WO2025153705A1 - Use of split intein for the treatment of myo7a-associated disease - Google Patents

Abstract

The present disclosure relates to the use of split inteins for expressing Myosin-VIIa protein encoded by MYO7A gene in a subject in need thereof for gene therapy in particular for the treatment of MYO7A associated disease, preferably Usher syndrome type 1B.

Description

USE OF SPLIT INTEIN FOR THE TREATMENT OF MYO7A-ASSOCIATED

DISEASE

TECHNICAL FIELD

The present disclosure relates to the use of split inteins for expressing Myosin- Vila protein encoded by MY07A gene in a subject in need thereof for gene therapy in particular for the treatment of MY07A associated disease, preferably Usher syndrome type IB.

BACKGROUND

Usher syndrome (USH) represents a group of genetically heterogenous autosomal recessive disorders, characterized by combined vision and sensorineural hearing loss, and in some cases vestibular dysfunction. It primarily affects the light-sensitive photoreceptor cells in the retina and the auditory hair cells in the cochlea. Vision loss in all cases is progressive, and manifests as retinitis pigmentosa (RP), characterized by the gradual degeneration of the photoreceptor cells. Peripheral rod function is lost first, leading to night blindness and constricted visual fields, followed by the death of cones and severe visual impairment (Dinculescu et al. Int Ophthalmol Clin. 2021 Fall; 61(4): 109-124).

Usher IB, the most prevalent form of Usher type 1, is caused by mutations in the gene encoding the unconventional molecular motor, MY07A.

A variety of different gene therapy approaches have been tested for retinal gene therapy to prevent the blindness of Usher IB.

One example is the lentiviral-mediated MYO7Agene delivery in USH1B model. This approach was shown to correct the melanosome mislocalization and opsin accumulation at the photoreceptor connecting cilium in the Myo7a-deficient shakerl mouse model of USH1B (Hashimoto T, et al.. Gene Then 2007;14:584-594). An equine infectious anemia virus-based lentiviral vector (equine infectious anemia virus-CMV-MYO7A, UshStat) was also used and subretinally delivered to USH1B patients with the goal to prevent or slow down the retinitis pigmentosa progression (ClinicalTrials.gov identifier: NCT01505062).

However, these integrating vector systems present a main disadvantage that is their potential risk of causing insertional mutagenesis. Adeno-associated viruses (AAV) have been widely used for viral delivery in gene therapy; however, the size of a gene that can be encapsulated into AAV has been reported to be limited to ~5 kb (Grieger and Samulski 2005, J Virol 79: 9933-9944). Due to its large size, MY07A gene of 100 Mb and a cDNA of nearly 7 kb cannot be encapsulated into a single AAV, and there remains a need to develop new strategies to deliver MY07A gene inside target cells.

One strategy to overcome this problem is to divide a gene or cDNA, such as MY07A, into two pieces and have each piece contained within a separate AAV vector. However, a major concern with the dual AAV system such as the hybrid dual vector was to achieve a high expression level of expression of MY07A.

The use of an AAV dual vector with overlapping sequences of MY07A was shown to produce non-functional MY07A protein in vivo (Lopes et al. 2013, Gene Therapy, 20(8): 824-33). In addition, the front-half fragment alone of the hybrid AAV dual vector was shown to be toxic.

Thus, there remains a need to develop new strategies to efficiently deliver MY07A gene in cells to prevent the formation of a toxic truncation fragment toxic.

Inteins are genetic elements that carry out trans-splicing, where two protein fragments bind to form a catalytically competent enzyme, then catalyze their own excision and the ligation of their flanking sequences. Split inteins have been mainly used to fuse different functional protein domains in protein purification system and labeling steps. In the last years several new inteins have been engineered based on consensus design (Stevens et al., J Am Chem Soc. 2016 Feb 24;138(7):2162-5; Stevens, Sekar, Gramespacher, Cowbum, & Muir, J Am Chem Soc. 2018 Sep 19; 140(37): 11791-11799) and shown to have superior properties than naturally occurring inteins.

The use of split intein-mediated protein trans-splicing to reconstitute therapeutic protein in vivo has been recently studied for few proteins (WO2021/191447 and W02020/079034).

SUMMARY

In the present application, the inventors showed for the first time that split intein-mediated protein trans-splicing can be used to reconstitute efficiently Myosin- Vila protein encoded by MY07A gene in a cell. Myosin- Vila reconstitution with split intein-mediated protein trans- splicing represents an efficient strategy to treat MYO7A-associated disease such as Usher syndrome type IB. The use of split-intein to reconstitute Myosin- Vila protein, in particular in combination with degrons, would make it possible to prevent the toxicity of the half-fragments in vivo.

The present disclosure relates to a combination of polynucleotides for use in the treatment of MYO7A-associated disease, preferably selected from the group consisting of: non-syndromic deafness DFNA11, non-syndromic deafness DFNB2, or Usher syndrome type IB, in a subject in need thereof wherein the combination comprises: i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’ : a N-terminal fragment of Myosin- Vila protein and N-split intein, fused directly or indirectly via a linker, ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in said subject generates Myosin- Vila by protein splicing.

In a preferred embodiment, said N-split intein is a N-Cfa-intein of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity to SEQ ID NO: 1; and said C-split intein is a C-Cfa intein of SEQ ID NO: 2 or any functional variant thereof having at least 90% identity to SEQ ID NO: 2, preferably wherein amino acid residues 20 to 22 of SEQ ID NO: 2 are GEP, more preferably C-Cfa_mut intein of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity to SEQ ID NO: 13.

In a specific embodiment, said N-split intein is a N-Npu-intein of SEQ ID NO: 3 or any functional variant thereof having at least 90% identity to SEQ ID NO: 3; and said C-split intein is a C-Npu intein of SEQ ID NO: 4 or any functional variant thereof having at least 90% identity to SEQ ID NO: 4, preferably wherein amino acid residues 20 to 22 of SEQ ID NO: 4 are GEP, more preferably C-Npu_mut intein of SEQ ID NO: 14 or any functional variant thereof having at least 90% identity to SEQ ID NO: 14.

In a preferred embodiment, said Myosin- Vila protein is human Myosin- Vila protein, preferably comprising or consisting of SEQ ID NO: 15 or any functional variant thereof having at least 90% identity to SEQ ID NO: 15.

In a more preferred embodiment, the N-terminal fragment of Myosin- Vila protein up to residue 823 and the C-terminal fragment of Myosin- Vila protein from residue 824 respectively, the N- terminal fragment of Myosin- Vila protein up to residue 1172 and the C-terminal fragment of Myosin- Vila protein from residue 1173 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C-terminal fragment of Myosin- Vila protein from residue 1198 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C-terminal fragment of Myosin- Vila protein from residue 1201 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C-terminal fragment of Myosin- Vlla protein from residue 1227 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C-terminal fragment of Myosin- Vila protein from residue 1284 respectively; or the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, more preferably, the N-terminal fragment of Myosin- Vlla protein up to residue 1172 and the C-terminal fragment of Myosin- Vila protein from residue 1173 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C-terminal fragment of Myosin- Vila protein from residue 1198 respectively, the N- terminal fragment of Myosin- Vila protein up to residue 1200 and the C-terminal fragment of Myosin- Vila protein from residue 1201 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C-terminal fragment of Myosin- Vila protein from residue 1227 respectively, or the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C-terminal fragment of Myosin- Vila protein from residue 1284 respectively, wherein said residue is numbered according to SEQ ID NO: 15.

In a more preferred embodiment, the first and second fusion proteins according to the present disclosure comprises amino acid sequences selected from any one of the following pairs: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, and, SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 30-43.

In a particular embodiment, the first fusion protein or second fusion protein may further comprise a degron, preferably selected from the group consisting of: SEQ ID NO: 44 to 119 or any combination thereof, or any functional variant thereof having at least 90% identity to any one of sequences SEQ ID NO: 44 to 119 or any combination thereof, more preferably the first fusion protein further comprises a degron located at the 3 ’end of the N-split-intein, and/or the second fusion protein further comprises a degron located at 5’end of the C-split-intein, fused directly or indirectly via a linker. According to the present disclosure, each polynucleotide of the combination further comprises some regulatory elements, for example, promoters, transcription termination sequences, translation termination sequences, introns, enhancers, signal peptides, and polyadenylation elements, preferably a promoter selected from the group consisting of: Cytomegalovirus (CMV) promoter (GenBank Accession number: AF396260.1, bp 150-812, last updated on August 13, 2001), chimeric reduced version of the CMV and chicken beta-actin (CEB A) promoter (GenBank Accession number: AF396260.1, bp 160-526, last updated on August 13, 2001; GenBank Accession number: X00182.1, bp 268-571, last updated on November 14, 2006), human phosphoglycerate kinase (hPGK) promoter (GenBank Accession number: AH002938.2, bp 2-516, last updated on August 01, 2016), chimeric CMV enhanced and human phosphoglycerate kinase (ePGK) promoter (GenBank Accession number: AF396260.1, pb 160- 500, last updated on August 13, 2001; GenBank Accession number AH002938.2, bp 2-516, last updated on August 01, 2016), photoreceptor-specific, human rhodopsin kinase (hGRKl) promoter, rod specific IRBP promoter, VMD2 (vitelliform macular dystrophy/Best disease) promoter, and EF 1 alpha promoter.

Preferably each polynucleotide is comprised within an expression vector, preferably a viral vector, preferably an adeno associated viral (AAV) vector, preferably said AAV vector comprises capsid protein of AAV selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or RhlO, preferably AAV2, AAV8 or AAV5.

In a preferred embodiment, said combination is administered in subject by a parenteral route, more preferably by intravenous, intraarterial, intramuscular, intranasal, intraocular, intravitreal, suprachoroidal or subretinal route.

The present disclosure also relates to a kit comprising:

(i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’ : a N-terminal fragment of Myosin- Vila protein and

- N-Cfa intein of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity to SEQ ID NO: 1, fused directly or indirectly via a linker,

(ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-Cfa intein of SEQ ID NO: 2 or C-Cfa_mut intein of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:

- the N-terminal fragment of Myosin- Vila protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively

- the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively; or

- the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vila fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, and, SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29, more preferably wherein the first and second fusion proteins comprises amino acid sequences selected from any one of the following pairs: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, and, SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 30-43. LEGEND FIGURES

Figure 1: Alignment of Myosin- Vila protein isoforms (isoform 1 (SEQ ID NO: 15, isoform 2 (SEQ ID NO: 135), isoform 3 (SEQ ID NO: 136), isoform 4 (SEQ ID NO: 137), isoform 5 (SEQ ID NO: 138), isoform 6 (SEQ ID NO: 139), isoform 7 (SEQ ID NO: 140), isoform 8 (SEQ ID NO: 141). The split position sites are indicated with arrows.

Figure 2: Splicing efficiency of Myosin-VIIa protein at position 1172, 1197, 1200 monitored by Western blot: Myosin-VIIa protein was first split at position 1172, 1197 or 1200 and the N-terminal fragment (residues 1-1172 or 1-1197 or 1-1200) recombinantly fused to N- inteins, and the C-terminal fragment (residues 1173-2215, 1198-2215 or 1201-2215) to the C- inteins. Cultured HEK293T cells were co-transfected with equimolar amounts of plasmids encoding for the N- and C-terminal fragments and splicing efficiency monitored by Western blotting. HEK293T cells transfected with a full-length Myosin-VIIa protein serve as control. Split position is numbered according to Myosin-VIIa human amino acid sequence (SEQ ID NO: 15).

Figure 3: Splicing efficiency of Myosin-VIIa protein at position 1226 or 1283 monitored by Western blot: Myosin-VIIa protein was first split at position 1172, 1197, 1200, 1226 or 1283 and the N-terminal fragment (residues 1-1172 or 1-1197 or 1-1200 or 1-1226 or 1-1283) recombinantly fused to N-inteins, and the C-terminal fragment (residues 1173-2215, 1198-2215 1201-2215, 1227-2215, or 1284-2215) to the C-inteins. Cultured HEK293T cells were cotransfected with equimolar amounts of plasmids encoding for the N- and C-terminal fragments and splicing efficiency monitored by Western blotting. HEK293T cells transfected with a full- length Myosin-VIIa protein serve as control. Split position is numbered according to Myosin- VIIa human amino acid sequence (SEQ ID NO: 15).

Figure 4. Comparison of splicing yields of MYO7A at split sites MYO7A-1172, MYO7A- 1197, MY07A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, MYO7A-1325 with full- length protein in HEK293T cell line. Cells were transfected with a full-length MY07A plasmid, or co-transfected with both MYO7A^N-IntN and IntC-MYO7A^c plasmids for each split site. Cells were lysed 48 hours after transfection and then analyzed by western blot. (A) Results blotted with a monoclonal antibody anti-flag tag and anti-tubulin. (B) Densitometric quantification of the western blots. Full-length and PTS-reconstituted myosin Vila protein levels were normalized with P-tubulin and the fold-change was computed with respect to the “FL” condition mean, expressed in percentage. Results are shown as the mean ± SD, n = 3-17. *In split site MYO7A-823, the smallest fragment is N-fragment as opposite to all other sites. FL, Full-Length. PTS, Protein Trans-Splicing.

Figure 5. Comparison of splicing yields of MYO7A at split sites MYO7A-1172, MYO7A- 1197, MY07A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, MYO7A-1325 with full- length protein in HeLa cell line. Cells were transfected with a full-length MY07A plasmid, or co-transfected with both MY07A^N-IntN and IntC-MY07A^c plasmids for each split site. Cells were lysed 48 hours after transfection and then analyzed by western blot. (A) Results blotted with a monoclonal antibody anti-flag tag and anti-tubulin. (B) Densitometric quantification of the western blots. Full-length and PTS-reconstituted myosin Vila protein levels were normalized with P-tubulin and the fold-change was computed with respect to the “FL” condition for each experiment, expressed in percentage. Results are shown as the mean ± SD, n = 4. *In split site MYO7A-823, the smallest fragment is N-fragment as opposite to all other sites. FL, Full-Length. PTS, Protein Trans-Splicing.

Figure 6. Maintenance of protein-protein interaction with SANS protein and reconstituted MYO7A via PTS in all different split sites (MYO7A used as bait protein). HEK293T cells were transfected with full-length (FL) MY07A plasmid, only N- or C-fragment, or cotransfected with both MY07A^N-IntN and IntC-MYO7A^c plasmids for each split site. Cells were lysed 48 hours after transfection and then analyzed by co-immunoprecipitation using MY07A as bait protein. Results above 50% of interaction were considered positive. Results are shown as the mean ± SD, n = 3-4. *C-fragment of split site MYO7A-823 includes entire MFI domain, so interaction with SANS is expected. C’: negative control consisting in cells transfected with SANS plasmid supplemented with empty pUC vector; FL, Full-Length; N, N- fragment; C, C-fragment; PTS, Protein Trans-Splicing.

Figure 7. Maintenance of protein-protein interaction with SANS protein and reconstituted MYO7A via PTS in all different split sites (SANS used as bait protein). HEK293T cells were transfected with full-length (FL) MY07A plasmid or co-transfected with both MY07A^N- IntN and IntC-MYO7A^c plasmids for each split site. Cells were lysed 48 hours after transfection and then analyzed by co-immunoprecipitation using SANS as bait protein followed by western blot. For detection of myosin VIIa-3xFLAG and SANS-HA proteins antibodies anti- FLAG and anti-HA were used, respectively. M, MY07A; FL, Full-Length; PTS, Protein Trans- Splicing; IP, input; E, elution. Figure 8. Maintenance of ATPase activity in reconstituted MYO7A protein via PTS in all different split sites. HEK293T cells were transfected with full-length (FL) MY07A plasmid, only C-fragment, or co-transfected with both MY07AN-IntN and IntC-MYO7AC plasmids for each split site. Cells were lysed 48 hours after transfection and then analyzed by ATPase activity assay. Results equal to or greater than 1.2 a.u. were considered positive, as a cutoff value of 1.2 a.u. was established based on the means of full-length positive control. Results are shown as the mean ± SD, n = 3-13. FL, Full-Length; PTS, Protein Trans-Splicing; a.u., arbitrary units; C+, positive control; C-, negative control.

Figure 9. Comparison of degradation effect mediated by different degron sequences in different split sites of MYO7A in HEK293T cells. Cells were co-transfected with both MY07AN-IntN and IntC-MYO7AC plasmids fused to selected degrons for each split site. Cells were lysed 48 hours after transfection and then analyzed by western blot. Results blotted with a monoclonal antibody anti-flag tag and anti-tubulin. Representative images are provided. FL, full-length.

Figure 10. Maintenance of protein-protein interaction with SANS protein and PTS reconstituted degron-containing MYO7A protein. HEK293T cells were transfected with full-length (FL) MY07A plasmid, or co-transfected with both MY07A^N-IntN and IntC- MY07A^c degron-containing plasmids. Cells were lysed 48 hours after transfection and then analyzed by co-immunoprecipitation using MY07A as bait protein followed by western blot. For detection of myosin VIIa-3xFLAG and SANS-HA proteins antibodies anti -FLAG and anti- HA were used, respectively. (A) Representative western blot image for analysis of some candidates. (B) Fold-change quantification of co-imunoprecipitation assay. Results above 50% of interaction were considered positive. Results are shown as the mean ± SD, n = 3-4. C’: negative control consisting in cells transfected with SANS plasmid supplemented with empty pUC vector; FL, Full-Length; M, MY07A; IP, input; E, elution.

Figure 11. Maintenance of ATPase activity in PTS reconstituted degron-containing MYO7A protein. HEK293T cells were transfected with full-length (FL) MY07A plasmid, only C-fragment, or co-transfected with both MYO7A^N-IntN and IntC-MYO7A^c degron- containing plasmids. Cells were lysed 48 hours after transfection and then analyzed by ATPase activity assay. Results equal to or greater than 1.2 a.u. were considered positive, as a cutoff value of 1.2 a.u. was established based on the means of full-length positive control. Results are shown as the mean ± SD, n = 3-13. FL, Full-Length; a.u., arbitrary units; C+, positive control; C-, negative control. Figure 12. Protein trans splicing reconstinutes MYO7A in vivo in the retina of WT mice. In vivo proof of concept studies in WT mice using GRK1 promoter after subretinal injection in adult WT mice. Studies conducted at (A) VHIO (Barcelona, Spain) and (B) EyeCRO (US). Subretinal delivery of AAVs expressing the N- and C-fragments of MY07A (split site MY07A-1172) conjugated to split inteins at a low dose (LD) of 5E+9 vg/eye and high dose (HD) of 1E+10 vg/eye, analysis of samples by western blot 4 weeks after injection. N-fragment unreacted starting material was barely detected only in few animals, while C-fragment accumulation was slightly detected. Each line corresponds to a different injected retina. LD, low dose; HD, high dose; NT, nontreated; C⁺, in vitro positive control of transfected HEK293T with N- and C-fragments of MY07A conjugated to split inteins.

DETAILED DESCRIPTION

Combination of polynucleotides encoding first and second fusion proteins

The limited cargo capacity of the AAV vectors precludes its use for delivering large genes such as MY07A gene in gene therapy. To deliver MY07A gene in patients, the inventors took the advantage of the intrinsic ability of split inteins to mediate protein trans-splicing to reconstitute large Myosin- Vila protein encoded by large MY07A gene following their fragmentation into two split-intein flanked polypeptides.

The present disclosure relates to a combination of polynucleotides comprising: i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’ : a N-terminal fragment of Myosin- Vila protein and N-split intein, fused directly or indirectly via a linker, ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in a cell generates full length Myosin- Vila protein by protein splicing.

According to the present disclosure, the combination of polynucleotides comprises a first polynucleotide encoding a first fusion protein and a second polynucleotide encoding a second fusion protein. According to the present disclosure, by the term “combination” is meant that the first and second polynucleotides according to the present disclosure can be formulated in a single or as separate formulations.

According to the present disclosure, the term “nucleic acid sequence”, “nucleic acid molecule”, “nucleotide sequence” or “polynucleotide” may be used interchangeably to refer to any molecule composed of or comprising monomeric nucleotides. A polynucleotide may be a DNA or RNA.

Herein, the terms "peptide", "oligopeptide", "polypeptide" and "protein" are employed interchangeably and refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain.

The term "amino acid" refers to naturally occurring and unnatural amino acids (also referred to herein as "non-naturally occurring amino acids"), e.g., amino acid analogues and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogues refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogues can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid. The terms "amino acid" and "amino acid residue" are used interchangeably throughout.

The term "fusion protein" as used herein, refers to a recombinant protein comprising two or more protein domains from at least two different proteins linked, preferably covalently. Said fusion protein is obtained or obtainable by genetic fusion, for example by genetic fusion of at least two gene fragments encoding separate domains of distinct proteins. In preferred embodiments, a fusion protein is a single chain polypeptide which may be fully encoded by a nucleic acid sequence and includes at least two protein domains directly covalently linked by peptidic bound or optionally covalently linked via a peptidic linker. According to the present disclosure, said first fusion protein comprises a N-terminal fragment of Myosin- Vila protein and N-split intein, fused directly or indirectly via a linker and said second fusion protein comprises a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker.

The term "linker" as used herein, refers to a chemical group or a molecule linking two adjacent molecules or moieties. In some embodiments, the polynucleotide encodes a linker selected from the group consisting of a (GGS)n (SEQ ID NO: 142), a (GGGGS)n (SEQ ID NO: 143), a (G)n (SEQ ID NO: 144), an (EAAAK)n (SEQ ID NO: 145), a XTEN-based linker, or an (XP)n motif, or a combination of any of these, wherein n is independently an integer between 1 and 50. In other embodiments, a linker is not used. Instead, e.g., the polynucleotide sequences comprise nucleic acids encoding a first and second protein domains and further comprise additional nucleic acids in at least one of their ends that make the function of linker.

According to the present disclosure, the expression of first and second polynucleotides encoding said first and second fusion proteins in a cell generates Myosin- Vila protein by protein splicing.

The term “protein trans-splicing” or “protein splicing” refers to the excision of a split-intein from a larger precursor polypeptide through the cleavage of two peptide bonds and, the concomitant ligation of the flanking protein fragments, also called exteins through the formation of a new peptide bond to form a mature protein and the free intein (Shah NH and Muir TW, Chem Sci. 2014; 5(1): 446-461. 2013).

As used herein, the term “intein” refers to a protein that is capable of ligating the flanking sequences (exteins) into a new protein.

As used herein, the term “split-inteins” or “trans-splicing inteins” means naturally occurring or engineered constructed protein fragments (i.e., N-intein and C-intein) which bind to form a catalytically competent enzyme capable of catalyzing a protein splicing reaction that excises the N- and C-intein sequences and joins flanking sequences (N- and C-exteins) with a peptide bond.

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

According to the present disclosure, the term “N-split intein”, “N-intein”, or “N-terminal split intein” refers to any N-terminal amino acid sequence of a split intein that is capable of associating with a C-terminal amino acid sequence of said split intein to form a functional split intein that is capable of catalyzing a protein splicing reaction that excises the N- and C-intein sequences and joins flanking sequences (N- and C-exteins) with a peptide bond.

According to the present disclosure, the term “C-intein”, “C-split intein” or “C-terminal split intein” refers to any C-terminal amino acid sequence of a split intein that is capable of associating with a N-terminal amino acid sequence of said split intein to form a functional split intein that is capable of catalyzing a protein splicing reaction that excises the N and C-intein sequences and joins flanking sequences (N- and C-exteins) with a peptide bond.

In a particular embodiment, said N- and C-split inteins according to the present disclosure comprised in the first and second fusion proteins respectively can derive from the catalytic subunit of DNA polymerase III (DriaE) gene from different organisms such as cyanobacteria including Nostoc punctiforme (Npu), Synechocystis sp. Strain PCC6803 (Ssp), Fischerella sp. PCC 9605, Scytonema tolypothrichoides, Cyanobacteria bacterium SW 9 47-5, Nodularia spumigena, Nostoc flagelliforme, Crocosphaera watsonii WH 8502, Chroococcidiopsis cubana CCALA 043, or Trichodesuium erythraeum; preferably from Npu or Ssp.

In another particular embodiment, the N- and C-split inteins can derive from the DnaB gene from Cyanobacteria including R. marinus (Rma), Synechocystis sp. PC6803 (Ssp), Porphyra purpurea chloroplast (Ppu), or can derive from gp41-l, gp41-8, NrdJ-1, or IMPDH-1 (Carvajal- Vallejos P. et al. J Biol Chem. 2012 Aug 17; 287(34): 28686-28696).

In a preferred embodiment, N- and/or C- split inteins according to the present disclosure comprised in the first and second fusion proteins respectively can be engineered N- and/or C- split inteins. Said engineered N- and/or C- split inteins can be engineered by introducing mutations in natural N- and/or C- split intein sequences, in particular to enhance protein splicing activity.

In a preferred embodiment, the N-split intein and/or C-split intein sequences comprised in the first and second fusion proteins respectively comprise or consist of amino acid sequences selected from any of N- and C-split inteins listed in Table 1 below:

Table 1: Examples of pairs of N- and C-split inteins that can be used according to the present disclosure.

Amino acids in bold can be replaced by GEP amino acids to improve extein tolerance.

In a particular embodiment, the N- and C-split inteins comprised in the first and second fusion proteins respectively according to the present disclosure are N- and C-split inteins comprising or consisting of amino acid sequences of SEQ ID No: 1 (Cfa-N split intein) and SEQ ID No: 2 (Cfa-C-split intein:), SEQ ID No: 3 (Npu-N) and 4 (Npu-C), SEQ ID No: 5 (Cat-N) and 6 (Cat- C), SEQ ID No: 7 (Gp41-N) and 8 (Gp41-C), SEQ ID No: 9 (ConN) and 10 (ConC) or SEQ ID No: 11 (Nrdj 1-N) and 12 (Nrdj 1-C) or any functional variant(s) thereof. As used herein, the term "variant" or “functional variant” refers to a polypeptide sequence that is derived from N- and/or C-split inteins as described above and comprises an alteration, i.e., a substitution, insertion, and/or deletion, at one or more positions, but retain the capacity when bound to form a functional enzyme to catalyze a protein splicing reaction that excises the N and C-intein sequences and joins flanking sequences (N- and C-exteins) with a peptide bond.

The variant may be obtained by various techniques well known in the art. Examples of techniques for altering the nucleotide sequence encoding the native protein, include, but are not limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction.

The protein splicing efficiency of N- and/or C-split intein functional variants may be assessed for instance by measuring the protein reconstitution efficiency in a cell. In particular, the protein reconstitution efficiency can be measured by expressing in a cell a combination of polynucleotides, said first polynucleotide encodes a N-terminal fragment of a reporter protein (e.g., GFP) fused to N-split intein and a second polynucleotide encodes a C-terminal fragment of said gene reporter fused to the C-split intein. The reconstitution efficiency of the reporter protein can then be monitored by determining the level of expression of reconstituted protein.

The expression level of reconstituted protein may be determined by any suitable methods known by skilled persons. The quantity of the protein may be measured, for example, by semi- quantitative Western blots, enzyme-labelled and mediated immunoassays, such as ELISAs, biotin/avidin type assays, radioimmunoassay, immunoelectrophoresis, mass spectrometry, or immunoprecipitation or by protein or antibody arrays. In a particular embodiment, when said reporter protein is a fluorescent protein, the quantity of protein may be measured by flow cytometry or fluorescence microscopy.

The expression level can then be compared to a control value. According to a preferred embodiment, the term "control value " refers to the expression level of protein reconstituted with the native N- and C- split inteins in a cell expressing a combination of polynucleotides, said first polynucleotide encodes a N-terminal fragment of a reporter protein (e.g., GFP) fused to native N-split intein and a second polynucleotide encodes a C-terminal fragment of said gene reporter fused to the native C-split intein.

The protein reconstitution efficiency of a functional variant is similar to that of native split intein in a cell when the expression level of said protein reconstituted with functional variants of N- and/or C-split intein(s) in a cell is similar than the control value (i.e., expression level of said protein reconstituted with native N- and C-split inteins), in particular the expression level varies by less than 40%, 30%, 20% or 10% of the control value.

As used herein, the term "variant" or “functional variant” may refer to a polypeptide having an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity any one of the N- and/or C-split intein(s) as described above, in particular in Table 1 and preferably retains protein splicing capacity of said polypeptide as described above.

In a particular embodiment, said N-split intein and C-split intein according to the present disclosure comprised in the first and second fusion proteins respectively comprises or consists 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(s) thereof, preferably having 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1 to 12.

As used herein, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (NEEDLEMAN, and Wunsch).

The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk, Rice et al 2000 Trends Genet 16 :276-277). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification. The term "variant" or “functional variant” may also refer to a polypeptide having an amino acid sequence that differs from a native sequence by less than 10, 9, 8, 7, 6, 5, 4 or 3 substitutions, insertions and/or deletions. In a preferred embodiment, the variant differs from the native sequence by one or more conservative substitutions, preferably by less than 10, 9, 8, 7, 6, 5, 4 or 3 conservative substitutions. Examples of conservative substitutions are within the groups of 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).

The inventors previously engineered N- and C-split intein with superior protein transplicing properties. They showed that Cfa-N and Cfa-C-split inteins have a higher protein splicing efficiency than Npu split inteins (WO2017/132582 and WO2021/191447).

In a preferred embodiment, the N- and C-split inteins according to the present disclosure comprised in the first and second fusion proteins respectively are Cfa-N- and Cfa-C-split inteins comprising or consisting of SEQ ID NO: 1 (Cfa-N split intein :) and SEQ ID NO: 2 (Cfa-C- split intein) or any functional variants thereof, preferably having 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 1 and/or 2.

In a preferred embodiment, the functional variant of Cfa N- and/or Cfa C-Split intein retain the functional splicing activity of native Cfa split intein, more preferably have a protein splicing efficiency higher than Npu split-intein.

The protein splicing efficiency of functional variants of Cfa-N- and/or Cfa- C-split inteins may be assessed as described above. In a preferred embodiment, the expression level of reconstituted protein with functional variant(s) of Cfa-N- and/or Cfa-C-Split inteins is then compared to a control value that refers to the expression level of protein reconstituted with a Npu intein.

The protein reconstitution efficiency is higher in a cell when the expression level of said protein reconstituted with engineered Cfa-N- and/or Cfa-C-split intein(s) in a cell is at least 1.5-fold higher, or 2, 3, 4, 5-fold higher or even more than in a control value (e.g., with Npu intein).

In a more preferred embodiment, the functional variant of Cfa N- and/or Cfa C-Split intein can also splice faster than Npu split intein, preferably at least 1.5-fold higher, or 2, 3-fold higher or even more than Npu split intein. Protein transplicing activity can be measured by incubating N and C-inteins individually in splicing buffer (e.g., lOOmM sodium phosphates, 150 mM NaCl, ImM EDTA, pH 7.2) with 2 mM tris(2-carboxyethyl)phosphine (TCEP) for 15 minutes. Splicing is initiated by mixing N- and C-inteins and quenched by the addition of 8M guanidine hydrochloride, 4% Trifluoroacetic acid TFA (3: 1 v/v). Splicing reactions progress can be monitored by RP-HPLC or SDS-PAGE. When using RP-HPLC, each individual peak is normalized against the total area of all peaks combined and reaction curves are plotted (see detailed protocol, paragraphs [00724]-[00731] of WO2017/132580 application. When using SDS-PAGE quantification of splicing product is performed by densitometry using P-tubulin as a loading control.

A major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site. Engineered versions of naturally split inteins, in particular C-split intein comprising GEP amino acids in positions 20, 21 and 22 wherein said residue is numbered according to SEQ ID NO: 2 possess improved extein tolerance.

According to the present disclosure, the C-split intein derived from the catalytic subunit of DNA polymerase III (DnaE) gene may comprise GEP amino acids in positions 20, 21 and 22 wherein said residue is numbered according to SEQ ID NO: 2 (see Stevens et al., J Am Chem Soc. 2016 Feb 24; 138(7): 2162-2165, or Fig. 7A and B, C-intein of SEQ ID NO: 5 -358 of WO2017/132580), in particular instead of amino acid positions indicated in bold in the Table 1.

In a preferred embodiment, the C-split intein is selected from any C-split inteins disclosed in Table 2.

Table 2: Engineered C-split intein with improved extein tolerance.

In a preferred embodiment, C-split intein is a mutated C-split intein having GEP amino acids in positions 20, 21 and 22 wherein said residue is numbered according to SEQ ID NO: 2 or any functional variant thereof, preferably retaining the functional splicing activity of split intein as described above, and more preferably having improved extein tolerance.

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

The extein tolerance can be assessed for instance in kanamycine resistance assay as described in WO2017/132580 p. 60, paragraphs [00735] - [00738] in which a nucleic acid construct coding for a fragmented aminoglycoside phosphotransferase fused to a split intein with F, G, R or E present at the position +2 of the C-extein was transformed in DH5a competent cells and cultured at various concentrations of kanamycin. The cell density at 650 nm at 24 hours end point is measured and IC50 value is determined and compared with Cfa-Cmut. A functional variant having improved extein tolerance is a C-split intein having a similar IC50 than the split intein having GEP amino acids in positions 20, 21 and 22 wherein said residue is numbered according to SEQ ID NO: 2.

In a preferred embodiment, the C-split intein is Cfa-Cmut split intein comprising or consisting of SEQ ID NO: 13 or any functional variant thereof having 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 13, preferably retaining the functional splicing activity of C- split intein as described above, and more preferably having improved extein tolerance, again more preferably having a similar IC50 than Cfa-Cmut comprising or consisting of SEQ ID NO: 2. In particular the IC50 varies by less than 40%, 30%, 20% or 10% of the positive control value (i.e., IC 50 of Cfa-c-mut comprising or consisting of SEQ ID NO: 13).

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N-terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa C-split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vlla protein, fused directly or indirectly via a linker.

The inventors took the advantage of the intrinsic ability of split inteins as described above to mediate protein trans-splicing to reconstitute large Myosin- Vila protein following their fragmentation into two split-intein flanked polypeptides. Unconventional myosin- Vila protein (also named herein MY07A protein) encoded by MY07A gene is a member of the myosin gene family. Myosins are mechanochemical proteins characterized by the presence of a motor domain, an actin-binding domain, a neck domain that interacts with other proteins, and a tail domain that serves as an anchor. This gene encodes an unconventional myosin with a very short tail.

In particular, Myosin- Vila protein is a human Myosin- Vila protein (UniProtKB accession number: Q13402; updated on September 13, 2023 encoded by MY07A gene (GENE ID: 4647, updated on September 7, 2023), also known as DFNB2; MYU7A; NSRD2; USH1B; DFNA11; MYOVIIA. Alternative splicing results in multiple protein isoforms for example, Myosin- Vila isoform 1 (NCBI reference sequence: NP_000251.3, updated on June 4, 2023, SEQ ID NO: 15) or Myosin- Vila isoform 2 (NCBI reference sequence: NP_001120652.1, updated on June 04, 2023, SEQ ID NO: 90). According to the present disclosure, the term “Myosin- Vila protein” encompasses all known protein isoforms known of the Myosin- Vila protein.

In a preferred embodiment, according to the present disclosure Myosin- Vila protein can be a human Myosin- Vila protein as disclosed above or any functional variant thereof.

Preferably, as used herein, the term "variant" or “functional variant” refers to a polypeptide having an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to the native sequence and retain function of said polypeptide, herein Myosin- Vila protein, preferably human Myosin- Vila protein (SEQ ID NO: 15), in particular actin-based motor activity of the human Myosin- Vila protein.

Myosin- Vila is a functional actin-based motor. Myosin- Vila associates with melanosomes to move along actin filaments to the apical retinal photoreceptor epithelium (RPE) and associates with RPE phagosomes and contributes to their motility.

Myosin- Vila activity of functional variant may be assessed by correcting the melanosome localization in the RPE and ciliary opsin distribution in Myo7a mutant Shaker- 1 mice model (Self T et al. development, 1998, Feb;125(4):557-66).

In a preferred embodiment, Myosin- Vila protein is human Myosin- Vila protein comprising or consisting of SEQ ID NO: 15 or any functional variant thereof having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 15.

More preferably, the term "variant" or “functional variant” refers to a polypeptide having an amino acid sequence that differs from a native sequence by less than 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10 or 5 substitutions, insertions and/or deletions. In a preferred embodiment, the functional variant differs from the native sequence by one or more conservative substitutions, preferably by less than 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10 or 5 conservative substitutions. In certain embodiments, the first methionine of the Myosin- Vila protein sequence can be removed. A number of different mammalians Myosin- Vlla are known including, but being not limited to, human, pig, chimpanzee, dog, cow, mouse, rabbit or rat, and can be easily found in sequence databases. The coding sequence may be easily determined by the skilled person based on the polypeptide sequence.

According to the present disclosure, a polynucleotide encoding the Myosin- Vila protein as described above is split into at least two nucleic acid sequences encoding N- and C-terminal Myosin- Vila protein fragments, each fragment being fused with at least N- and C-split inteins as described above to form a first and a second fusion protein, respectively, in such a manner that following expression of said first and second fusion proteins, a protein splicing reaction can occur in a cell and induces the excision of the N and C-split intein sequences and the ligation of Myosin- Vila fragment flanking sequences (N- and C-terminal Myosin- Vila fragments, also named N- and C-exteins) with a peptide bond to reconstitute the full-length of Myosin- Vila protein as described above in a cell.

According to the present disclosure, the Myosin- Vila protein or any functional variant thereof as described above can be split at any positions into a N- and C-terminal Myosin- Vila fragments. However, the inventors have shown that some specific split positions are particularly advantageous to increase the efficiency of Myosin- Vila protein reconstitution. In particular, the Myosin- Vila protein or any functional variant thereof as described above can be split into bland C-terminal Myosin- Vila fragments at the split position 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 residue is numbered according to SEQ ID NO: 15. It will be easy for a person skilled in the art to determine the split positions in the Myosin- Vila isoforms or Myosin- Vila functional variants of Myosin- Vila, in particular by sequence alignment as illustrated in Figure 1.

In a preferred embodiment, the N-terminal Myosin- Vila fragment ends up to residue 823 and the C-terminal fragment of Myosin- Vila protein starts from residue 824 respectively wherein said residue is numbered according to SEQ ID NO: 15. In another preferred embodiment, the N-terminal Myosin- Vila fragment ends up to residue 1172 and the C-terminal fragment of Myosin- Vila protein starts from residue 1173 respectively wherein said residue is numbered according to SEQ ID NO: 15.

In another preferred embodiment, the N-terminal Myosin- Vila fragment ends up to residue 1197 and the C-terminal fragment of Myosin- Vila protein starts from residue 1198 respectively wherein said residue is numbered according to SEQ ID NO: 15.

In another preferred embodiment, the N-terminal Myosin- Vila fragment ends up to residue 1200 and the C-terminal fragment of Myosin- Vila protein starts from residue 1201 respectively wherein said residue is numbered according to SEQ ID NO: 15.

In another preferred embodiment, the N-terminal Myosin- Vila fragment ends up to residue 1226 and the C-terminal fragment of Myosin- Vila protein starts from residue 1227 respectively wherein said residue is numbered according to SEQ ID NO: 15.

In another preferred embodiment, the N-terminal Myosin- Vila fragment ends up to residue 1283 and the C-terminal fragment of Myosin- Vila protein starts from residue 1284 respectively wherein said residue is numbered according to SEQ ID NO: 15.

In another preferred embodiment, the N-terminal Myosin- Vila fragment ends up to residue 1325 and the C-terminal fragment of Myosin- Vila protein starts from residue 1326 respectively wherein said residue is numbered according to SEQ ID NO: 15.

In a particular embodiment, the first and second fusion proteins according to the present disclosure comprise a N-terminal Myosin- Vila fragment and a C-terminal Myosin- Vila fragment respectively consisting of amino acid sequences selected from the Table 3, preferably from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27 and, SEQ ID NO: 28 and 29, 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 having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of SEQ ID NO: 16 to 29, preferably SEQ ID NO: 18 to 27 and preferably wherein reconstituted functional variant of Myosin- Vila protein retains the function of said Myosin- Vila, in particular actin-based motor activity of the human Myosin- Vila protein. The function of Myosin- VII a functional variant can be assessed for example by the capacity of said variant of correcting the melanosome localization in the RPE and ciliary opsin distribution in Myo7a mutant Shaker- 1 mice model (Self T et al. development, 1998, Feb;125(4):557-66).

Table 3: Examples of N-and C-terminal Myosin- Vila fragments. Highlighted amino acids in Full-length Myosin- Vila represent preferred split positions. The first methionine of the Myosin- VII and N-terminal fragment sequences is indicated in the sequences listed in table 3. First methionine can be maintained or removed in certain embodiments. In a preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:

- the N-terminal fragment of Myosin- Vila protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively; or

- the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-C-split intein of SEQ ID NO: 2 or Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:

- the N-terminal fragment of Myosin- Vila protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively; or

- the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and

SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises polynucleotides encoding a first fusion protein and a second fusion protein comprising amino acid sequences selected from the pairs disclosed in Table 4, preferably selected from the pairs consisting of: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, and SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 30-43.

Table 4: Preferred first and second protein fusions according to the present disclosure.

Cfa-N-Split intein and Cfa-C-mut-split inteins sequences are indicated in bold. In certain embodiments, first methionine of the fusion proteins is indicated and can be maintained or removed.

To prevent the accumulation of undesired starting materials and to eliminate the excised intein fragments, a degron can be fused directly or indirectly via a linker to the first and/or second fusion proteins to mediate degradation of the excised intein.

A “protein degradation signal” or “degron” refers to a peptide fragment that induces degradation of the protein that contains the fragment. Protein degradation can happen through any of the many known protein degradation pathways, including but not limited to, ubiquitination, lysosomal degradation or autophagy. In a preferred embodiment, the degron targets protein to the ubiquitin-proteasome pathway. In a more preferred embodiment, the degron according to the present disclosure comprises amino acid sequence having less than less than 200, 190, 180, 170, 150, 130, 125, 110, 100, 95, 90, 85, 80, 75 amino acids. In a particular embodiment, said degron is fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, preferably said degron is located at the 3’-end of the N-Split intein or at the 5’end of the C-split intein.

In a particular embodiment, said degron is fused, directly or indirectly via a linker, to the N- split intein and C-split intein comprised in the first and second fusion proteins, respectively, preferably said degron is located at the 3’-end of the N-Split intein and at the 5’end of the C- split intein.

In a preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein or at the 5’end of the C-split intein.

In a preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and at the 5’end of the C-split intein. In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ a Cfa-c-Split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, and wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3’-end of the N-Split intein or at the 5’end of the C-split intein.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ a Cfa-c-Split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, and wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3’-end of the N-Split intein and at the 5’end of the C-split intein. In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:

- the N-terminal fragment of Myosin- Vila protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively; or

- the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively, wherein said residue is numbered according to SEQ ID NO: 15, or

- the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29 and wherein said first or second fusion protein further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein or at the 5 ’end of the C-split intein.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:

- the N-terminal fragment of Myosin- Vila 1 protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively; or

- the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively; wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29 and wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and at the 5 ’end of the C-split intein.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-c-Split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila 1 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:

- the N-terminal fragment of Myosin- Vila 1 protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively; - the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively; or

- the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively; wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29 and wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein or at the 5 ’end of the C-split intein.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-c-Split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:

- the N-terminal fragment of Myosin- Vila 1 protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively; or - the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively; or

- the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively; wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29 and wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and at the 5 ’end of the C-split intein.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure encode a first fusion protein and a second fusion protein comprising amino acid sequences selected from the pairs consisting of: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41; and SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 30-43 and wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein or at the 5 ’end of the C-split intein. In a more preferred embodiment, the combination of polynucleotides according to the present disclosure encode a first fusion protein and a second fusion protein comprising amino acid sequences selected from the pairs consisting of: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41; and SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 30-43 and wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and at the 5’end of the C-split intein.

According to the present disclosure, said degron can be selected as non-limiting examples in the degrons listed in Table 5 below.

Table 5: Examples of degrons and corresponding sequences.

In a preferred embodiment, the degron comprised in the first and/or second fusion protein according to the present disclosure can be selected from the group consisting of: CL1 (SEQ ID NO: 44), Degl (SEQ ID NO: 45), PEST (SEQ ID NO: 46), DD1 (SEQ ID NO: 47), DD2 (SEQ ID NO: 48), VI 5 (SEQ ID NO: 49), Ml (SEQ ID NO: 50), M2 (SEQ ID NO: 51), SopE (SEQ ID NO: 52), SopEl-78 (SEQ ID NO: 53), SopE 15-78 (SEQ ID NO: 54), SopE-15-50 (SEQ ID NO: 55), L2 (SEQ ID NO: 56), L6 (SEQ ID NO: 57), L9 (SEQ ID NO: 58), LIO (SEQ ID NO: 59), Lil (SEQ ID NO: 60), L12 (SEQ ID NO: 61), L15 (SEQ ID NO: 62), L16 (SEQ ID NO: 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: 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), CL 10+CL 1 (SEQ ID NO: 76), CL 1+CL 10 (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: 82), DD I 3 (SEQ ID NO: 83), CHAI (SEQID NI: 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), RBI (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), DegVl (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), Ndcl0-2p (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), Sul A (SEQ ID NO: 116), HdiR (SEQ ID NO: 117), YopE (SEQ ID NO: 118) and HdiR-flip (SEQ ID NO: 119), preferably DDI, DD3, PEST, SopE, V12, M4, L2, L9, more preferably SopE, L2, L9, M4 or V12 or any combination thereof or any functional variant thereof that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of the excised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., Myosin- Vila protein) preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 44 to 119, or any combination thereof.

In a preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and/or at the 5’end of the C-split intein, and wherein the degron comprised in the first and/or second fusion protein according to the present 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., that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of the excised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., Myosin- Vila protein)), preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 44 to 119 or any combination thereof.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ a Cfa-c-Split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein said first and/or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and/or at the 5’end of the C-split intein, and wherein the degron comprised in the first and/or second fusion protein according to the present 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., that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of the excised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., Myosin- Vila protein)), , preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 44 to 119 or any combination thereof.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:

- the N-terminal fragment of Myosin- Vila protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively, wherein said residue is numbered according to SEQ ID NO: 15,

- the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively, wherein said residue is numbered according to SEQ ID NO: 15, or

- the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29 and wherein said first and/or second fusion protein further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3’-end of the N-Split intein and/or at the 5’end of the C- split intein, and wherein the degron comprised in the first and/or second fusion protein according to the present 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., that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of the excised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., Myosin- Vila protein)), preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 44 to 119 or any combination thereof.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-c-Split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila 1 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:

- the N-terminal fragment of Myosin- Vila 1 protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively;

- the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively; or

- the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively; wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21 or SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25 and SEQ ID NO: 26 and 27 and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29 and wherein said first and/or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and/or at the 5 ’end of the C-split intein, and wherein the degron comprised in the first and/or second fusion protein according to the present 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., that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of the excised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., Myosin- Vila protein)), preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 44 to 119 or any combination thereof.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure encode a first fusion protein and a second fusion protein comprising amino acid sequences selected from the pairs consisting of: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41; and SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 30-43 and wherein said first and/or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and/or second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and/or at the 5’end of the C-split intein, and wherein the degron comprised in the first and/or second fusion protein according to the present 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., that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of the excised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., Myosin- Vila protein)), preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 44 to 119 or any combination thereof.

The functional variant of the degron as described above induces degradation of the excised intein that is fused to the degron, and also of the starting material fusion protein comprising the protein of interest, the intein and the degron and preferably does not interfere with the reconstitution of the protein of interest (i.e., Myosin- Vila protein).

The degradation of the starting material consisting of the fusion protein comprising the protein of interest, the intein and the degron and the degradation of the excised intein that is fused to the degron can be tested according to Example 3 of WO2021181447, in particularly said degron is fused to a first fusion protein comprising a N-Split intein and a N-terminal fragment of a protein, said degron being fused to the 3’ end of N-Split intein and/or to a second fusion protein comprising a C-Split intein and a C-terminal fragment of a protein, said degron being fused to the 5 ’-end of the C-Split intein of a protein. The polynucleotides encoding said first and second protein is transfected in a cell, and the amount of starting material and excised intein is determined for example by Western blot. The degron induces degradation of starting material (i.e., the fusion protein comprising the protein of interest, the intein and the degron) and excised intein(s) when the amount of the starting material and excised intein is lower than the starting material and excised intein amount in a cell transfected with fusion proteins without degrons, preferably when the amount of intein is at least 1.2, 1.3, 1.4, 1.5, 1.8 or 2.0 fold lower than the starting material and intein amount in a cell transfected with fusion proteins without degrons. The expression level of reconstituted protein may be determined by any suitable methods known by skilled persons. The quantity of the reconstituted protein may be measured, for example, by semi-quantitative Western blots, enzyme-labelled and mediated immunoassays, such as ELISAs, biotin/avidin type assays, radioimmunoassay, immunoelectrophoresis, mass spectrometry, or immunoprecipitation or by protein or antibody arrays.

In another aspect, the present disclosure relates to i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N-terminal fragment of a protein of interest and N-split intein, fused directly or indirectly via a linker, ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of said protein of interest, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in a cell generates full length protein of interest by protein splicing, wherein said first and/or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and/or at the 5 ’end of the C-split intein, and wherein the degron comprised in the first and/or second fusion protein according to the present disclosure is 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%, 99% sequence identity to any one of sequences SEQ ID Nos: 44 to 119, preferably SEQ ID NO: 75-119.

In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of protein of interest and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-C-split intein of SEQ ID NO: 2 or Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of the protein of interest, fused directly or indirectly via a linker, wherein said first and/or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and/or at the 5 ’end of the C-split intein, and wherein the degron comprised in the first and/or second fusion protein according to the present disclosure is 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%, 99% sequence identity to any one of sequences SEQ ID Nos: 44 to 119, preferably SEQ ID NO: 75-119.

In some embodiments, each first and second polynucleotides according to the present disclosure encoding the first and second fusion proteins, respectively, as described above may be a nucleic acid construct.

In a particular embodiment, said first and second polynucleotides may be an optimized sequence encoding the first and second fusion proteins. The term "codon optimized" means that a codon that expresses a bias for human (i.e. is common in human genes but uncommon in other mammalian genes or non-mammalian genes) is changed to a synonymous codon (a codon that codes for the same amino acid) that does not express a bias for human. Thus, the change in codon does not result in any amino acid change in the encoded protein.

The term “nucleic acid construct” as used herein refers to a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleic acid molecule, either single- or double-stranded, which has been modified to contain segments of nucleic acids sequences, which are combined and juxtaposed in a manner, which would not otherwise exist in nature. A nucleic acid construct usually is a “vector”, i.e., a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell.

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

The nucleic acid construct may also comprise additional regulatory elements such as, for example, enhancer sequences, a polylinker sequence facilitating the insertion of a DNA fragment within a vector and/or splicing signal sequences.

According to a preferred embodiment, said nucleic acid construct may comprise a SV40 intron.

In one embodiment, the polynucleotide or nucleic acid construct according to the present disclosure comprises a promoter. Said promoter initiates transgene expression upon introduction into a host cell.

In a preferred embodiment, the promoter according to the present disclosure can be selected from the group consisting of: Cytomegalovirus (CMV) promoter, chimeric reduced version of the CMV and chicken beta-actin (CEBA) promoter, human phosphoglycerate kinase (hPGK) promoter, chimeric CMV enhanced and human phosphoglycerate kinase (ePGK) promoter, photoreceptor-specific, human rhodopsin kinase (hGRKl) promoter, rod specific IRBP promoter, VMD2 (vitelliform macular dystrophy /Best disease) promoter, and EFl alpha promoter. However, any suitable promoter known in the art may be used.

As used herein, the term "promoter" refers to a regulatory element that directs the transcription of a nucleic acid to which it is operably linked. A promoter can regulate both rate and efficiency of transcription of an operably linked nucleic acid. A promoter may also be operably linked to other regulatory elements which enhance ("enhancers") or repress ("repressors") promoterdependent transcription of a nucleic acid. These regulatory elements include, without limitation, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter, including e.g., attenuators, enhancers, and silencers. The promoter is located near the transcription start site of the gene or coding sequence to which it is operably linked, on the same strand and upstream of the DNA sequence (towards the 5' region of the sense strand). A promoter can be about 100-3000 base pairs long. Positions in a promoter are designated relative to the transcriptional start site for a particular gene (i.e., positions upstream are negative numbers counting back from -1, for example -100 is a position 100 base pairs upstream).

As used herein, the term “operably linked” refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically but not necessarily contiguous; where it is necessary to join two protein encoding regions, they are contiguous and in reading frame.

In a preferred embodiment, each polynucleotide or nucleic acid construct according to the present disclosure may be comprised in an expression vector.

As used herein, the term "expression vector" refers to a nucleic acid molecule used as a vehicle to transfer genetic material, and in particular to deliver a nucleic acid into a host cell, either in vitro or in vivo. Expression vector also refers to a nucleic acid molecule capable of effecting expression of a gene (transgene) in host cells or host organisms compatible with such sequences. Expression vectors typically include at least suitable transcription regulatory sequences and optionally 3 ’-transcription termination signals.

Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements able to respond to a precise inductive signal (endogenous or chimeric transcription factors) or specific for certain cells, organs or tissues. Vectors include, but are not limited to, plasmids, phasmids, cosmids, transposable elements, viruses, and artificial chromosomes (e.g., YACs).

Preferably, the vectors of the disclosure is vectors suitable for use in gene or cell therapy, and in particular is suitable to target retinal or hair cells. In some embodiments, the expression vector is a viral vector, such as vectors derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV or SNV, lentiviral vectors (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)), adenoviral (Ad) vectors, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors.

As is known in the art, depending on the specific viral vector considered for use, suitable sequences should be introduced in the vector of the disclosure for obtaining a functional viral vector, such as AAV ITRs for an AAV vector, or LTRs for lentiviral vectors. In a particular embodiment, said vector is an AAV vector.

AAV has arisen considerable interest as a potential vector for human gene therapy. Among the favorable properties of the virus are its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and the wide range of cell lines derived from different tissues that can be infected. The AAV genome is composed of a linear, single-stranded DNA molecule which contains 4681 bases (Berns and Bohenzky, 1987, Advances in Virus Research (Academic Press, Inc.) 32:243-307). The genome includes inverted terminal repeats (ITRs) at each end, which function in cis as origins of DNA replication and as packaging signals for the virus. The ITRs are approximately 145 bp in length. The internal non-repeated portion of the genome includes two large open reading frames, known as the AAV rep and cap genes, respectively. These genes code for the viral proteins involved in replication and packaging of the virion. In particular, at least four viral proteins are synthesized from the AAV rep gene, Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight. The AAV cap gene encodes at least three proteins, VP1, VP2 and VP3. For a detailed description of the AAV genome, see, e.g., Muzyczka, N. 1992 Current Topics in Microbiol, and Immunol. 158:97- 129.

Thus, in one embodiment, the polynucleotides, nucleic acid constructs or expression vectors according to the present disclosure thereof further comprises a 5’ITR and a 3TTR sequences, preferably a 5’ITR and a 3’ ITR sequences of an adeno-associated virus.

As used herein the term “inverted terminal repeat (ITR)” refers to a nucleotide sequence located at the 5’-end (5’ITR) and a nucleotide sequence located at the 3’-end (3’ITR) of a virus, that contain palindromic sequences and that can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication. They are also needed for viral genome integration into the host genome; for the rescue from the host genome; and for the encapsidation of viral nucleic acid into mature virions. The ITRs are required in cis for the vector genome replication and its packaging into the viral particles.

In one embodiment, the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure further comprises a 5’ITR and a 3TTR of an AAV, preferably of a serotype AAV2.

The polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins as described above may be packaged into a virus capsid to generate a "viral particle", also named “viral vector particle”. In a particular embodiment, the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure is packaged into an AAV-derived capsids to generate an "adeno- associated viral particles" or "AAV particles". The present disclosure relates to viral particles comprising the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure and preferably comprising capsid proteins of adeno-associated virus.

The construction of recombinant AAV viral particles is generally known in the art and has been described for instance in US 5,173,414 and US5,139,941; WO 92/01070, WO 93/03769, Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533- 539; Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.

Thus, in AAV viral particle according to the present disclosure, the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins as described above including ITR(s) of a given AAV serotype can be packaged, for example, into: a) a viral particle constituted of capsid proteins derived from the same or different AAV serotype [e.g. AAV2 ITRs and AAV5 capsid proteins; AAV2 ITRs and AAV8 capsid proteins; AAV2 ITRs and Anc80 capsid proteins; AAV2 ITRs and AAV9 capsid proteins]; b) a mosaic viral particle constituted of a mixture of capsid proteins from different AAV serotypes or mutants [e.g. AAV2 ITRs with AAV1 and AAV5 capsid proteins]; c) a chimeric viral particle constituted of capsid proteins that have been truncated by domain swapping between different AAV serotypes or variants [e.g. AAV2 ITRs with AAV5 capsid proteins with AAV3 domains].

The skilled person will appreciate that the AAV viral particle for use according to the present disclosure may comprise capsid proteins from any AAV serotype including AAV1, AAV2, AAV3 (including types 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, synthetic AAV variants such as NP40, NP59, NP84 (Paulk et al. Mol then 2018.26(l):289-303), LK03 (Wang L et al. Mol Then 2015. 23(12): 1877-87), AAV3-ST (Vercauteren et al. Mol Then 2016.24(6): 1042-1049), Anc80 (Zinn E et al., Cell Rep. 2015;12(6): 1056-68), AAVrhlO and any other AAV serotype now known or later discovered.

Thus, in a further aspect, the present disclosure relates to a viral particle comprising the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins as described above and preferably comprising capsid proteins of adeno-associated virus such as capsid proteins from AAV9 and AAV-PHP.B, AAV2, AAV8 and AAV5.

Therapeutic use

According to the present disclosure, the 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 is administered in a subject in need thereof for use in gene therapy, preferably for the treatment of MYO7A-associated disease.

As used herein, "gene therapy" refers to the administration of a gene-therapy vector to treat a disease caused by a change in the subject DNA sequence, in particular a disease caused by a mutation in at least one gene (i.e., genetic disease) such as MY07A into a subject in need thereof.

As used herein, the term "treatment", "treat" or "treating" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease. In certain embodiments, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease. According to the present disclosure, examples of symptoms associated with MYO7A-associated disease are deafness, reduced vestibular function, and retinal degeneration.

The term “subject” or “patient” as used herein, refers to mammals. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, nonhuman primates such as apes, chimpanzees, monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like. In particular embodiment, said subject is a human patient.

The combination of polynucleotides, nucleic acid constructs, expression vectors or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure will be typically included in a pharmaceutical composition or medicament, optionally in combination with a pharmaceutical carrier, diluent and/or adjuvant. Such composition or medicinal product comprises the product of the disclosure in an effective amount, sufficient to provide a desired therapeutic effect, and a pharmaceutically acceptable carrier or excipient.

As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency or recognized pharmacopeia such as European Pharmacopeia, for use in animals and/or humans. The term "excipient" refers to a diluent, adjuvant, carrier, or vehicle with which the therapeutic agent is administered. As is well known in the art, pharmaceutically acceptable excipients are relatively inert substances that facilitate administration of a pharmacologically effective substance and can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to use. For example, an excipient can give form or consistency, or act as a diluent. Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolality, encapsulating agents, pH buffering substances, and buffers.

In one embodiment, the pharmaceutical composition is a parenteral pharmaceutical composition, including a composition suitable for intravenous, intraarterial, intramuscular, intranasal, intraocular, intravitreal, suprachoroidal or subretinal administration. These pharmaceutical compositions are exemplary only and do not limit the pharmaceutical compositions suitable for other parenteral and non-parenteral administration routes. The pharmaceutical compositions described herein can be packaged in single unit dosage or in multidosage forms. Myosin- Vila is a type of motor protein that moves along actin filaments and transports various cargoes within the cell. It is especially important for the function of sensory cells, such as inner ear hair cells and retinal photoreceptors. Myosin- Vila is involved in the development and maintenance of the hair cell stereocilia, which are the structures that detect sound vibrations. Myosin- Vila is also involved in the transport of melanosomes, which are pigment-containing organelles, in the retinal pigment epithelium (RPE) cells. Melanosomes protect the retina from light damage and help with visual adaptation.

Mutations in the MYO 7 A gene, cause deafness and retinal degeneration. These mutations can affect the motor domain or the tail domain of Myosin- Vila, and result in different phenotypes depending on the severity and location of the mutation. Some mutations cause Usher syndrome type IB (USH1B), which is a form of deaf-blindness that also involves vestibular dysfunction. Other mutations cause non-syndromic deafness (DFNB2 or DFNA11 which is isolated hearing loss without other symptoms. The deafness caused by MY07A mutations is due to the disruption of hair cell function and the progressive loss of hair cells. The retinal degeneration caused by MY07A mutations is due to the abnormal distribution of melanosomes in the RPE cells, which leads to photoreceptor damage and impaired visual adaptation.

In a more preferred embodiment, the present disclosure relates to the 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 or pharmaceutical composition thereof for use in the treatment of a MYO7A-associated disease, preferably Usher syndrome type IB, non-syndromic deafness DFNB2 or DFNA11.

The present disclosure also relates to the use of the 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 or pharmaceutical composition thereof for the manufacture of a medicament for treating a MY07A associated disease.

The disclosure also provides a method for treating a MYO7A-associated disease as described above in a patient in need thereof comprising administering to said patient a therapeutically effective amount of the polynucleotides, nucleic acid constructs, expression vectors or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins as described above or pharmaceutical composition thereof.

As used herein a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the products of the disclosure may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the product or pharmaceutical composition to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also typically one in which any toxic or detrimental effect of the product or pharmaceutical composition is outweighed by the therapeutically beneficial effects. According to the present disclosure, a therapeutically effective amount allows to reduce for example the deafness, retinal degeneration and improve vestibular function.

According to the present disclosure, said combination of polynucleotides can be administered into the subject in a single formulation or as separate formulations simultaneously, sequentially or separately. Such administration encompasses co-administration of the first and second polynucleotides in a substantially simultaneous manner, such as in a single formulation having a fixed ratio of the polynucleotides or in separate formulations for each polynucleotide. In addition, such administration also encompasses use of each polynucleotide in a sequential or separate manner, either at approximately the same time or at different times. Regardless of whether the polynucleotides are administered as a single formulation or in separate formulations, the first and second polynucleotides are administered to the same subject as part of the same course of therapy. In any case, the treatment regimen will provide beneficial effects in treating the diseases described herein.

In one embodiment, the combination of polynucleotides, nucleic acid constructs, expression vectors or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure for its therapeutic use is administered to the subject or patient by a parenteral route, in particularly by intravenous, intraarterial, intramuscular, intranasal, intraocular, intravitreal, suprachoroidal or subretinal route.

The amount of product of the disclosure that is administered to the subject or patient may vary depending on the particular circumstances of the individual subject or patient including, age, sex, and weight of the individual; the nature and stage of the disease, the aggressiveness of the disease; the route of administration; and/or concomitant medication that has been prescribed to the subject or patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For any particular subject, specific dosage regimens may be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions. The dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.

Kit

In another aspect, the disclosure further relates to a kit, preferably for use in the treatment of MYO7A-associated disease as described above, preferably Usher syndrome type IB comprising a combination of polynucleotides as described above, comprising: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, optionally wherein said first and/or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3’-end of the N-Split intein and/or at the 5’end of the C- split intein, again 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 the protein that contains the fragment, preferably having at least at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 44 to 119 or any combination thereof.

In a more preferred embodiment, the kit comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : Cfa C-split intein of SEQ ID NO: 2 or Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, and optionally wherein said first and/or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C- split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3’-end of the N-Split intein and/or at the 5’end of the C-split intein, again 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 the protein that contains the fragment, preferably having at least at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 44 to 119 or any combination thereof.

In a more preferred embodiment, the kit comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:

- the N-terminal fragment of Myosin- Vila protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively,

- the N-terminal fragment of Myosin- Vila rotein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1225 and the C- terminal fragment of Myosin- Vila protein from residue 1226 respectively

- the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively; or

- the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vila fragment respectively consists of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NOs: 16 to 29 and optionally wherein said first and/or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C- split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3’-end of the N-Split intein and/or at the 5’end of the C-split intein, again 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 the protein that contains the fragment, preferably having at least at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 44 to 119 or any combination thereof.

In a more preferred embodiment, the kit comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Myosin- Vila protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa C-split intein of SEQ ID NO: 2 or Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:

- the N-terminal fragment of Myosin- Vila protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively,

- the N-terminal fragment of Myosin- Vila rotein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively, - the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively,

- the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively

- the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively; or

- the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vila fragment respectively consists of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, and SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NOs: 16 to 29 and optionally wherein said first and/or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C- split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3’-end of the N-Split intein and/or at the 5’end of the C-split intein, again 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 the protein that contains the fragment, preferably having at least at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 44 to 119 or any combination thereof,.

In a more preferred embodiment, the kit comprises a combination of polynucleotides encoding a first fusion protein and a second fusion protein comprising amino acid sequences selected from the groups of pairs consisting of: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, and, SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% identity to any one of sequences SEQ ID NO: 30-43, and optionally wherein said first and/or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and/or second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and/or at the 5’end of the C-split intein, again 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 the protein that contains the fragment, preferably having at least at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 44 to 119 or any combination thereof.

In another aspect, the present disclosure relates to a kit comprising a combination of polynucleotides comprising a first polynucleotide encoding a first fusion protein comprising from 5 ’ to 3 ’ : a N-terminal fragment of a protein of interest and N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of said protein of interest, fused directly or indirectly via a linker, wherein said first and/or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and/or at the 5’end of the C-split intein, and wherein the degron comprised in the first and/or second fusion protein according to the present disclosure is 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%, 99% sequence identity to any one of sequences SEQ ID Nos: 44 to 119, preferably SEQ ID NO: 75-119.

In a more preferred embodiment, the present disclosure relates to a kit comprising a combination of polynucleotides comprising: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of protein of interest and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-C-split intein of SEQ ID NO: 2 or Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of the protein of interest, fused directly or indirectly via a linker, wherein said first and/or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and/or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and/or at the 5 ’end of the C-split intein, and wherein the degron comprised in the first and/or second fusion protein according to the present 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%, 99% sequence identity to any one of sequences SEQ ID Nos: 44 to 119, preferably SEQ ID NO: 75- 119 or any combination thereof.

In another embodiment, the present disclosure relates to a kit comprising a nucleic acid construct comprising a degron comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 75-119, or any combination thereof or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 75-119.

The kit may include instructions or packaging materials that describe how to administer the polynucleotides contained within the kit to a patient.

Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In certain embodiments, the kits may include one or more ampoules or syringes that contain the products of the invention in a suitable liquid or solution form.

The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

EXAMPLES

1. Materials and Methods

Materials: Oligonucleotides were purchased from Eurofins genomics. Synthetic genes were purchased from GENEWIZ. Pfu Ultra fusion polymerase for cloning and all restriction enzymes were purchased from Thermofisher Scientific. High-competency cells used for cloning were generated from XLIO-Gold chemically competent E. coli. HEK293T and HeLa cells were purchased from ATCC. DNA purification kits were purchased from Thermofisher Scientific. All plasmids were sequenced by Macrogen. Luria Bertani (LB) media, and all buffering salts were purchased from Thermofisher Scientific. Coomassie brilliant blue, NH4HCO3, DTT, formic acid, fetal bovine serum and asolectin from soybean were purchased from Sigma- Aldrich. Acetonitrile (ACN) was purchased from Carlo-Erba. EDTA-free complete protease inhibitors were purchased from Roche. Lipofectamine 2000 transfection reagent, DMEM high glucose GlutaMAX supplement, RPMI 1640 medium GlutaMAX supplement, RIPA lysis and extraction buffer, BCA protein assay kit, MES-SDS running buffer, pre-stained protein ladder and SDS-PAGE (Bis-tris and Tris-acetate gels) were purchased from Thermofisher Scientific. The primary antibodies used were anti-flag tag mouse monoclonal antibody (Sigma and Abeam), anti-MYO7A rabbit monoclonal antibody (Abeam), anti -HA tag rabbit monoclonal antibody (Thermo Fisher Scientific), and anti-tubulin rabbit polyclonal antibody (Thermo Fisher Scientific). The secondary goat anti-mouse IgG (H+L) highly cross-adsorbed Alexa fluor plus 488 antibody, secondary goat anti-rabbit IgG (H+L) antibody highly cross-adsorbed Alexa fluor 633 antibody, and 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, DI 306) were purchased from Thermo Fisher Scientific. HRP-conjugated secondary antibody was purchased from Vitro. Dodecyl maltoside (D310) and cholesteryl hemisuccinate (CH210) solution were purchased from Anatrace. Trypsin was purchased from Promega. ADP-GloTM Kinase Assay was purchased from Promega. SuperSignal ELISA Femto Maximum Sensitivity Substrate was purchased from Thermo Fisher Scientific.Equipment:

Gels and Western-blots were imaged with a LI-COR Odyssey Infrared Imager. Cell lysis was carried out using a SFX550 Branson sonifier. ATP/ADP signal was acquired by a luminescence microplate reader (Synergy HIM, BioTek). Mouse eye examination was conducted by retinography and optical coherence tomography (OCT).

Cloning of Recombinant DNA

Synthetic genes to prepare constructs MY07A-l-1200-CfaN-3FT (SEQ ID NO: 124), MY07A- 1201-2215-CfaCmut-3FT (SEQ ID NO: 125) were purchased and introduced into plasmid expression vectors using Kpnl and Notl restriction enzymes. Constructs MYO7A-l-1172-CfaN-3FT (SEQ ID NO: 120), MYO7A-1173-2215-CfaCmut- 3FT (SEQ ID NO: 121), MYO7A-l-1197-CfaN-3FT (SEQ ID NO: 122), MYO7A-1198-2215- CfaCmut-3FT (SEQ ID NO: 123), MYO7A-l-1226-CfaN-3FT (SEQ ID NO: 126), MYO7A- 1227-2215-CfaCmut-3FT (SEQ ID NO: 127), MYO7A-l-1283-CfaN-3FT (SEQ ID NO: 128), MYO7A-1284-2215-CfaCmut-3FT (SEQ ID NO: 129) MYO7A-3FT (SEQ ID NO: 134) were prepared by restriction enzymes free cloning.

The identity of all recombinant plasmids was confirmed through sequencing and the corresponding protein sequences are reported in Table 6.

Table 6: Protein sequences of constructs used in the examples. (1) Said residues are numbered according to SEQ ID NO: 15 (full-length Myosin- Vila)

Transfection of intein plasmids in HEK293T and HeLa cells:

HEK293T cells (ATCC, CRL-3216) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S) at 37°C in a 5% CO2 atmosphere. HeLa cells (ATCC, ATCC-CCL-2) were maintained in DMEM with 10% FBS and 1% P/S in a 5% CO2 cell culture humidified incubator at 37°C. Cells were co-transfected at around 70-80% confluence using Lipofectamine 2000 and 1 pg of each plasmid in 6-well plate format, 0.25 pg of total plasmid DNAin 48-well plates for degron identification, and 1.75 pg of total plasmid DNA in 12-well plates for co-immunoprecipitation and ATPase assays. For the experiments where the plasmid encoded the full-length gene was used, a scramble plasmid was co-transfected with the full-length plasmid to achieve the same amount of DNA transfected when two intein plasmid were used. Cells were harvested after 48 h post-transfection and levels of protein was analyzed by Western blot. Western blot analysis:

MY07 A transfected cells (HEK293T) were lysed in 0,1% n-Dodecyl-P-D-Maltopyranoside (D310S) and 1% cholesteryl hemisuccinate (CH210) solution in 25 mM Tris-HCl pH=7.5 and 150 mM NaCl supplemented with protease inhibitors and 1 mM phenylmethylsulfonyl. Retinal tissue was mechanically homogenized with a pestle in 150 pL of cell lysis buffer (IM Tris-HCl, 0.5M NaCl, 0.2M EDTA, 10% SDS, 200 mM PMSF, Triton X-100, Glycerol, pH=7.8). After lysis, MY07A samples were quantified by BCA protein assay kit. Samples with 25 pg of total protein, or 20 pL of samples, were denatured at 95°C for 5 minutes in IX Laemmli sample buffer containing 2.5 mg/ml of asolectin. Lysates were separated by 3-8% Tris-acetate SDS- PAGE gels for 1.5 h at 150 V. The antibodies used for immuno-blotting were either anti -flag tag to detect the MY07A protein and anti-P-tubulin as loading control. The quantification of MY07A bands detected by Western blot was performed using LI-COR Odyssey Infrared Imager.

To quantify the degron efficiency, the PTS, N- and C-fragment bands were selected to obtain the band signal. The same procedure was done for the P-tubulin signal band. To normalize, the PTS, N- and C-fragment band signals were divided by the P-tubulin band signal per each sample. To determine how the expression of the PTS, N- and C-fragment changed compared to the sample with-out degron, a fold change was performed taking the non-degron sample as control and set to 100%.

Co-immunoprecipitation assay for interaction ofMYO7A and SANS protein:

Transfected HEK293T cells were resuspended in a mixure of lysis buffer (0,1% n-Dodecyl-P- D-Maltopyranoside (D310S) and 1% cholesteryl hemisuccinate (CH210) solution in 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl supplemented with protease inhibitors and 1 mM phenylmethyl sulfonyl) and incubated 15 minutes at 4°C. Protein extracts were recovered after centrifugation, and then the lysate was incubated with anti-FLAG magnetic beads for 2 hours at room temperature in rotation. Samples were washed with 500 mM NaCl in lx PBS (pH 7.4) three times and then eluted by boiling for 5 minutes in lx Laemmli sample buffer containing 2.5 mg/ml of asolectin. Input samples were also denatured at 95°C for 5 minutes in lx Laemmli sample buffer containing 2.5 mg/ml of asolectin. Input and elution samples were analyzed by 3-8% Tris-acetate SDS-PAGE and transferred onto PVDF membranes. The antibodies used for immuno-blotting were either anti -flag tag to detect the MY07A protein, anti -HA to detect SANS, and anti-P-tubulin as loading control. The quantification of MY07A bands detected by Western blot was performed using LLCOR Odyssey Infrared Imager.

ATPase activity assay:

The ATPase assay was performed using an ELISA-like approach, where transfected samples were enriched for myosin Vila by using a coated 96-well plate with antibody anti-C-terminus MY07A. The amount of immobilized myosin VIIa-3xFLAG was quantified via a sandwich ELISA assay by using anti-Flag tag antibody as detection antibody, and ATPase activity was measured using a luciferase-based commercial kit (ADP-Glo™ Kinase Assay (Promega) following the manufacturer’s instruction.

Animals:

Wild-type C57B1/6 mice were purchased from Charles River and maintained at VHIO animal facility (Barcelona, Spain) or EyeCRO animal facility (Oklahoma, US). All mice were fed ad libitum with a standard diet and were maintained under a 12-hour light-dark cycle.

Recombinant AAV vectors:

The generation of AAV expression cassettes was done by cloning the cDNA of interest into single-stranded AAV backbone plasmids using restriction enzymes. Production of AAV vectors was conducted by helper virus-free transfection of HEK293 cells followed by iodixanol gradient purification.

Administration of AAV vectors into mice:

For subretinal (SRI) administration conducted at VHIO (Barcelona, Spain), adult mice aged 6- 7 weeks were anesthetized by intraperitoneal injection (Ketamine/Xylazine at 85 mg/kg and 14 mg/kg, respectively). After, pupils were dilated with tropicamide (10 mg/mL Colorcusi Tropicamida®, Alcon Cusi Laboratories) and topically anesthetized with Oxibuprocaina (0.4%, Lavoratorios Llorens). Under a surgical microscope, simple sutures were performed in the upper eyelid and upper limbal conjunctiva with ProleneTM 7/0 Ethicon (lohnson & lohnson) to expose the superior bulbar conjunctiva, and 1 mm sclerotomy was performed using a 30- gauge blade, 5 mm from the limbus. Bilateral subretinal administration of 1 pl/eye was performed using a 36-gauge blunt or beveled needle attached to a 10 pL Nanofil syringe (World Precision Instruments), which was introduced tangentially into the subretinal space through the sclerotomy. Finally, the needle and securing sutures were carefully removed. A drop of tobramycin and dexamethasone (Tobradex® 3 mg/mL + 1 mg/mL, Alcon Cusi Laboratories) was topically administered just after surgery as a local anti-inflammatory and antibiotic prophylaxis. The bleb in the sub-retinal space was assessed by fundus imaging.

For subretinal (SRI) administration conducted at EyeCRO (Oklahoma, US), adult mice aged 4- 6 weeks were anesthetized by intraperitoneal injection (Ketamine/Xylazine at 85 mg/kg and 14 mg/kg, respectively). After, mouse eyes were dilated and mice were placed on a regulated heating pad, and the posterior pole will be visualized under magnification. A 12.7mm 30-gauge insulin syringe was used to puncture the cornea just above the corneal limbus, avoiding any contact with the sclera and lens. The transvitreal subretinal injections was performed using a 10 pL Hamilton syringe with a 33-gauge blunt needle inserted through the corneal puncture across the vitreous, with the shaft aimed at the back of the eyecup, avoiding any trauma to the lens or iris. A total volume of 1 pL was delivered. Following injection, a small amount of Neomycin, polymyxin B, and dexa. 3.5g (Bausch and Lomb) was applied.

Retinal sample obtention:

Four weeks after vector administration via SRI in mice, animals were euthanized and eyes were enucleated. Retinas were individually dissected and snap-frozen in liquid nitrogen.

2. Results and discussion

In the last years several new inteins have been engineered based on consensus design (Stevens et al., 2016; Stevens, Sekar, Gramespacher, Cowburn, & Muir, 2018) and shown to have superior properties than naturally occurring inteins. One of these inteins, termed Cfa, has faster kinetics, higher expression levels and high tolerance to extreme conditions such as high temperature and concentration of denaturing agents. Interestingly, Cfa variants with degrees of homology from 90% or higher display similar properties. A major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site. Deviation from this preferred sequence context leads to a marked reduction in splicing activity, limiting the applicability of protein trans-splicing (PTS)-based methods. Recently engineered versions of naturally split inteins that possess greatly improved extein tolerance have been developed (Stevens et al., 2017). This intein is termed Cfa_mut.

In order to demonstrate that Cfa_mut allow the reconstitution of proteins such as MY07A, via protein trans-splicing for gene therapy application, a study using different split sites was performed. MY07A is a large protein, which is mutated in USHER1B syndrome. Usher syndrome is an inherited disease that causes severe hearing loss and retinitis pigmentosa, an eye disorder that causes vision to deteriorate over time. It is the most common condition that affects both hearing and vision. Reconstitution of MY07A has been proposed as a viable strategy to treat the disease. Several approaches based on AAV gene therapy are currently being explored to reconstitute it. Due to its large size MY07A gene cannot be encapsulated into a single AAV, and so different strategies have been proposed to reconstitute the function of this protein inside the target cells. Initial experiments were performed using three sites (SEQ ID NO: 120 and 121, SEQ ID NO: 122 and 123, and SEQ ID NO: 124 and 125). Two of the sites did not contain a Phe on the position +2 of the split site. These three sites that were tested were position 1173, which corresponds to a Gin at the +2 site, position 1198 which corresponds to a Vai at the +2 site and position 1201 which corresponds to a Phe at the +2 site. Constructs were cloned and tested as shown above. Specifically, MYO7A(l-1172)-CfaN, CfaCmut- MY07A (1173-2215) constructs were used to evaluate splicing MY07A at position 1172 (SEQ ID NO: 120 and SEQ ID NO: 121), MY07A (1-1197)-CfaN and CfaCmut- MY07A(l 198-2215) to evaluate splitting MY07A at position 1197 (SEQ ID NO: 122 and SEQ ID NO: 123), and MY07A (1-1200)- CfaN, CfaCmut-MYO7A(1201-2215) constructs were used to evaluate splicing MY07A at position 1200 (SEQ ID NO: 124 and SEQ ID NO: 125). Sites were selected based on the topological structure of Myosin- Vila and taking into consideration the presence of folded domains. Sites were selected outside of well-defined folded domains. Briefly, constructs were co-transfected into HEK293T cells as well as full-length Myo7A (SEQ ID NO: 134) was transfected to serve as control. Cells were lysed and protein reconstitution yields determined by Western Blot to compare the yields of reconstituted protein versus the full-length protein. Results showed that the yield for these three sites are comparable to the protein obtained only with the full-length protein (Figure 2).

Two additionally sites were tested (1226 and 1283): MYO7A(l-1226)-CfaN and CfaCmut- MYO7A(1227-2215) constructs (SEQ ID NO: 126 and SEQ ID NO: 127) and MY07A(l- 1283)-CfaN, CfaCmut-MYO7A(1284-2215) constructs (SEQ ID NO: 128 and SEQ ID NO: 129) were co-transfected into HEK293T cells as well as full-length MYO7A(SEQ ID NO: 134) was transfected to serve as control. Cells were lysed and protein reconstitution yields determined by Western Blot to compare the yields of reconstituted protein versus the full-length protein. Results showed that the yield for these two sites are comparable to the protein obtained only with the full-length protein (Figure 3).

Furthermore, two additionally sites were tested (823 and 1325): MYO7A(l-823)-CfaN and CfaCmut-MYO7A(824-2215) constructs (SEQ ID NO: 130 and SEQ ID NO: 131) and MYO7A(l-1325)-CfaN, CfaCmut-MYO7A(1326-2215) constructs (SEQ ID NO: 132 and SEQ ID NO: 133) were co-transfected into HEK293T cells as well as full-length MY07A (SEQ ID NO: 89) was transfected to serve as control as well as all previous different split sites evaluated to compare all of them. Cells were lysed and protein reconstitution yields determined by Western Blot to compare the yields of reconstituted protein versus the full-length protein. Results demonstrated that the yield for all seven different sites are comparable to the protein obtained only with the full-length protein (Figure 4).

In order to confirm the high performance of all seven different split sites, constructs were also characterized in HeLa cell lines. To this end, cells were transfected either with full-length MY07A plasmid or co-transfected with both N-fragment and C-fragment of MY07A plasmids. Cells were lysed using a non-cationic detergent mixture as lysis buffer. Expression levels were analyzed by western blot. For detection of protein anti-flag tag and anti-tubulin antibodies were used. As previously, results showed excellent in vitro reconstitution of MY07A by protein trans-splicing reaching equivalent levels of expression as full-length MY07A construct in HeLa cell lines (Figure 5).

To study the functionality of the reconstituted MY07A protein, a specific coimmunoprecipitation (CoIP) assay between myosin Vila protein and a relevant interactor was developed. To this end, SANS protein, a scaffold protein associated with the microtubule system, was selected as key interactor since it is well known that it interacts with MY07A through the MFI domain (Reiners J, et. al., Experimental Eye Research, 2006. 83:97-119). The CoIP assay was developed using MY07A as bait protein in anti-FLAG magnetic beats.

With the aim to evaluate if interaction of the reconstituted MY07A with SANS protein was maintained after PTS, pairs of interest were evaluated in vitro in HEK293T cells. To this end, cells were transfected either with full-length MY07A plasmid or co-transfected with both N- fragment and C-fragment of MY07 A plasmids for all seven different split sites MY07A-1172, MYO7A-1197, MY07A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, MYO7A-1325. Cells were lysed using a non-cationic detergent mixture as lysis buffer. CoIP assay was performed using anti-FLAG magnetics beads and analyzed by western blot. For detection of myosin VIIa-3xFLAG and SANS-HA proteins antibodies anti-FLAG and anti -HA were used respectively. Results showed that positive interactions with both full-length and PTS MY07A protein were achieved by using all seven different split sites MYO7A-1172, MYO7A-1197, MY07A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, MYO7A-1325 (Figure 6). No interactions with individual fragments (either N- or C-fragment) were observed, with the expected exception of the C-fragment of split site MYO7A-823 due to the splitting position in MFI domain.

In addition, the protocol was validated by reverse co-immunoprecipitation assays by using SANS as bait protein. The data obtained confirmed the interaction between SANS protein and reconstituted myosin Vila from all seven different split sites identified (MYO7A-1172, MYO7A-1197, MY07A-1200, MYO7A-1226, MYO7A-1283, MYO7A-823, MYO7A-1325) because MY07A was successfully pulled down as well as a clear enrichment of SANS protein was detected in elution samples (Figure 7).

To further study the functionality of the reconstituted MY07A protein, a specific ATPase activity assay was developed. This assay was based on an ELISA-like system to enrich MY07A sample followed by the use of a commercial kit to detect ATPase activity and using only C- fragment sample as negative control of the assay due to its lack of ATPase activity as well as its capacity to inhibit it (Umeki N, et. al., Proc. Natl. Acad. Set. U.S.A., 2009. 106:8483-8488.

Aiming to evaluate if ATPase activity of reconstituted MY07A was maintained after PTS reconstitution, pairs of interest were assessed in vitro in HEK293T cells. To this end, cells were transfected either with full-length MY07A plasmid, only C-fragment of split site MY07A- 1325 plasmid (being the more C-terminal construct available), or co-transfected with both N- fragment and C-fragment of MY07 A plasmids of interest. Cells were lysed using a non-cationic detergent mixture as lysis buffer. Then, samples were immobilized in a Maxisorp 96-well plate by using an antibody that specifically recognized the C-terminus of MY07A protein. Next, ATPase assay was carried out by means of a luciferase assay to estimate the concentration of ADP using the ADP-Glo kit. Finally, the sandwich ELISA was completed by adding a detection antibody anti-Flag tag into the wells of the Maxisorp 96-well plate to detect the amount of myosin Vila and fragments. Proportion between full-length, reconstituted MY07A protein and individual fragments were evaluated also by western blot using anti-Flag and anti-tubulin antibodies. Results showed that functional ATPase activity was detected in both full-length and PTS MY07 A protein for all seven different split sites MY07A-1172, MY07A-1197, MY07A- 1200, MYO7A-1226, MYO7A-1283, MYO7A-823, MYO7A-1325; while no activity was recorded in individual C-fragment for all aforementioned split sites (Figure 8).

Once the technology was successfully validated in all the different split sites mentioned above, the approach was taken a step further by incorporating degradation sequence technology into the constructs. The requirement of this design was the elimination of N- and C-fragments that did not react to form a functional full-length MY07A protein, as well as the excised inteins. To that end, a set of degron sequences were evaluated in vitro in HEK293T cells. Cells were transfected either with full-length MY07 A plasmid or co-transfected with both N-fragment and C-fragment of MY07A plasmids containing degron sequences under evaluation. Cells were lysed using a non-cationic detergent mixture as lysis buffer. Expression levels were analyzed by western blot. For detection of protein anti-flag tag and anti -tubulin antibodies were used. After evaluating more than a thousand and five hundred (1500) different combinations between split sites and degron sequences at N- and C-fragment, several degradation sequences were identified and successfully combined with different MY07A split sites, leading to degradation of individual half-parts while maintaining yields of reconstituted protein trans-splicing (Figure

9). The wide range of degron-containing candidates identified included degrons of different size, different families as well as different mechanism of action (e.g. proteosome mediated or chaperone-mediated autophagy) (Table 7).

Table 7: Degradation effect achieved by combination of several degradation sequences with different MYO7A split sites. SD, standard deviation; n, Number of biological replicates; Degron, degradation sequence.

To ensure that the addition of degron sequences did not interfere in the functionality of reconstituted MY07A protein, degron-containing pair candidates were evaluated at functionality level in vitro. First, candidates were tested by co-immunoprecipitation assay based on MY07A as bait protein, and all candidates assessed showed positive interaction with SANS (Figure 10 and Table 7). Second, candidates were evaluated by ATPase activity assay, and again all candidates tested showed positive ATPase activity (Figure 11 and Table 7).

Finally, in order to assess whether protein trans-splicing with MY07A gene efficiently occurs in the retina after co-administration, AAV8 vectors coding for N- and C-split MYO7A gene (MYO7A-1172 cleavage site) (SEQ ID NO: 120 and 121) were assessed through subretinal (SRI) administration into C57B1/6 WT adult mice. Results showed that reconstitution of the full-length MY07 A protein was achieved in the retina after SRI injection into WT mice (Figure 12). Noteworthy, the inventors not only provide evidence that the PTS technology successfully works in vivo, but also that the reconstituted MY07A protein occurs efficiently in vivo after SRI performed in two different Clinical Research Organization sites by two methods of delivery, trans-corneal and trans-sclera.

Claims

1. A combination of polynucleotides for use in the treatment of MYO7A-associated disease in a subject in need thereof wherein the combination comprises: i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’ : a N- terminal fragment of Myosin- Vila protein and N-split intein, fused directly or indirectly via a linker, ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in said subject generates Myosin- Vlla protein by protein splicing.

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

3. A combination of polynucleotides for use of claim 1 or 2 wherein said C-split intein is C-Cfamut intein of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity to SEQ ID NO: 13.

4. The combination for use according to any one of claims 1 to 3 wherein said Myosin- Vlla protein is human Myosin- Vila protein, preferably comprising or consisting of SEQ ID NO: 15 or any functional variant thereof having at least 90% identity to 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 comprises: the N-terminal fragment of Myosin- Vila protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively, the N-terminal fragment of Myosin- Vila rotein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively; or the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15.

6. The combination for use according to any one of claims 1 to 5 wherein the first and second fusion proteins comprises amino acid sequences selected from any one of the following pairs: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, and, SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 30-43.

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

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

9. The combination for use of claim 7 or 8 wherein said degron is selected from the group consisting of: SEQ ID NO: 44 to 119, or any combination thereof, or any functional variant thereof having at least 90% identity to any one of sequences SEQ ID NO: 44 to 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 the CMV and chicken beta-actin (CEBA) promoter, human phosphoglycerate kinase (hPGK) promoter, chimeric CMV enhanced and human phosphoglycerate kinase (ePGK) promoter, photoreceptor-specific, human rhodopsin kinase (hGRKl) promoter, rod specific IRBP promoter, VMD2 (vitelliform macular dystrophy/Best disease) promoter, and EF 1 alpha promoter.

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

12. The combination for use according to claim 11 wherein said expression vector is a viral vector, preferably an adeno associated viral (AAV) vector, preferably said AAV vector comprises capsid protein of AAV selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or rhl0.

13. The combination for use according to any one of claims 1 to 12 wherein said MYO7A-associated disease is non-syndromic deafness DFNA11, non-syndromic deafness DFNB2, or Usher syndrome type IB, preferably wherein said disease is Usher syndrome type IB, more preferably wherein said combination is administered in subject by a parenteral route, more preferably by intravenous, intraarterial, intramuscular, intranasal, intraocular, intravitreal, suprachoroidal or subretinal route.

14. A kit compri sing :

(i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N-terminal fragment of Myosin- Vila protein and

N-Cfa intein of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity to SEQ ID NO: 1, fused directly or indirectly via a linker,

(ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-Cfa intein of SEQ ID NO: 2 or C-Cfa_mut intein of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of Myosin- Vila protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises: the N-terminal fragment of Myosin- Vila protein up to residue 823 and the C- terminal fragment of Myosin- Vila protein from residue 824 respectively, the N-terminal fragment of Myosin- Vila rotein up to residue 1172 and the C- terminal fragment of Myosin- Vila protein from residue 1173 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1197 and the C- terminal fragment of Myosin- Vila protein from residue 1198 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1200 and the C- terminal fragment of Myosin- Vila protein from residue 1201 respectively, the N-terminal fragment of Myosin- Vila protein up to residue 1226 and the C- terminal fragment of Myosin- Vila protein from residue 1227 respectively the N-terminal fragment of Myosin- Vila protein up to residue 1283 and the C- terminal fragment of Myosin- Vila protein from residue 1284 respectively; or the N-terminal fragment of Myosin- Vila protein up to residue 1325 and the C- terminal fragment of Myosin- Vila protein from residue 1326 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Myosin- Vila fragment and the C-terminal Myosin- Vlla fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, and, SEQ ID NO: 28 and 29 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 29.

15. The kit of claim 14 wherein the first and second fusion proteins comprises amino acid sequences selected from any one of the following pairs: SEQ ID NO: 30 and 31, SEQ ID NO: 32 and 33, SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, and, SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 30-43.