity with any of the sequences SEQ ID NO: 30 to 43, and wherein the first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage inteptide or C-cleavage inteptide contained in the first and / or second fusion protein, more preferably the degrader is located at the 3' end of the N-cleavage inteptide and the 5' end of the C-cleavage inteptide, and wherein the degrader contained in the first and / or second fusion protein according to the present disclosure is selected from 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 with any of the sequences SEQ ID NO: 30 to 43, and wherein the first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage int The group of NO: 44-119, or any combination thereof, preferably SEQ ID NO: 47, 49, 46, 52, 67, 65, 56 or 58, more preferably SEQ ID NO: 52, 56, 58, 65 or 67 or any functional variant thereof (e.g., inducing degradation of a fusion protein comprising the target protein, an integrin and a degrader (i.e., starting material), and also inducing degradation of the cleaved integrin fused with the degrader, preferably while maintaining the recombinant of the target protein (i.e., myosin VIIa protein, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity with any of the sequences in SEQ ID NO: 44 to 119, or any combination thereof).

[0131] The functional variants of the degrader described above induce the degradation of the cleaved integument fused with the degrader, as well as the degradation of the fusion protein containing the target protein, the integument, and the starting material of the degrader, and preferably do not interfere with the recombination of the target protein (i.e., myosin VIIa protein).

[0132] The degradation of the starting material, consisting of a fusion protein containing a target protein, an integrin, and a degrader, as well as the degradation of the excised integrin fused with the degrader, can be tested according to Example 3 of WO 2021181447, specifically the fusion of the degrader with a first fusion protein containing an N-terminal fragment of an N-terminal integrin and a protein, wherein the degrader is fused to the 3' end of the N-terminal integrin, and / or the fusion of the degrader with a second fusion protein containing a C-terminal fragment of a C-terminal integrin and a protein, wherein the degrader is fused to the 5' end of the C-terminal integrin. Polynucleotides encoding the first and second proteins are transfected into cells, and the amounts of starting material and excised integrins are determined by, for example, Western blotting. When the amounts of starting material and excised integuments in cells transfected with the fusion protein without the degrader are lower than those of the starting material and excised integuments, preferably when the amount of integuments in cells transfected with the fusion protein without the degrader is at least 1.2, 1.3, 1.4, 1.5, 1.8, or 2.0 times lower than the amounts of starting material and integuments, the degrader induces degradation of the starting material (i.e., the fusion protein containing the target protein, integuments, and degrader) and the excised integuments. The expression level of the reconstituted protein can be determined by any suitable method known to those skilled in the art. The amount of reconstituted protein can be measured, for example, by semi-quantitative Western blotting, enzyme labeling, and mediated immunoassays such as ELISA, biotin / antibiotin proteophoresis, radioimmunoassay, immunoelectrophoresis, mass spectrometry, or immunoprecipitation, or by protein or antibody arrays.

[0133] On the other hand, this disclosure relates to i) a first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of the target protein and an N-cleavage inteptide, which are fused directly or indirectly via a linker; and ii) a second polynucleotide encoding a second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of the target protein, which are fused directly or indirectly via a linker. The expression of the first and second polynucleotides in the cell generates the full-length target protein through protein splicing. The first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage inteptide and / or a C-cleavage inteptide contained in the first and second fusion proteins, more preferably the degrader is located at the 3' end of the N-cleavage inteptide and / or the 5' end of the C-cleavage inteptide. Furthermore, the degraders included in the first and / or second fusion proteins according to this disclosure are selected from the group consisting of: SEQ ID NO: 44-119, preferably SEQ ID NO: 75-119, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one of the sequences SEQ ID NO: 44 to 119, preferably SEQ ID NO: 75-119.

[0134] In a more preferred embodiment, the polynucleotide combination according to the present disclosure comprises: - A first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of the target protein and the Cfa N-cleavage inpeptide of SEQ ID NO: 1 or any functional variant having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with 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': the Cfa-C-cleavage inteptide of SEQ ID NO: 2 or the Cfa-Cmut-cleavage inteptide of SEQ ID NO: 13 or any functional variant having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of the target protein, which is fused directly or indirectly via a linker. The first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage inteptide and / or a C-cleavage inteptide contained in the first and second fusion proteins, more preferably the degrader is located at the 3' end of the N-cleavage inteptide and / or the 5' end of the C-cleavage inteptide. Furthermore, the degraders included in the first and / or second fusion proteins according to this disclosure are selected from the group consisting of: SEQ ID NO: 44-119, preferably SEQ ID NO: 75-119, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one of the sequences SEQ ID NO: 44 to 119, preferably SEQ ID NO: 75-119.

[0135] In some embodiments, the first and second polynucleotides according to this disclosure that encode the first and second fusion proteins as described above may each be nucleic acid constructs.

[0136] In a particular embodiment, the first and second polynucleotides may be optimized sequences encoding the first and second fusion proteins. The term "codon optimization" means changing a codon that expresses human preference (i.e., is common in human genes but uncommon in other mammalian or non-mammalian genes) to a synonymous codon (encoding the same amino acid) that does not express human preference. Therefore, the codon change does not result in any change in the encoded protein.

[0137] As used in this article, the term "nucleic acid construct" refers to an artificial nucleic acid molecule produced using recombinant DNA technology. Nucleic acid constructs are single-stranded or double-stranded nucleic acid molecules that have been modified to contain segments of nucleic acid sequences combined and juxtaposed in a manner not normally found in nature. Nucleic acid constructs are often "carriers," meaning nucleic acid molecules used to deliver exogenously generated DNA into host cells.

[0138] The nucleic acid construct contains one or more control sequences required for the expression of the coding sequence. Typically, the nucleic acid construct contains a coding sequence and regulatory sequences preceding (5' non-coding) and following (3' non-coding) the coding sequence, which are required for the expression of the selected gene product. Therefore, the nucleic acid construct typically contains a promoter sequence, a coding sequence, and a 3' untranslated region that typically contains a polyadenylation site and / or a transcription terminator. In a preferred embodiment, the polyadenylation site is bovine growth hormone polyadenylation signal (bGH), synthetic polyadenylation signal (SynpA), and / or late or full-length simian virus 40 (SV40) polyadenylation signal (SV40).

[0139] Nucleic acid constructs may also contain additional regulatory elements, such as enhancer sequences, multi-adaptor sequences that facilitate the insertion of DNA fragments into the vector, and / or splicing signal sequences.

[0140] According to a preferred embodiment, the nucleic acid construct may contain SV40 introns.

[0141] In one embodiment, the polynucleotide or nucleic acid construct according to this disclosure includes a promoter. The promoter initiates transgenic expression upon introduction into a host cell.

[0142] In a preferred embodiment, the promoters according to this disclosure may be selected from the group consisting of: cytomegalovirus (CMV) promoters, chimeric reduced versions of CMV and chicken β-actin (CEBA) promoters, human phosphoglycerate kinase (hPGK) promoters, chimeric enhanced CMV and human phosphoglycerate kinase (ePGK) promoters, photoreceptor-specific human rhodopsin kinase (hGRK1) promoters, rod cell-specific IRBP promoters, VMD2 (vitiligoma dystrophy / Best disease) promoters, and EF1α promoters. However, any suitable promoter known in the art may be used.

[0143] As used herein, the term "promoter" refers to a regulatory element that directs the transcription of a nucleic acid operatively linked to it. A promoter can regulate the rate and efficiency of transcription of the operatively linked nucleic acid. A promoter can also be operatively linked to other regulatory elements that enhance ("enhancer") or repress ("repressor") promoter-dependent transcription of the nucleic acid. These regulatory elements include, but are not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that directly or indirectly regulate the amount of promoter transcription, including, for example, attenuators, enhancers, and silencers. A promoter is located near the transcription start site of the gene or coding sequence operatively linked to it, on the same strand and upstream of the DNA sequence (towards the 5' region of the sense strand). A promoter can be approximately 100–3000 base pairs in length. The position in the promoter is specified relative to the transcription start site of a particular gene (i.e., the upstream position is a negative number counting backwards from -1; for example, -100 is 100 base pairs upstream).

[0144] As used herein, the term "operably ligated" refers to the connection of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably ligated" when it is in a functional relationship with another nucleic acid sequence. For example, if a promoter or transcriptional regulatory sequence affects the transcription of a coding sequence, it is operably ligated to the coding sequence. Operable ligation means that the DNA sequences being ligated are usually, but not necessarily, contiguous; when it is necessary to ligate two protein-coding regions, they are contiguous and within the reading frame.

[0145] In a preferred embodiment, each polynucleotide or nucleic acid construct according to this disclosure may be contained in an expression vector.

[0146] As used herein, the term "expression vector" refers to a nucleic acid molecule used as a carrier for transferring genetic material, particularly a nucleic acid molecule that delivers nucleic acids into host cells in vitro or in vivo. Expression vectors also refer to nucleic acid molecules capable of influencing the expression of genes (transgenics) in host cells or host organisms compatible with these sequences. Expression vectors typically include at least a suitable transcriptional regulatory sequence and an optional 3'-transcriptional termination signal.

[0147] Other factors that are necessary or helpful in influencing expression may also be present, such as expression enhancers that respond to precise inducing signals (endogenous or chimeric transcription factors) or that are specific to certain cells, organs, or tissues. Vectors include, but are not limited to, plasmids, phagemids, visceral particles, transposable elements, viruses, and artificial chromosomes (e.g., YAC).

[0148] Preferably, the vector disclosed herein is a vector suitable for gene or cell therapy, particularly suitable for targeting the retina or hair cells.

[0149] In some implementations, the expression vector is a viral vector, such as a vector derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, or SNV; a lentiviral vector (e.g., derived from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), or equine infectious anemia virus (EIAV)); an adenovirus (Ad) vector; adeno-associated virus (AAV) vector; simian virus 40 (SV-40) vector; bovine papillomavirus vector; Epstein-Barr virus; herpesvirus vector; vaccinia virus vector; Harvey murine sarcoma virus vector; murine mammary tumor virus vector; and Rous sarcoma virus vector.

[0150] As is known in the art, depending on the specific viral vector to be used, a suitable sequence should be introduced into the vector of this disclosure to obtain a functional viral vector, such as an AAV ITR of an AAV vector or an LTR of a lentiviral vector. In a particular embodiment, the vector is an AAV vector.

[0151] AAV has attracted considerable interest as a potential vector for human gene therapy. Advantageous properties of the virus include its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and its wide range of cell lines derived from various tissues from which it can be infected. The AAV genome consists of a linear single-stranded DNA molecule containing 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 the origin of DNA replication and as a packaging signal for the virus. The ITRs are approximately 145 bp in length. The internal non-repetitive portion of the genome comprises two large open reading frames, designated AAV rep and cap genes. These genes encode viral proteins involved in virion replication and packaging. Specifically, at least four viral proteins, named according to their apparent molecular weight, are synthesized from the AAV rep genes Rep78, Rep68, Rep52, and Rep40. The AAV cap gene encodes at least three proteins, VP1, VP2, and VP3. For a detailed description of the AAV genome, see, for example, Muzyczka, N. 1992 Current Topics in Microbiol. and Immunol. 158:97-129.

[0152] Therefore, in one embodiment, the polynucleotide, nucleic acid construct or expression vector according to this disclosure further comprises 5'ITR and 3'ITR sequences, preferably 5'ITR and 3'ITR sequences of adeno-associated virus.

[0153] As used herein, the term "inverted terminal repeat (ITR)" refers to the nucleotide sequences located at the 5' end (5' ITR) and the 3' end (3' ITR) of a virus, which contain palindromic sequences and can fold to form T-shaped hairpin structures that act as primers during DNA replication initiation. These are also necessary for viral genome integration into the host genome; for rescue from said host genome; and for capsidating viral nucleic acids into mature viral particles. Cis-ITRs are required for vector genome replication and its packaging into viral particles.

[0154] In one embodiment, the polynucleotide, nucleic acid construct, or expression vector comprising the nucleic acid sequence encoding the first and second fusion proteins disclosed herein further comprises AAV, preferably the 5'ITR and 3'ITR of serotype AAV2.

[0155] Polynucleotides, nucleic acid constructs, or expression vectors containing nucleic acid sequences encoding the first and second fusion proteins described above can be packaged into a viral capsid to produce "viral particles," also known as "viral vector particles." In certain embodiments, polynucleotides, nucleic acid constructs, or expression vectors containing nucleic acid sequences encoding the first and second fusion proteins of this disclosure are packaged into an AAV-derived capsid to produce "adeno-associated virus particles" or "AAV particles." This disclosure relates to viral particles comprising polynucleotides, nucleic acid constructs, or expression vectors containing nucleic acid sequences encoding the first and second fusion proteins of this disclosure, and preferably containing adeno-associated virus capsid proteins.

[0156] The construction of recombinant AAV viral particles is generally known in the art and has been described, for example, in US 5,173,414 and US 5,139,941; WO92 / 01070, WO93 / 03769, Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, BJ (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, RM (1994) Human Gene Therapy 5:793-801.

[0157] Therefore, in the AAV viral particles according to this disclosure, a polynucleotide, nucleic acid construct, or expression vector comprising a nucleic acid sequence encoding a first and second fusion protein encoding an ITR of a given AAV serotype as described above can be packaged into, for example: a) viral particles composed of capsid proteins derived from the same or different AAV serotypes [e.g., AAV2 ITR and AAV5 capsid proteins; AAV2 ITR and AAV8 capsid proteins; AAV2 ITR and AAV8 capsid proteins; AAV2 ITR and AAV9 capsid proteins]; b) chimeric viral particles composed of a mixture of capsid proteins from different AAV serotypes or mutants [e.g., AAV2 ITR with AAV1 and AAV5 capsid proteins]; c) chimeric viral particles composed of capsid proteins truncated through domain exchange between different AAV serotypes or variants [e.g., AAV2 ITR with AAV5 capsid protein having an AAV3 domain].

[0158] Those skilled in the art will understand that the AAV viral particles used according to this disclosure may contain capsid proteins from any AAV serotype, including AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, sheep AAV, synthetic AAV variants such as NP40, NP59, NP84 (Paulk et al. Mol ther. 2018.26(1):289-303), LK03 (Wang L et al. Mol Ther. 2015. 23(12):1877-87), AAV3-ST (Vercauteren et al. Mol Ther.2016.24(6):1042-1049), and Anc80 (Zinn E et al., Cell Rep. 2015;12(6):1056-68), AAVrh10 and any other AAV serotypes currently known or later discovered.

[0159] Therefore, in another aspect, this disclosure relates to viral particles comprising polynucleotides, nucleic acid constructs or expression vectors, which contain nucleic acid sequences encoding the first and second fusion proteins as described above, and preferably contain capsid proteins of adeno-associated viruses, such as capsid proteins from AAV9 and AAV-PHP.B, AAV2, AAV8 and AAV5.

[0160] Therapeutic uses According to this disclosure, a combination of polynucleotides, nucleic acid constructs, expression vectors, or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins as described above are administered to subjects in need of gene therapy, preferably for the treatment of MYO7A-related diseases.

[0161] As used in this article, “gene therapy” refers to the administration of gene therapy vectors to subjects in need to treat diseases caused by alterations in the subject’s DNA sequence, particularly diseases caused by mutations in at least one gene (i.e., genetic diseases) such as MYO7A.

[0162] As used herein, the terms “treatment,” “treat,” or “treating” refer to any action aimed at improving a patient’s health condition, such as therapy, prevention, prophylaxis, and delay of a disease. In some embodiments, the term refers to the improvement or eradication of a disease or disease-related symptoms. Examples of symptoms associated with MYO7A-related diseases according to this disclosure include deafness, decreased vestibular function, and retinal degeneration.

[0163] As used herein, the terms "subject" or "patient" refer to a mammal. Mammal species that may benefit from the disclosed treatment methods include, but are not limited to, humans, non-human primates such as apes, chimpanzees, monkeys, and orangutans, domesticated animals including dogs and cats, and livestock such as horses, cattle, pigs, sheep, and goats, or other mammal species including, but not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc. In a particular embodiment, the subject is a human patient.

[0164] Combinations of multinucleotides, nucleic acid constructs, expression vectors, or viral particles containing nucleic acid sequences encoding the first and second fusion proteins according to this disclosure are generally included in pharmaceutical compositions or medicaments, optionally in combination with a pharmaceutical carrier, diluent, and / or adjuvant. The composition or pharmaceutical product contains an effective amount of the product of this disclosure and a pharmaceutically acceptable carrier or excipient sufficient to provide the desired therapeutic effect.

[0165] As used herein, the term "pharmaceuticalally acceptable" means approved by a regulatory authority or recognized by a pharmacopoeia (e.g., the European Pharmacopoeia) for use in animals and / or humans. The term "excipient" refers to a diluent, adjuvant, carrier, or medium administered with a therapeutic agent. As is well known in the art, a pharmaceutically acceptable excipient is a relatively inert substance that facilitates the administration of a pharmacologically effective substance and may be provided as a liquid solution or suspension, as an emulsion, or as a solid form suitable for dissolution or suspension in a liquid prior to use. For example, an excipient may provide form or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting agents and emulsifiers, salts for altering osmotic concentration by weight molar, encapsulating agents, pH buffers, and buffers.

[0166] In one embodiment, the pharmaceutical composition is a parenteral pharmaceutical composition, including compositions suitable for intravenous, intra-arterial, intramuscular, intranasal, intraocular, intravitreal, suprachoroidal, or subretinal administration. These pharmaceutical compositions are merely exemplary and do not limit the suitability for other parenteral and non-parenteral administration routes. The pharmaceutical compositions described herein may be packaged in single-unit or multi-unit forms.

[0167] Myosin VIIa is a type of motor protein that moves along actin filaments and transports various goods within cells. It is particularly important for the function of sensory cells, such as inner ear hair cells and retinal photoreceptors. Myosin VIIa participates in the development and maintenance of the stomatal cilia of hair cells, which are structures that detect sound vibrations. Myosin VIIa also participates in the transport of melanosomes (pigmented organelles) in retinal pigment epithelium (RPE) cells. Melanosomes protect the retina from light damage and aid in visual adaptation.

[0168] MYO7A Gene mutations can cause deafness and retinal degeneration. These mutations can affect the motor or tail domains of myosin VIIa and result in different phenotypes depending on the severity and location of the mutation. Some mutations cause Usher syndrome type 1B (USH1B), a form of deafness and blindness that also involves vestibular dysfunction. Other mutations cause non-syndromic deafness (…). DFNB2 or DFNA11 This refers to isolated hearing loss without other symptoms. MYO7A Deafness caused by mutations is due to the destruction of hair cell function and the progressive loss of hair cells. MYO7A The retinal degeneration caused by the mutation is due to the abnormal distribution of melanosomes in RPE cells, which leads to damage to photoreceptors and impaired visual adaptation.

[0169] In a more preferred embodiment, this disclosure relates to a combination of polynucleotides, nucleic acid constructs, expression vectors or viral particles, or pharmaceutical compositions thereof, comprising nucleic acid sequences encoding the first and second fusion proteins as described above, for the treatment of MYO7A-related diseases, preferably Usher syndrome type 1B, nonsyndromic deafness DFNB2 or DFNA11.

[0170] This disclosure also relates to the use of a combination of polynucleotides, nucleic acid constructs, expression vectors or viral particles, or pharmaceutical compositions thereof, comprising nucleic acid sequences encoding the first and second fusion proteins as described above, in the preparation of a medicament for treating MYO7A-related diseases.

[0171] This disclosure also provides a method for treating MYO7A-related diseases as described above in patients in need, comprising administering to the patient a therapeutically effective amount of a polynucleotide, nucleic acid construct, expression vector, or viral particle or pharmaceutical composition thereof that encodes the first and second fusion proteins as described above.

[0172] As used herein, a "therapeutic effective amount" refers to the amount effective at the dose and for the time period necessary to achieve the desired therapeutic outcome. Therapeutic effective amounts of the products of this disclosure may vary depending on factors such as an individual's disease state, age, sex, and weight, and the ability of the product or pharmaceutical composition to elicit the desired response in an individual. Dosing regimens may be adjusted to provide an optimal therapeutic response. A therapeutic effective amount is generally also the amount in which the beneficial therapeutic effect outweighs any toxic or adverse effects of the product or pharmaceutical composition. According to this disclosure, therapeutic effective amounts may, for example, reduce hearing loss, retinal degeneration, and improve vestibular function.

[0173] According to this disclosure, the combination of polynucleotides can be administered to a subject simultaneously, sequentially, or separately, either as a single formulation or as separate formulations. Such administration encompasses co-administering the first and second polynucleotides in a substantially simultaneous manner, for example, in a single formulation of the polynucleotides having a fixed ratio or in separate formulations of each polynucleotide. Furthermore, such administration also includes administering each polynucleotide sequentially or separately, at approximately the same time or at different times. Regardless of whether the polynucleotides are administered as a single formulation or as separate formulations, the first and second polynucleotides are administered to the same subject as part of the same treatment regimen. In any case, the treatment regimen will provide a beneficial effect in treating the disease described herein.

[0174] In one embodiment, a 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 therapeutic use, is administered to a subject or patient via a parenteral route, particularly via intravenous, intra-arterial, intramuscular, intranasal, intraocular, intravitreal, suprachoroidal, or subretinal route.

[0175] The amount of the product of this disclosure administered to a subject or patient may vary depending on the specific circumstances of the individual subject or patient, including the individual's age, sex, and weight; the nature and stage of the disease, the aggressiveness of the disease; the route of administration; and / or any concomitant medications prescribed to the subject or patient. Dosing regimens may be adjusted to provide the best therapeutic response.

[0176] For any given subject, the specific dosage regimen may be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the composition. The dosage ranges described herein are merely exemplary and do not limit the dosage ranges that a licensed physician may choose.

[0177] Reagent test kit In another aspect, this disclosure also relates to a kit comprising a combination of polynucleotides as described above, preferably for treating MYO7A-related diseases as described above, preferably Usher syndrome type 1B, said kit comprising: - A first polynucleotide encoding the first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein, an N-cleavage inteptide, which is fused directly or indirectly via a linker, and - A second polynucleotide encoding a second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. Optionally, the first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage inteptide and / or a C-cleavage inteptide contained in the first and second fusion proteins, more preferably the degrader is located at the 3' end of the N-cleavage inteptide and / or the 5' end of the C-cleavage inteptide, and even more preferably selected from the group consisting of SEQ ID NO: 44 to 119 or any combination thereof or any functional variant thereof that induces degradation of proteins containing the fragment, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity with any one or any combination of sequences SEQ ID NO: 44 to 119.

[0178] In a more preferred embodiment, the kit comprises: - A first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and the Cfa N-cleavage inteptin of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with 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': the Cfa C-cleavage inteptide of SEQ ID NO: 2 or the Cfa Cmut-cleavage inteptide of SEQ ID NO: 13, or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of myosin VIIa protein, which is fused directly or indirectly through a linker. Optionally, the first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage inteptide and / or a C-cleavage inteptide contained in the first and second fusion proteins, more preferably the degrader is located at the 3' end of the N-cleavage inteptide and / or the 5' end of the C-cleavage inteptide, and even more preferably any functional variant selected from SEQ ID NO: 44 to 119 or any combination thereof or inducing degradation of proteins containing the fragment, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity with any one or any combination of sequences SEQ ID NO: 44 to 119.

[0179] In a more preferred embodiment, the kit comprises: - A first polynucleotide encoding the first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein, an N-cleavage inteptide, which is fused directly or indirectly via a linker, and - A second polynucleotide encoding a second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. The first and second fusion proteins contain: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1225, and the C-terminal segment of myosin VIIa protein begins at residue 1226. - Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284; or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO: 15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are composed of amino acid sequences selected from the following pairs: 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%, or 99% sequence identity with any one of sequences SEQ ID NO: 16 to 29, and Optionally, the first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage inteptide and / or a C-cleavage inteptide contained in the first and second fusion proteins, more preferably the degrader is located at the 3' end of the N-cleavage inteptide and / or the 5' end of the C-cleavage inteptide, even more preferably any functional variant selected from SEQ ID NO: 44 to 119 or any combination thereof or inducing degradation of proteins containing the fragment, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity with any one or any combination of sequences SEQ ID NO: 44 to 119.

[0180] In a more preferred embodiment, the kit comprises: - A first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and the Cfa N-cleavage inteptin of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with 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': the Cfa C-cleavage inteptide of SEQ ID NO: 2 or the Cfa Cmut-cleavage inteptide of SEQ ID NO: 13, or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of myosin VIIa protein, which is fused directly or indirectly through a linker. The first and second fusion proteins contain: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. - Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284; or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO: 15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are composed of amino acid sequences selected from the following pairs: 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%, or 99% sequence identity with any one of sequences SEQ ID NO: 16 to 29, and Optionally, the first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage inteptide and / or a C-cleavage inteptide contained in the first and second fusion proteins, more preferably the degrader is located at the 3' end of the N-cleavage inteptide and / or the 5' end of the C-cleavage inteptide, even more preferably any functional variant selected from SEQ ID NO: 44 to 119 or any combination thereof or inducing degradation of proteins containing the fragment, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity with any one or any combination of sequences SEQ ID NO: 44 to 119.

[0181] In a more preferred embodiment, the kit comprises a polynucleotide combination encoding a first fusion protein and a second fusion protein, comprising amino acid sequences selected from 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 80%, 85%, 90%, 95%, 98%, or 99% identity with any one of sequences SEQ ID NO: 30 to 43, and optionally wherein the first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage inteptide and / or C-cleavage inteptide contained in the first and / or second fusion protein, more preferably the degrader is located at the 3' end of the N-cleavage inteptide and / or the 5' end of the C-cleavage inteptide, and even more preferably selected from SEQ ID NO: SEQ ID NO: 44 to 119 or any combination thereof or any functional variant thereof that induces the degradation of proteins containing the said fragment, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity with any one or any combination of sequences SEQ ID NO: 44 to 119.

[0182] In another aspect, this disclosure relates to a kit comprising a polynucleotide combination, said combination comprising a first polynucleotide encoding a first fusion protein, wherein from 5' to 3' it comprises: an N-terminal fragment of a target protein and an N-cleavage inteptide, which are fused directly or indirectly via a linker; and a second polynucleotide encoding a second fusion protein, wherein from 5' to 3' it comprises: a C-cleavage inteptide and a C-terminal fragment of said target protein, which are fused directly or indirectly via a linker. The first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage inteptide and / or a C-cleavage inteptide contained in the first and second fusion proteins, more preferably the degrader is located at the 3' end of the N-cleavage inteptide and / or the 5' end of the C-cleavage inteptide. Furthermore, the degradative component included in the first and / or second fusion protein according to this disclosure is selected from SEQ ID NO:44-119, preferably SEQ ID NO:75-119, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one of SEQ ID NO:44 to 119, preferably SEQ ID NO:75-119.

[0183] In a more preferred embodiment, this disclosure relates to a kit comprising a polynucleotide combination, said combination comprising: - A first polynucleotide encoding a first fusion protein, comprising from 5' to 3': an N-terminal fragment of the target protein and the Cfa N-cleavage inteptide of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with 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': the Cfa-C-cleavage inteptide of SEQ ID NO: 2 or the Cfa-Cmut-cleavage inteptide of SEQ ID NO: 13, or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98, or 99% identity with SEQ ID NO: 2 or 13, and a C-terminal fragment of the target protein, which is fused directly or indirectly via a linker. The first and / or second fusion protein further comprises a degrader, preferably fused directly or indirectly via a linker to an N-cleavage inteptide and / or a C-cleavage inteptide contained in the first and second fusion proteins, more preferably the degrader is located at the 3' end of the N-cleavage inteptide and / or the 5' end of the C-cleavage inteptide. Furthermore, the degrader contained in the first and / or second fusion protein according to this disclosure comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 44-119, preferably SEQ ID NO: 75-119, or any combination thereof or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one of the sequences SEQ ID NO: 44 to 119, preferably SEQ ID NO: 75-119 or any combination thereof.

[0184] In another embodiment, this disclosure relates to a kit comprising a nucleic acid construct containing a degrader 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%, or 99% sequence identity with any one of the sequences SEQ ID NO: 75-119.

[0185] The kit may include instructions or packaging materials describing how to administer the polynucleotides contained in the kit to a patient.

[0186] The reagent kit container can be any suitable material, such as glass, plastic, metal, etc., and can be any suitable size, shape, or configuration. In some embodiments, the reagent kit may include one or more ampoules or syringes containing the product of this disclosure in a suitable liquid or solution form.

[0187] The following embodiments are provided as illustrations and are not intended to limit this disclosure. Furthermore, this disclosure covers all possible combinations of the specific and preferred embodiments described herein.

[0188] Example 1. Materials and Methods Material: Oligonucleotides were purchased from Eurofins Genomics. Synthetic genes were purchased from GENEWIZ. Pfu Ultra fusion polymerase and all restriction enzymes used for cloning were purchased from Thermofisher Scientific. Highly competent cells used for cloning were derived from XL10-Gold chemocompetent *E. coli*. HEK293T and HeLa cells were purchased from ATCC. DNA purification kits were purchased from Thermofisher Scientific. All plasmids were sequenced using Macrogen. Luria Bertani (LB) medium and all buffer salts were purchased from Thermofisher Scientific. Coomassie Brilliant Blue, NH4HCO3, DTT, formic acid, fetal bovine serum, and soybean lecithin 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 electrophoresis buffer, pre-stained protein ladder, and SDS-PAGE (bis-tris and tris-acetate gels) were purchased from Thermofisher Scientific. The primary antibodies used were anti-flag-labeled mouse monoclonal antibodies (Sigma and Abcam), anti-MYO7A rabbit monoclonal antibody (Abcam), anti-HA-labeled rabbit monoclonal antibody (Thermo Fisher Scientific), and anti-microtubule-containing rabbit polyclonal antibody (Thermofisher Scientific). Highly cross-linked Alexa fluor +488 antibody against secondary goat anti-mouse IgG (H+L), highly cross-linked Alexa fluor 633 antibody against secondary goat anti-rabbit IgG (H+L), and 4',6-diamidindo-2-phenylindole dihydrochloride (DAPI, D1306) were purchased from Thermofisher Scientific. HRP-conjugated secondary antibody was purchased from Vitro. Dodecyl maltodextrin (D310) and cholesterol hemisuccinate (CH210) solutions were purchased from Anatrace. Trypsin was purchased from Promega. ADP-Glo™ kinase assay was purchased from Promega. SuperSignal ELISA Femto maximum sensitivity substrate was purchased from Thermo Fisher Scientific.

[0189] equipment: Gel and Western blot imaging were performed using a LI-COR Odyssey infrared imager. Cell lysis was performed using an SFX550 Branson sonicator. ATP / ADP signals were acquired using a luminescent microplate reader (Synergy H1M, BioTek). Mouse eye examination was performed using retinal photography and optical coherence tomography (OCT).

[0190] Recombinant DNA Cloning Purchase synthetic genes for preparing constructs MYO7A-1-1200-CfaN-3FT (SEQ ID NO: 124) and MYO7A-1201-2215-CfaCmut-3FT (SEQ ID NO: 125), and introduce them into plasmid expression vectors using KpnI and NotI restriction endonucleases.

[0191] The constructs MYO7A-1-1172-CfaN-3FT (SEQ ID NO: 120), MYO7A-1173-2215-CfaCmut-3FT (SEQ ID NO: 121), MYO7A-1-1197-CfaN-3FT (SEQ ID NO: 122), MYO7A-1198-2215-CfaCmut-3FT (SEQ ID NO: 123), MYO7A-1-1226-CfaN-3FT (SEQ ID NO: 126), MYO7A-1227-2215-CfaCmut-3FT (SEQ ID NO: 127), MYO7A-1-1283-CfaN-3FT (SEQ ID NO: 128), and MYO7A-1284-2215-CfaCmut-3FT (SEQ ID NO: 128) were prepared by unrestricted enzyme cloning. 129) MYO7A-3FT (SEQ IDNO: 134).

[0192] Sequencing confirmed the identity of all recombinant plasmids, and the corresponding protein sequences are listed in Table 6.

[0193] Table 6: Protein sequences of the constructs used in the examples. (1) The residues are numbered according to SEQ ID NO: 15 (full length of myosin VIIa).

[0194] Transfection of peptide-containing plasmids into HEK293T and HeLa cells: HEK293T cells (ATCC, CRL-3216) were maintained in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin / streptomycin (P / S) at 37°C and 5% CO2. HeLa cells (ATCC, ATCC-CCL-2) were maintained in a humidified cell culture incubator containing 10% FBS and 1% P / S at 37°C and 5% CO2. Cells were co-transfected at approximately 70-80% confluence using Lipofectamine 2000, with 1 µg of each plasmid in 6-well plates, 0.25 µg of total plasmid DNA for degrader identification in 48-well plates, and 1.75 µg of total plasmid DNA for immunoprecipitation and ATPase assays in 12-well plates. For experiments using plasmids encoding full-length genes, scrambled plasmids were co-transfected with the full-length plasmids to achieve the same amount of transfected DNA when using two peptide-containing plasmids. Cells were harvested 48 hours post-transfection, and protein levels were analyzed by Western blotting.

[0195] Western blot analysis : MYO7A-transfected cells (HEK293T) were lysed in 25 mM Tris-HCl (pH=7.5) and 150 mM NaCl supplemented with protease inhibitors and 1 mM benzylsulfonyl fluoride, containing 0.1% n-dodecyl-β-D-maltopyranoside (D310S) and 1% cholesterol hemisuccinate (CH210). Retinal tissue was mechanically homogenized in 150 μL of cell lysis buffer (1 M Tris-HCl, 0.5 M NaCl, 0.2 M EDTA, 10% SDS, 200 mM PMSF, Triton X-100, glycerol, pH=7.8). After lysis, MYO7A samples were quantified using a BCA protein assay kit. Samples containing 25 µg of total protein or 20 µL of sample were denatured at 95 °C for 5 min in 1×Laemmli sample buffer containing 2.5 mg / ml soybean lecithin. The lysates were separated by 3-8% Tris-acetic acid SDS-PAGE gel at 150V for 1.5 hours. The antibodies used for immunoblotting were anti-Flag tag antibody to detect MYO7A protein and anti-β-tubulin antibody (as a loading control). Western ImprintThe MYO7A bands were quantitatively analyzed using a LI-COR Odyssey infrared imager.

[0196] To quantify degrader efficiency, PTS, N-, and C-fragment bands were selected to obtain band signals. The same procedure was performed on the β-tubulin signal band. For standardization, the PTS, N-, and C-fragment band signals for each sample were divided by the β-tubulin band signal. To determine changes in PTS, N-, and C-fragment expression compared to samples without degraders, samples without degraders were used as controls and set to 100% for fold changes.

[0197] Immunoprecipitation assay of the interaction between MYO7A and SANS protein: Transfected HEK293T cells were resuspended in a lysis buffer mixture (supplemented with protease inhibitors and 1 mM benzyl sulfonyl fluoride in 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl containing 0.1% n-dodecyl-β-D-maltodextrin (D310S) and 1% cholesterol hemisuccinate (CH210)) and incubated at 4°C for 15 min. After centrifugation, the protein extract was recovered, and the lysate was then incubated with anti-FLAG magnetic beads at room temperature for 2 h. Samples were washed three times with 500 mM NaCl in 1x PBS (pH 7.4) and then eluted by boiling for 5 min in 1x Laemmli sample buffer containing 2.5 mg / ml soybean lecithin. Input samples were also denatured at 95°C for 5 min in 1x Laemmli sample buffer containing 2.5 mg / ml soybean lecithin. Input and eluted samples were analyzed by 3-8% Tris-acetic acid SDS-PAGE and transferred to a PVDF membrane. Antibodies used for immunoblotting included anti-flag tag for detecting MYO7A protein, anti-HA for detecting SANS, and anti-β-tubulin as a loading control. MYO7A band quantification was performed using a LI-COR Odyssey infrared imager.

[0198] ATPase activity assay: ATPase assays were performed using an ELISA-like method, in which myosin VIIa in transfected samples was enriched using 96-well plates coated with anti-C-terminal MYO7A antibody. The amount of immobilized myosin VIIa-3xFLAG was quantified using a sandwich ELISA assay with anti-Flag-tagged antibody as the detection antibody, following the manufacturer's instructions, using a commercial luciferase-based kit (ADP-Globe). TM The Promega kinase assay measures ATPase activity.

[0199] animal: Wild-type C57Bl / 6 mice were purchased from Charles River and maintained at either the VHIO Animal Facility (Barcelona, ​​Spain) or the EyeCRO Animal Facility (Oklahoma, USA). All mice were given free access to standard feed and maintained under a 12-hour light-dark cycle.

[0200] Recombinant AAV vector : AAV expression cassettes were generated by cloning the target cDNA into a single-stranded AAV backbone plasmid using restriction enzymes. AAV vector production was performed by transfecting HEK293 cells without helper-free virus, followed by gradient purification with iodixanol.

[0201] AAV vector was administered to mice. : For subretinal intradermal administration (SRI) performed at VHIO (Barcelona, ​​Spain), 6–7-week-old adult mice were anesthetized via intraperitoneal injection (ketamine / toluidine 85 mg / kg and 14 mg / kg, respectively). Subsequently, the pupils were dilated with tropicamide (10 mg / mL Colorcusi tropicamide®, Alcon Cusí Laboratories) and locally anesthetized with Oxibuprocaína (0.4%, Lavoratorios Llorens). Under a surgical microscope, a simple suture was made in the upper eyelid and superior limbal conjunctiva using a Prolene™ 7 / 0 Ethicon (Johnson & Johnson) to expose the superior bulbar conjunctiva, and a 1 mm scleral incision was performed 5 mm from the limbus using a 30-gauge blade. A 36-gauge blunt or beveled needle connected to a 10 μL Nanofil syringe (World Precision Instruments) was introduced tangentially into the subretinal space via the scleral incision for bilateral subretinal administration, 1 μl per eye. Finally, the needle was carefully removed and the sutures were secured. Immediately after surgery, tobradex® 3 mg / mL + 1 mg / mL drops (Alcon Cusí Laboratory) were applied topically as local anti-inflammatory and antibiotic prophylaxis. Bubble structures in the subretinal space were assessed using fundus imaging.

[0202] For subretinal intravitreal (SRI) administration performed at EyeCRO (Oklahoma, USA), 4–6 week old adult mice were anesthetized via intraperitoneal injection (ketamine / toluidine 85 mg / kg and methaqualone 14 mg / kg, respectively). Subsequently, the mice's eyes were dilated, and they were placed on an adjustable heating pad for magnified observation of the posterior pole. A 12.7 mm 30-gauge insulin syringe was used to puncture the cornea above the limbus, avoiding any contact with the sclera or lens. A 10 μL Hamilton syringe with a 33-gauge blunt needle was used for transvitreal subretinal injection via corneal puncture, with the needle shaft pointing towards the posterior part of the eyeball to avoid any trauma to the lens or iris. The total delivery volume was 1 μL. Following injection, small doses of neomycin, polymyxin B, and dexamethasone 3.5 g (Bausch and Lomb) were administered.

[0203] Retinal sample acquisition: Four weeks after administration of the vector to mice via SRI, the animals were sacrificed and their eyes were removed. The retina was dissected separately and flash-frozen in liquid nitrogen.

[0204] 2. Results and Discussion In recent years, several novel intepids have been engineered based on shared design (Stevens et al., 2016; Stevens, Sekar, Gramespacher, Cowburn, & Muir, 2018), demonstrating superior properties compared to naturally occurring intepids. One such intepid, called Cfa, exhibits faster kinetics, higher expression levels, and greater tolerance to extreme conditions such as high temperatures and denaturant concentrations. Interestingly, Cfa variants with 90% or higher homology show similar properties. A key hint from splicing-based approaches is that all characterized intepids exhibit sequence preference at exopeptide residues adjacent to the splice site. Deviations from this preferred sequence lead to a significant reduction in splicing activity, limiting the applicability of protein trans-splicing (PTS)-based approaches. Recently, an engineered form of a naturally occurring splitting intepid with greatly improved exopeptide tolerance has been developed (Stevens et al., 2017). This intepid is named Cfa. mut .

[0205] To prove CFA mutRecombination of proteins such as MYO7A via protein transsplicing for gene therapy applications has been studied using different cleavage sites. MYO7A is a large protein that is mutated in Usher1B syndrome. Usher syndrome is a genetic disorder that causes severe hearing loss and retinitis pigmentosa, an eye disease that worsens vision over time. It is one of the most common diseases affecting hearing and vision. Recombination of MYO7A has been proposed as a feasible strategy for treating this disease. Several AAV-based gene therapy approaches are currently being explored to recombine it. Due to its large size, MYO7A Since the gene cannot be encapsulated into a single AAV, different strategies have been proposed to reconstitute the function of the protein within target cells.

[0206] The initial experiments used three sites (SEQ ID NO: 120 and 121, SEQ ID NO: 122 and 123, and SEQ ID NO: 124 and 125). Two of these sites did not contain Phe at the +2 position of the cleavage site. The three sites tested were position 1173 corresponding to the +2 Gln, position 1198 corresponding to the +2 Val, and position 1201 corresponding to the +2 Phe. The construct was cloned and tested as described above. Specifically, splicing of MYO7A at position 1172 (SEQ ID NO: 120 and SEQ ID NO: 121) was evaluated using the MYO7A(1-1172)-CfaN and CfaCmut-MYO7A(1173-2215) constructs; splitting of MYO7A at position 1197 (SEQ ID NO: 122 and SEQ ID NO: 123) was evaluated using the MYO7A(1-1197)-CfaN and CfaCmut-MYO7A(1198-2215) constructs; and splicing of MYO7A at position 1200 (SEQ ID NO: 124 and SEQ ID NO: 125) was evaluated using the MYO7A(1-1200)-CfaN and CfaCmut-MYO7A(1201-2215) constructs. Site selection was based on the topological structure of myosin VIIa, taking into account the presence of folded domains. Sites were selected outside the well-defined folding domains. In short, the construct was co-transfected into HEK293T cells, with full-length Myo7A (SEQ ID NO: 134) as a control. Cells were lysed, and recombinant protein yield was determined by Western blotting to compare the yield of recombinant protein with that of full-length protein. The results showed that the yield at these three sites was comparable to that obtained using only full-length protein. Figure 2 ).

[0207] Two additional sites (1226 and 1283) were tested: MYO7A(1-1226)-CfaN and CfaCmut-MYO7A(1227-2215) constructs (SEQ ID NO: 126 and SEQ ID NO: 127) and MYO7A(1-1283)-CfaN and CfaCmut-MYO7A(1284-2215) constructs (SEQ ID NO: 128 and SEQ ID NO: 129) were co-transfected into HEK293T cells, and full-length MYO7A (SEQ ID NO: 134) was transfected as a control. Cells were lysed, and the recombinant protein yield was determined by Western blotting to compare the yield of recombinant protein with that of full-length protein. The results showed that the yield at these two sites was comparable to that obtained using only full-length protein. Figure 3 ).

[0208] In addition, two other sites (823 and 1325) were tested: co-transfection of HEK293T cells with MYO7A(1-823)-CfaN and CfaCmut-MYO7A(824-2215) constructs (SEQ ID NO: 130 and SEQ ID NO: 131) and MYO7A(1-1325)-CfaN and CfaCmut-MYO7A(1326-2215) constructs (SEQ ID NO: 132 and SEQ ID NO: 133) and full-length MYO7A (SEQ ID NO: 89) as a control, and all previously assessed different mitotic sites were compared. Cells were lysed, and recombinant protein yield was determined by Western blotting to compare the yield of recombinant protein with that of full-length protein. The results showed that the yields at all seven different sites were comparable to those obtained using only full-length protein. Figure 4 ).

[0209] To confirm the high performance of all seven different cleavage sites, the construct was also characterized in the HeLa cell line. For this purpose, cells were transfected with the full-length MYO7A plasmid or with co-transfected cells using the N- and C-fragments of the MYO7A plasmid. Cells were lysed using a non-cationic detergent mixture as lysis buffer. Expression levels were analyzed by Western blotting. Protein detection was performed using anti-flag tag and anti-tubulin antibodies. As before, the results showed that the excellent in vitro recombination of MYO7A via protein trans-splicing achieved expression levels comparable to the full-length MYO7A construct in the HeLa cell line. Figure 5 ).

[0210] To investigate the functionality of recombinant MYO7A protein, a specific co-immunoprecipitation (CoIP) assay for myosin VIIa protein and its related interacting proteins was developed. For this purpose, SANS protein (a scaffold protein associated with the microtubule system) was chosen as the key interacting protein because it is well known to interact with MYO7A through its MF1 domain (Reiners J, et al., Experimental Eye Research, 2006. 83:97-119). The CoIP assay was developed using anti-FLAG magnetic beads with MYO7A as the decoy protein.

[0211] To assess whether the interaction between recombinant MYO7A and SANS proteins was maintained after PTS, the pairings of interest were evaluated in vitro in HEK293T cells. For this purpose, cells were co-transfected with the full-length MYO7A plasmid or with N- and C-fragments of the MYO7A plasmid at all seven different cleavage sites: MYO7A-1172, MYO7A-1197, MYO7A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, and MYO7A-1325. Cells were lysed using a non-cationic detergent mixture as lysis buffer. CoIP assays were performed using anti-FLAG magnetic beads, and analysis was performed by Western blot. Myosin VIIa-3xFLAG and SANS-HA proteins were detected using anti-FLAG and anti-HA antibodies, respectively. The results showed that positive interactions were achieved with both full-length and PTSMYO7A proteins using all seven different cleavage sites: MYO7A-1172, MYO7A-1197, MYO7A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, and MYO7A-1325. Figure 6 No interactions between individual segments (N- or C-segments) were observed, except for the C-segment at the MYO7A-823 split site, which is due to the splitting location being within the MF1 domain.

[0212] Furthermore, the protocol was validated using reverse immunoprecipitation assays with SANS as the bait protein. The obtained data confirmed the interaction between the SANS protein and recombinant myosin VIIa (MYO7A-1172, MYO7A-1197, MYO7A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, MYO7A-1325) identified at all seven different cleavage sites, as MYO7A was successfully pulled down, and significant enrichment of the SANS protein was detected in the eluted sample. Figure 7 ).

[0213] To further investigate the functionality of recombinant MYO7A protein, a specific ATPase activity assay was developed. This assay was based on an ELISA-like system to enrich MYO7A samples, followed by the detection of ATPase activity using a commercial kit. Only C-fragment samples were used as a negative control due to their lack of ATPase activity and their ability to inhibit it (Umeki N, et al., Proc. Natl. Acad. Sci. USA, 2009. 106:8483-8488).

[0214] To assess whether the ATPase activity of recombinant MYO7A was maintained after PTS recombination, the pair of interest was evaluated in vitro in HEK293T cells. For this purpose, cells were co-transfected with the full-length MYO7A plasmid, the C-fragment of the MYO7A-1325 plasmid cleavage site alone (as the most C-terminal construct available), or the N- and C-fragments of the target MYO7A plasmid. Cells were lysed using a non-cationic detergent mixture as lysis buffer. Samples were then immobilized in Maxisorp 96-well plates using an antibody that specifically recognizes the C-terminus of the MYO7A protein. Next, ATPase assays were performed using the ADP-Glo ​​kit via luciferase assay to estimate ADP concentration. Finally, a sandwich ELISA was performed by adding anti-Flag tag detection antibody to the wells of the Maxisorp 96-well plates to detect the amount of myosin VIIa and the fragment. The ratio between full-length, recombinant MYO7A protein and single fragments was also assessed by Western blotting using anti-Flag and anti-microtubule antibodies. The results showed that functional ATPase activity was detected in the full-length PTS MYO7A protein and in all seven different cleavage sites (MYO7A-1172, MYO7A-1197, MYO7A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, and MYO7A-1325); however, no activity was recorded in the individual C-fragments of all the aforementioned cleavage sites. Figure 8 ).

[0215] Once all the techniques for different cleavage sites were successfully validated, the approach was further advanced by incorporating degradation sequence techniques into the construct. The design required the elimination of unreacted N- and C-fragments that form the functional full-length MYO7A protein, as well as excised integuments. To this end, a set of degradation sequences were evaluated in vitro in HEK293T cells. Cells were transfected with the full-length MYO7A plasmid or co-transfected with the N- and C-fragments of the MYO7A plasmid containing the evaluated degradation sequences. Cells were lysed using a non-cationic detergent mixture as the lysis buffer. Expression levels were analyzed by Western blotting. Protein detection was performed using anti-flag tags and anti-tubulin antibodies. After evaluating over 1,500 (1,500) different combinations of cleavage sites with degradation sequences at the N- and C-fragments, several degradation sequences were identified and successfully combined with different MYO7A cleavage sites, resulting in the degradation of a single half while maintaining the yield of trans-splicing of the recombinant protein. Figure 9 The large number of candidates containing degradants identified include degradants of different sizes, families, and mechanisms of action (e.g., proteasome-mediated or molecular chaperone-mediated autophagy) (Table 7).

[0216] Table 7: Degradation effects achieved by combinations of several degradation sequences with different MYO7A cleavage sites. SD, standard deviation; n, number of biological replicates; Degron, degradation sequence.

[0217] To ensure that the addition of the degrader sequence did not interfere with the functionality of the recombinant MYO7A protein, the functional level of the paired candidates containing the degrader was assessed in vitro. First, the candidates were tested by immunoprecipitation assay using MYO7A as the bait protein; all evaluated candidates showed positive interaction with SANS (Figure 10 and Table 7). Second, the candidates were assessed by ATPase activity assay; again, all tested candidates showed positive ATPase activity. Figure 11 (and Table 7).

[0218] Finally, in order to evaluate MYO7A Whether protein transsplicing of genes occurs effectively in the retina after co-administration was assessed by subretinal (SRI) administration to adult C57Bl / 6 WT mice encoding N- and C-mitotic proteins. MYO7AThe AAV8 vector of the MYO7A-1172 cleavage site (SEQ ID NO: 120 and 121) gene. Results showed that, after SRI injection into WT mice, recombination of the full-length MYO7A protein was achieved in the retina. Figure 12 It is noteworthy that the inventors not only provided evidence that the PTS technology worked successfully in vivo, but also that the recombinant MYO7A protein was effectively administered via SRI at two different clinical research sites using two delivery methods (transcorneal and transscleral).

Claims

1. A polynucleotide combination for the use of in treating MYO7A-related diseases in subjects who require it, wherein said combination comprises: i) A first polynucleotide encoding the first fusion protein, comprising from 5' to 3': an N-terminal fragment of myosin VIIa protein and an N-cleavage inteptide, which are fused directly or indirectly through a linker. ii) A second polynucleotide encoding the second fusion protein, comprising from 5' to 3': a C-cleavage inteptide and a C-terminal fragment of myosin VIIa protein, which are fused directly or indirectly through a linker. The expression of the first and second polynucleotides in the subjects generates myosin VIIa protein through protein splicing.

2. The polynucleotide combination for use according to claim 1, wherein: - The N-cleavage inteptide is the N-Cfa-inteptide of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity with SEQ ID NO: 1; and - The C-split inteptide is the C-Cfa inteptide of SEQ ID NO: 2 or any functional variant thereof having at least 90% identity with SEQ ID NO: 2, preferably wherein amino acid residues 20-22 of SEQ ID NO: 2 are GEP.

3. The polynucleotide combination for use according to claim 1 or 2, wherein the C-cleavage inteptide is C-Cfa of SEQ ID NO:

13. mut Intended peptides or any functional variant thereof having at least 90% identity with SEQ ID NO:

13.

4. The combination for use according to any one of claims 1 to 3, wherein the myosin VIIa protein is human myosin VIIa protein, preferably comprising or consisting of SEQ ID NO: 15 or any functional variant thereof having at least 90% identity with SEQ ID NO:

15.

5. The combination for use according to any one of claims 1 to 4, wherein the first and second fusion proteins comprise: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. - Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284; or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO:

15.

6. The combination for use according to any one of claims 1 to 5, wherein the first and second fusion proteins comprise an amino acid sequence 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 with any one of sequences SEQ ID NO: 30-43.

7. The combination for use according to any one of claims 1 to 6, wherein the first fusion protein or the second fusion protein further comprises a degradant.

8. The combination for use according to any one of claims 1 to 7, wherein: i) The first fusion protein further comprises a degrader located at the 3' end of the N-splitting inteptide, and / or ii) The second fusion protein further comprises a degrader located at the 5' end of the C-cleavage inteptide.

9. The combination for use according to claim 7 or 8, wherein the degrader is selected from the group consisting of: SEQ ID NO:44-119 or any combination thereof, or any functional variant thereof having at least 90% identity with any one of the sequences SEQ ID NO:44-119 or any combination thereof.

10. The combination for use according to any one of claims 1 to 9, wherein each polynucleotide further comprises a promoter selected from the group consisting of: cytomegalovirus (CMV) promoter, chimeric reduced version of CMV and chicken β-actin (CEBA) promoter, human phosphoglycerate kinase (hPGK) promoter, chimeric enhanced CMV and human phosphoglycerate kinase (ePGK) promoter, photoreceptor-specific human rhodopsin kinase (hGRK1) promoter, rod cell-specific IRBP promoter, VMD2 (vital-like macular dystrophy / Best disease) promoter, and EF1α promoter.

11. The combination for use according to any one of claims 1 to 10, wherein each polynucleotide is contained within an expression vector.

12. The combination for use according to claim 11, wherein the expression vector is a viral vector, preferably an adeno-associated virus (AAV) vector, and preferably the AAV vector contains an AAV capsid protein selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or rh10.

13. The combination for use according to any one of claims 1 to 12, wherein the MYO7A-related disease is non-syndromic hearing loss DFNA11, non-syndromic hearing loss DFNB2, or Usher syndrome type 1B, preferably wherein the disease is Usher syndrome type 1B, more preferably wherein the combination is administered to the subject via a parenteral route, and even more preferably via an intravenous, intra-arterial, intramuscular, intranasal, intraocular, intravitreal, suprachoroidal, or subretinal route.

14. A kit comprising: (i) The first polynucleotide encoding the first fusion protein, comprising from 5' to 3': - N-terminal fragment of myosin VIIa protein and - The N-Cfa inteptide of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity with SEQ ID NO: 1, It fuses directly or indirectly through a joint. (ii) A second polynucleotide encoding the second fusion protein, comprising from 5' to 3': -C-Cfa intima-peptide of SEQ ID NO: 2 or C-Cfa of SEQ ID NO: 13 mut Intended peptides or any functional variant thereof having at least 90% identity with SEQ ID NO:2 or 13, and - The C-terminal fragment of myosin VIIa protein, It fuses directly or indirectly through a joint. The first and second fusion proteins respectively comprise: Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 823, and the C-terminal segment of myosin VIIa protein begins at residue 824. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1172, and the C-terminal segment of myosin VIIa protein begins at residue 1173. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1197, and the C-terminal segment of myosin VIIa protein begins at residue 1198. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1200, and the C-terminal segment of myosin VIIa protein begins at residue 1201. Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1226, and the C-terminal segment of myosin VIIa protein begins at residue 1227. - Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1283, and the C-terminal segment of myosin VIIa protein begins at residue 1284; or Specifically, the N-terminal segment of myosin VIIa protein terminates at residue 1325, and the C-terminal segment of myosin VIIa protein begins at residue 1326. The residues thereon are numbered according to SEQ ID NO:

15. Preferably, the N-terminal myosin VIIa fragment and the C-terminal myosin VIIa fragment are each composed of amino acid sequences selected from the following pairs: 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%, or 99% sequence identity with any one of SEQ ID NO: 16 to 29.

15. The kit of claim 14, wherein the first and second fusion proteins comprise amino acid sequences selected from any 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 with any one of sequences SEQ ID NO: 30-43.

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