Use of split intein for the treatment of SCN1a associated disease

Abstract

The present invention relates to the use of split inteins for expressing Nav1.1 protein encoded by SCN1A gene in a subject in need thereof for gene therapy in particular for the treatment of SCN1A associated disease, preferably Dravet syndrome.

Description

USE OF SPLIT INTEIN FOR THE TREATMENT OF SCN1 A ASSOCIATED DISEASETECHNICAL FIELDThe present invention relates to the use of split inteins for expressing Navl.l protein encoded by SCN1 A gene in a subject in need thereof for gene therapy, in particular for the treatment of SCN1 A associated disease, preferably Dravet syndrome.BACKGROUNDDravet syndrome (DS) is a severe developmental and epileptic encephalopathy characterized by the onset of prolonged febrile and afebrile seizures in infancy and evolving to drug-resistant epilepsy with accompanying cognitive, behavioral, and motor impairment. Most cases are now known to be caused by pathogenic variants in the sodium channel gene SCN1A, which encodes the a-subunit of the voltage-gated ion channel Navl .1.AAVs have been shown to be highly efficient when treating neurological disease. However, the limited cargo capacity of the AAV vectors precludes its use for delivering large genes such as SCNIA in gene therapy. Indeed, due to its large size SCNIA gene cannot be encapsulated into a single AAV, and there remains a need to develop new strategies to deliver SCN1 A gene inside target cells.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 only for few proteins (WO2021 / 191447 and W02020 / 079034). However, the reconstitution of a complex functional protein such as voltage-gated ion channel has never been shown in vivo.SUMMARYIn the present application, the inventors showed for the first time that split intein-mediated protein trans-splicing can be used to reconstitute efficiently complex Navl.l protein encoded by SCN1A gene in a cell. In vitro, the Navl.l channel sodium is localized correctly in the cell membrane and is active as a sodium channel. Moreover, in vivo Navl .1 reconstitution by protein trans-splicing is efficient and mediates efficacy in Dravet syndrome mouse model. Navl. l reconstitution with split intein-mediated protein trans-splicing represents a viable strategy to treat SCNIA-associated disease.The present disclosure relates to a combination of polynucleotides for use in the treatment of Sodium channel protein type 1 subunit alpha (SCN1A) associated disease, preferably selected from the group consisting of: Dravet syndrome, SCNIA-associated Non-dravet syndrome Epilepsy such as generalized epilepsy with febrile seizures-plus, Malignant migrating foal seizures of infancy, West syndrome, Lennox-Gastaut syndrome, Rett syndrome, Developmental and Epileptic, and SNC1 -associated nonepileptic disease such as familial hemiplegic migraine, Autism spectrum disorder, Arthrogryposis Multiplex Congenita, more preferably wherein said disease is Dravet syndrome, 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 Navl. l 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 Navl. l protein, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in said subject generates Navl .1 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-Cfamut intein of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity to SEQ ID NO: 13.In a preferred embodiment, said Navl. l protein is human Navl. l 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 Navl .1 protein ends up to residue 925 and the C-terminal fragment of Navl.l protein starts from residue 926 respectively, the N- terminal fragment of Navl.l protein ends up to residue 1059 and the C-terminal fragment of Navl. l protein starts from residue 1060 respectively; or the N-terminal fragment of Navl. l protein ends up to residue 1178 and the C-terminal fragment of Navl. l protein starts from residue 1179 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 comprise amino acid sequences selected from any one of the following pairs: SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 22-27.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: 28 to 58 or any functional variant thereof having at least 90% identity to any one of sequences SEQ ID NO: 28 to 58, 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.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), chimeric Synapsin I and CMV (Syn-CMV) promoter (GenBankAccession number: AH006533.2, bp 1538-2006, last updated August 01, 2016; GenBank Accession number: AF396260.1, bp 160-728, last updated on August 13, 2001), minimal neuronal-specific enolase (NSEmin) promoter (GenBank Accession number: AH002215.2, bp 854-1153, last updated August 01, 2016), chimeric murine SCN1A E2 enhancer and minimal CMV (E2min) promoter (Vormstein-Schneider et. al. 2020).Preferred polyadenylation elements are bovine growth hormone polyadenylation signal (bGH), a synthetic polyadenylation signal (SynpA), simian virus 40 (SV40) late or full-length polyadenylation signal (SV40). Preferred intron is simian virus (SV40) intron.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.In a preferred embodiment, said combination is administered in subject by a parenteral route, more preferably by intravenous, intraarterial, intracerebral, intrathecal, intra-ci sterna magma, intracerebral spinal fluid or intracerebroventricular 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 Nav1.1 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-Cfamut 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 Nav1.1 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:- the N-terminal fragment of Navl .1 protein up to residue 925 and the C-terminal fragment of Navl. l protein from residue 926 respectively,- the N-terminal fragment of Navl .1 protein up to residue 1059 and the C-terminal fragment of Navl. l protein from residue 1060 respectively; or- the N-terminal fragment of Navi .1 protein up to residue 1178 and the C-terminal fragment of Navl. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Navl. l fragment and the C-terminal Navl. l 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 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 21, more preferably wherein the first and second fusion proteins comprises amino acid sequences selected from any one of the following pairs: SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, and SEQ ID NO: 26 and 27.LEGEND FIGURESFigure 1: Splicing efficiency of Navl.l protein at position 926 monitored by Western blot. Navl . 1 protein was first split at position 926 (Site 1) and the N-terminal fragment (residues 1-925, S IN) recombinantly fused to N-inteins, and the C-terminal fragment (residues 926-2008, S 1C) 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. Split position is numbered according to Navl . 1 human amino acid sequence (SEQ ID NO: 15).Figure 2: Splicing efficiency of Navl.l protein at position 948 monitored by Western blot. Navl . 1 protein was first split at position 948 (Site 2) and the N-terminal fragment (residues 1-947, S2N) recombinantly fused to N-inteins, and the C-terminal fragment (residues 948-2008, S2C) 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 Navl . 1 serve as control. Split position is numbered according to Navl . 1 human amino acid sequence (SEQ ID NO: 15).Figure 3: Splicing efficiency of Navl.l protein at position 1060 and 1179 monitored by Western blot. Navl . l protein was first split at position 1060 (Site 3) or 1179 (Site 4) and the N- terminal fragment (residues 1-1059 or 1-1178) recombinantly fused to N-inteins, and the C-terminal fragment (residues 1060-2008 or 1179-2008) to the C-inteins. Cultured HEK293T cells were cotransfected with equimolar amounts of plasmids encoding for the N- and C-terminal fragments andsplicing efficiency monitored by Western blotting. HEK293T cells transfected with a full-length Navl . 1 serve as control. Split position is numbered according to Navl . 1 human amino acid sequence (SEQ ID NO: 15).Figure 4: HEK293T cells transfected with SCNIA-intein plasmids comprising Navl.l split at site 3 (1060) (A) and site 4 (1179) (B) analyzed by confocal microscopy. Cells were analyzed by confocal microscopy after 48 h post-transfection by specific antibodies to properly label flag tag and Navl.l protein. DAPI was used to counterstain nuclei.Figure 5: Patch Clamp assay on HEK293T cells transfected with SCNIA-intein plasmids comprising Navl.l split at site 3 (1060, PTS-3) and site 4 (1179, PTS-4). Electrophysiology analysis by patch clamp was performed 48h post-transfection. Non-transfected HEK293T cells, HEK293T cells transfected with full-length SCN1A gene and HEK-Navl.l stable cell line were used as controls.Figure 6: Schematic representation of the constructs according to the present disclosure including degron (DD). The N-terminal fragment of the protein of interest is fused to the IntN, a linker, which in this case is a flag tag, and a degron. The C-terminus of the protein is fused to the IntC, which in its N-terminus is linked to a degron. Splicing reaction results in the formation of the desired full-length protein, without any degron fused to it, and the cleaved inteins, each of them bearing a degradation signal.Figure 7: Combination of inteins and degrons to reconstitute protein Navl.l. HEK293T cells were transfected with the indicated constructs, lysed and analyzed by western blot using an anti -HA tag mAb. Results indicate that the use of constructs combining CfaN or C inteins with a degradation signal (L9) resulted in disappearance of any signal arising from starting materials (CfaC-SCNIA -HA).Figure 8: Detection of Navl.l protein in hippocampus (A) and cortex (B) samples of WT mice treated at 4-5 weeks-of-age. Western blot of hippocampus (A) and cortex (B) protein extraction of wild-type (WT) mice intracerebroventricular-injected (ICV) at the age of 4-5 weeks with dual AAV9 vectors (N- and C-vectors encoding for split site 3, 4.4E+10 vg / mouse) under the control of different promoters with different cell specificity. CMV, cytomegalovirus promoter; CEBA, chimeric reduced version of the CMV and chicken beta-actin promoter; NSEmin, minimal neuronal-specific enolase promoter; hSyn-CMV, chimeric human Synapsin I and CMV promoter; E2min, chimeric murine SCN1A E2 enhancer and minimal CMV promoter. Samples obtained 1-month post injection. Results blotted with a monoclonal antibody anti -HA tag. Each line corresponds to a different treated animal. Navl. l protein was successfully reconstituted in CNS by all promoters evaluated with different specificity.Figure 9: Detection of Navl.l protein in CNS samples of WT mice treated at the age of PIO (A) and P15 (B) days Western blot of protein extraction from different Central Nervous System (CNS) areas (hippocampus, cortex, striatum and hypothalamus) of wild-type (WT) mice treated at the age of 10 post-natal days (PIO) (A) and 15 post-natal days (P15) (B) by ICV-injection with dual AAV9 vectors (N- and C-vectors encoding for split site 3, 4.4E+10 vg / mouse) under the control of CEBA promoter. Samples obtained 1-month post injection. Results blotted with a monoclonal antibody anti- HA tag. Each line corresponds to a different treated animal. Navl.l protein was successfully reconstituted in all CNS areas analyzed after ICV administration of dual AAV approach at the early age of PIO and also P15.Figure 10: Reconstituted Navl.l protein in the CNS behaves as the endogenous wild-type protein. In vivo samples (hippocampus) obtained 1-month post ICV injection of WT mice with dual AAV9 vectors (N- and C-vectors encoding for split site 3, 4.4E+10 vg / mouse) under the control of CEBA promoter (A), or from non-treated (NT) WT littermates used as controls (B). In vitro sample obtained 48 hours after co-transfection of HEK293T cells with both N-fragment and C-fragment plasmids for split site 3. Results blotted with a monoclonal antibody anti -HA tag (A) or a monoclonal antibody anti-Navl.l protein (B). Each line corresponds to a different treated animal. NT, non-treated. Reconstituted Navl.l protein migrates in western blot as the endogenous wild-type Navl.l protein.Figure 11: Co-localization signal of reconstituted Navl.l protein and endogenous protein in hippocampus sample of WT treated mice. Western blot of representative hippocampus protein extraction of WT mice ICV-injected at the age of 4-5 weeks with individual C-vector as control (SCNlAc-CfaCmut) or with dual N- and C- AAV9 vectors (PTS split site 3, 4.4E+10 vg / mouse), compared to negative control of non-injected mouse (NT). Results blotted with a monoclonal anti- Navl.l (red signal) and a monoclonal anti-HA antibody (green signal). NT, non-treated.Figure 12: No detection of accumulation of unreacted fragments after PTS reaction in vivo. Western blot of representative hippocampus protein extraction ofWT mice ICV-injected at the age of 4-5 weeks with individual N- or C-vector as control (SCNlAN-CfaN or SCNlAc-CfaCmut, respectively) or with dual N- and C- AAV9 vectors (PTS split site 3, 4.4E+10 vg / mouse), compared to negative control of non-injected mouse (NT). (A) Results blotted with a monoclonal anti-3FT, that specifically recognize the tag used for the N-fragment. (B) Results blotted with a polyclonal anti-Sodium Channel Pan antibody that specifically recognize an epitope located at C-terminus of Navprotein. NT, non-treated. Each line corresponds to a different treated animal.Figure 13: Long-term expression of reconstituted Navl.l protein in CNS. Western blot of hippocampus (A) and cortex (B) protein extraction of WT mice ICV-injected at the age of 4-5 weekswith dual AAV9 vectors (N- and C-vectors encoding for split site 3, 4.4E+10 vg / mouse) under the control of CEBA promoter. Samples obtained 1-month post injection (1-mpi) and 3-months post injection (3-mpi). Results blotted with a monoclonal antibody anti-HA tag. Each line corresponds to a different treated animal. Graphs show quantification of western blot. Reconstituted Navl. l protein expression was sustained 3-mohts post injection in CNS.Figure 14: Efficacy of dual AAV approach in Dravet syndrome mouse model. Survival (A) and epileptogenic thermal threshold (B) of Dravet syndrome (DS) mice intravenously injected (IV) at the age of 0-3 post-natal days (P0-P3) with dual AAV9 vectors (N- and C-vectors encoding for split site 3, 6E+10 vg / mouse) under the control of CEBA promoter. Saline-injected DS littermate mice were used as controls. Analysis of survival (A) by Logrank test, p=0.0056. Analysis of hyperthermia induced seizures (B) 2-months post-injection. Mean temperature of induction of seizures at 38.85 °C in saline- treated DS mice in comparison to 39.47°C in dual AAV-treated DS mice (p=0.0353, Log-rank Mantel- Cox test). Complete protection in survival and partial but statistically significant efficacy in reduction of thermal induced seizures were achieved when administering dual AAV approach in D S mouse model at P0-P3 by IV.DETAILED DESCRIPTIONCombination of polynucleotides encoding first and second fusion proteinsThe limited cargo capacity of the AAV vectors precludes its use for delivering large genes such as SCN1A gene in gene therapy. To deliver SCN1A gene in patients, the inventors took the advantage of the intrinsic ability of split inteins to mediate protein trans-splicing to reconstitute large Navl .1 protein encoded by large SCN1 Agene 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 Navl. l 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 Navl. l protein, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in a cell generates full length Navl. l 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 preferredembodiments, 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 Navl.l 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 Navl. l 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: 68), a (GGGGS)n (SEQ ID NO: 69), a (G)n (SEQ ID NO: 70), an (EAAAK)n (SEQ ID NO: 71), a XTEN-based linker, or an (XP)n motif (SEQ ID NO: 72), 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 Navl.l 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 querysequence 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

[0724] -

[0731] 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 toleranceIn 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 anyfunctional 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

[0735] -

[0738] 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: 13. 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 Navl.l 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 Navl.l 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 Navl .1 protein following their fragmentation into two split-intein flanked polypeptides.Sodium channel protein type 1 subunit alpha (Navl. l) protein (also named herein SCN1A protein) encoded by SCNIAgene (UniProtKB accession number: P35498; updated on May 03, 2023) mediates the voltage-dependent sodium ion permeability of excitable membranes. Voltage-dependent sodium channels are heteromeric complexes that regulate sodium exchange between intracellular and extracellular spaces and are essential for the generation and propagation of action potentials in muscle cells and neurons. Each sodium channel is composed of a large pore-forming, glycosylated alpha subunit and two smaller beta subunits. Navl.l has four homologous domains, each of which contains six transmembrane regions. Navl.l plays a key role in brain, probably by regulating the moment when neurotransmitters are released in neurons.In particular, Navl.l protein is a human Navl. l protein (UniProtKB accession number: P35498; updated on May 03, 2023, SEQ ID NO: 15 (human Navl. l amino acid sequence without first methionine) encoded by SCN1A gene (GENE ID: 6323, updated on May 15, 2023), also known asEIEE6, FEB3, FEB3A, FHM3, GEFSP2, HBSCI, NAC1, Navl.l, SCN1, SMEI, DRVT, DEE6, DEE6A, DEE6B.In a preferred embodiment, according to the present disclosure Navl. l protein can be a human Navl. l 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 Navl. l protein, preferably human Navl .1 protein (SEQ ID NO: 15), in particular sodium channel activity of the human Navl .1 protein.Navl. l mediates the voltage-dependent sodium ion permeability of excitable membranes. Sodium channel activity of functional variant may be assessed by electrophysiology analysis by patch clamp as illustrated in Example part (Material and Methods / Patch Clamp) of the present application. For instance, cells are transfected with functional variant of Navl. l andelectrophysiology analysis by patch clamp was performed, in particular as described in example (Material and Methods / Patch Clamp) by using extracellular solution (140 mM NaCl, 4 mM KC1, 2 mM CaC12, 1 mM MgC12, 10 mM HEPES, 5 mM Glucose, pH 7.4 adjusted with NaOH, approximately 298 mOsm / L) and intracellular solution (120 mM CsF, 10 mM NaCl, 10 mM EGTA, 10 mM HEPES, 2 mM NaATP, pH 7.2 ajusted with CsOH, approximately 314 mOsm / 1). Untransfected cells can be used as negative control and Navl.l stable cell line or cell expressing human SNC1 A (SEQ ID NO: 15) can be used as positive control.In a preferred embodiment, the current amplitude of Navl. l functional variant is higher than the current amplitude of the negative control and / or similar or higher than the value of positive control value. In particular, the value is higher than the negative control and preferably varies by less than 40%, 30%, 20% or 10% of the positive control value.In a preferred embodiment, Navl. l protein is human Navl. l 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.A number of different mammalians Navl. l 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 Navl .1 protein as described above is split into at least two nucleic acid sequences encoding N- and C-terminal Navl. l 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 Navl. l fragment flanking sequences (N- and C-terminal Navl. l fragments, also named N- and C-exteins) with a peptide bond to reconstitute the full-length of Navl .1 protein as described above in a cell.According to the present disclosure, the Navl. l protein or any functional variant thereof as described above can be split at any positions into a N- and C-terminal Navl. l fragments. However, the inventors have showed that some specific split positions are particularly advantageous to increase the efficiency of Navl. l protein reconstitution. In particular, the Navl .1 protein or any functional variant thereof as described above can be split into N- and C- terminal Navl.l fragments at the split position between amino acids 925-926; 1059-1060 or 1178-1179, 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 functional variants, homologs or isoforms of Navl.l, in particular by sequence alignment.In a preferred embodiment, the N-terminal Navl. l fragment ends up to residue 925 and the C- terminal fragment of Navl.l protein starts from residue 926 respectively wherein said residue is numbered according to SEQ ID NO: 15.In another particular embodiment, the N-terminal fragment of Navl.1 protein ends up to residue 1059 and the C-terminal fragment of Navl.l protein starts from residue 1060 respectively wherein said residue is numbered according to SEQ ID NO: 15.In another particular embodiment, the N-terminal fragment of Navl.1 protein ends up to residue 1178 and the C-terminal fragment of Navl.l protein starts from residue 1179 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 Navl.l fragment and a C-terminal Navl.l 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 and SEQ ID NO: 20 and 21 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 21 and preferably wherein reconstituted functional variant of Navl.l protein retains the function of said Navl. l, in particular sodium channel activity of the human Navl.l protein.Table 3: Examples of N-and C-terminal Navl.l fragments. Highlighted amino acids in Full- length Navl.l represent preferred split positions. The first methionine of the Navl. l and N- terminal fragment sequences is not indicated in the sequences listed in the table. A first methionine can be added to the sequence to initiate protein synthesis. Split positions are numbered according to Navl .1 full-length sequence without methionine.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 Navl.1 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 Navl. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:- the N-terminal fragment of Navl .1 protein up to residue 925 and the C-terminal fragment of Navl. l protein from residue 926 respectively,- the N-terminal fragment of Navl .1 protein up to residue 1059 and the C-terminal fragment of Navl.l protein from residue 1060 respectively; or- the N-terminal fragment of Navl .1 protein up to residue 1178 and the C-terminal fragment of Navl. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Navl .1 fragment and the C-terminal Navl .1 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 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 21.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 Navl.l 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 Navl. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:- the N-terminal fragment of Navl .1 protein up to residue 925 and the C-terminal fragment of Navl.l protein from residue 926 respectively,- the N-terminal fragment of Navl .1 protein up to residue 1059 and the C-terminal fragment of Navl.l protein from residue 1060 respectively; or - the N-terminal fragment of Navl .1 protein up to residue 1178 and the C-terminal fragment of Navl.l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Navl .1 fragment and the C-terminal Navl .1 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 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 21.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 from the pairs consisting of: SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27 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: 22-27.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. The first methionine of the fragment sequences is not indicated in the sequences listed in the table. A first methionine can be added to the sequence to initiate protein synthesis. Split positions are numbered according to Navl. l full-length sequence without methionine.To prevent the accumulation of undesired starting materials and to eliminate the excised intein fragments, a degron can be added 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 100, preferably less than 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 specific 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 Navl. l 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 Navl. l 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 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 specific 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 Navl.l 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 Navl. l 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 Navl. l protein and Cfa N-split intein of SEQ ID NO: 1 or anyfunctional 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 Navl. l 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 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 Navl.l 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 Navl. l 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 Navl. l protein, N-split intein, fused directly or indirectly via a linker, anda second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of Navl. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:- the N-terminal fragment of Navl.1 protein up to residue 925 and the C-terminal fragment of Navl. l protein from residue 926 respectively,- the N-terminal fragment of Navl.1 protein up to residue 1059 and the C-terminal fragment of Navl. l protein from residue 1060 respectively; or- the N-terminal fragment of Navl .1 protein up to residue 1178 and the C-terminal fragment of Navl. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Navl .1 fragment and the C-terminal Navl .1 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 and SEQ ID NO: 20 and 21 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 21 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 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 Navl. l 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 Navl. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:- the N-terminal fragment of Navl.1 protein up to residue 925 and the C-terminal fragment of Navl. l protein from residue 926 respectively,- the N-terminal fragment of Navl.1 protein up to residue 1059 and the C-terminal fragment of Navl. l protein from residue 1060 respectively; or- the N-terminal fragment of Navl .1 protein up to residue 1178 and the C-terminal fragment of Navl. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Navl .1 fragment and the C-terminal Navl .1 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 and SEQ ID NO: 20 and 21 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 21 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 Navl.l 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 Navl. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:- the N-terminal fragment of Navl .1 protein up to residue 925 and the C-terminal fragment of Navl.l protein from residue 926 respectively,- the N-terminal fragment of Navl .1 protein up to residue 1059 and the C-terminal fragment of Navl. l protein from residue 1060 respectively; or- the N-terminal fragment of Navl .1 protein up to residue 1178 and the C-terminal fragment of Navl. l protein from residue 1179 respectively,wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Navl .1 fragment and the C-terminal Navl .1 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 and SEQ ID NO: 20 and 21 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 21 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 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 Navl.l 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 Navl. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:- the N-terminal fragment of Navl .1 protein up to residue 925 and the C-terminal fragment of Navl. l protein from residue 926 respectively,- the N-terminal fragment of Navl .1 protein up to residue 1059 and the C-terminal fragment of Navl. l protein from residue 1060 respectively; or- the N-terminal fragment of Navl .1 protein up to residue 1178 and the C-terminal fragment of Navl. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Navl .1 fragment and the C-terminal Navl .1 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 and SEQ ID NO: 20 and 21 or any functionalvariant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NOs: 16 to 21 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: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27 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: 22-27 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 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: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27 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: 22-27 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 amino acid 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: 28), Degl (SEQ ID NO: 29), PEST (SEQ ID NO: 30), DD1 (SEQ ID NO: 31), DD2 (SEQ ID NO: 32), DD3 (SEQ ID NO: 33), Ml (SEQ ID NO: 34), M2 (SEQ ID NO: 35), SopE (SEQID NO: 36), SopEl-78 (SEQ ID NO: 37), SopE 15-78 (SEQ ID NO: 38), SopE-15-50 (SEQ ID NO: 39), L2 (SEQ ID NO: 40), L6 (SEQ ID NO: 41), L9 (SEQ ID NO: 42), LIO (SEQ ID NO: 43), Lil (SEQ ID NO: 44), L12 (SEQ ID NO: 45), L15 (SEQ ID NO: 46), L16 (SEQ ID NO: 47), M3 (SEQ ID NO: 48), M4 (SEQ ID NO: 49), M5 (SEQ ID NO:50), V12 (SEQ ID NO: 51), DD4 (SEQ ID NO: 52), DD5 (SEQ ID NO: 53), DD6 (SEQ ID NO: 54), DD7 (SEQ IDNO: 55), T1 (SEQ ID NO: 56), T2 (SEQ ID NO: 57) and, T3 (SEQ ID NO: 58), preferablyDD1, DD3, PEST, SopE, V12, M4, L2, L9, more preferably SopE, L2, L9, M4 or V12 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., Navl.l protein) preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 28 to 58.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., Navl.l 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 materiel 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 some embodiments, each first and second polynucleotides according to the present disclosure encoding the first and second fusion proteins, respectively, as described above may a nucleic acid construct.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, chimeric Synapsin I and CMV (Syn-CMV) promoter, minimal neuronal-specific enolase (NSEmin) promoter (AH002215.2, bp 854-1153), chimeric murine SCN1A E2 enhancer and minimal CMV (E2min) promoter (Vormstein-Schneider et. al. 2020). 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 neuronal 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 favourable 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 3’ITR 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 andsecond 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 and AAV5.Therapeutic useAccording 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 SCN1 A 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 SCN1 Ainto 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 SCN1A associated disease, such as prolonged seizures often triggered by high body temperature (hyperthermia), developmental delay, speech impairment, ataxia, hypotonia, sleep disturbances, and other health problems.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 human patient, preferably children under the age of 18.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, intracerebral, intracerebral spinal fluid, intra-ci sterna magma, intrathecal or intracerebroventricular 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.Voltage-gated sodium (Nav) channels such as Navl. l, encoded by SCNIA gene, are crucial in the initiation and propagation of electrical signals (action potentials) in excitable neuronal cells. SCN1A is one of the most common epilepsy genes and the SNC1A gene mutation or protein dysfunction is responsible of abnormal neuronal cell excitability (Ding et al. Front Neurol. 2021; 12: 743726). 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 SCN1A associated disease, preferably selected from the group consisting of: Dravet syndrome, SCNIA-associated Non- dravet syndrome Epilepsy such as generalized epilepsy with febrile seizures-plus, Malignant migrating foal seizures of infancy, West syndrome, Lennox-Gastaut syndrome, Rett syndrome, Developmental and Epileptic, and SCNIA-associated nonepileptic disease such as familial hemiplegic migraine, Autism spectrum disorder, Arthrogryposis Multiplex Congenita, preferably wherein said disease is Dravet syndrome.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 SCN1 A associated disease.The disclosure also provides a method for treating a SCN1A 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 therapeuticallyeffective 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 sudden unexpected death in epilepsy (SUDEP) and both spontaneous and thermic inducible seizures.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, intracerebral, intracerebral spinal fluid, intrathecal, intra-ci sterna magma or intracerebroventricular 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. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.KitIn another aspect, the disclosure further relates to a kit, preferably for use in the treatment of SNC1A associated disease as described above, preferably Dravet syndrome 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 Navl.l 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 Navl. l protein, fused directly or indirectly via a linker, optionally wherein said first and second fusion proteins further comprise a degron, preferably fused 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: 28 to 58 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: 28 to 58.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 Navl.l 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 Navl. l protein, fused directly or indirectly via a linker,and optionally 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 / 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: 28 to 58 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: 28 to 58.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 Navl.l 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 Navl. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:- the N-terminal fragment of Navl .1 protein up to residue 925 and the C-terminal fragment of Navl. l protein from residue 926 respectively,- the N-terminal fragment of Navl .1 protein up to residue 1059 and the C-terminal fragment of Navl. l protein from residue 1060 respectively; or- the N-terminal fragment of Navl .1 protein up to residue 1178 and the C-terminal fragment of Navl. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably the N-terminal Navl. l fragment and the C-terminal Navl.l 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 and SEQ ID NO: 20 and 21 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 21 and optionally 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 / 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:28 to 58 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: 28 to 58.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 Navl.l 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 Navl. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:- the N-terminal fragment of Navl .1 protein up to residue 925 and the C-terminal fragment of Navl. l protein from residue 926 respectively,- the N-terminal fragment of Navl .1 protein up to residue 1059 and the C-terminal fragment of Navl. l protein from residue 1060 respectively; or- the N-terminal fragment of Navl .1 protein up to residue 1178 and the C-terminal fragment of Navl. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Navl. l fragment and the C-terminal Navl. l 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 and SEQ ID NO: 20 and 21 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 21, and optionally 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 / 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:28 to 58 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: 28 to 58.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: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27 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: 22-27, and optionally 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 / 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: 28 to 58 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: 28 to 58.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.EXAMPLES1. Materials and MethodsMaterials: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 competentE. coli. HEK293T cells were purchased fromATCC. 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), anti-Navl.l rabbit polyclonal antibody (Alomone), anti-HA tag rabbit monoclonal antibody (Cell Signaling), 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, D1306) were purchased from Thermo Fisher Scientific. Dodecyl maltoside (D310) and cholesteryl hemisuccinate (CH210) solution were purchased from Anatrace. Trypsin was purchased from Promega.Equipment:Gels and Western-blots were imaged with a LI-COR Odyssey Infrared Imager. Cell lysis was carried out using a SFX550 Branson sonifier. Confocal microscope images were captured using Andor DragonFly 505 Nikon confocal microscopy. Patch clamp assay to assess Navl .1 current was performed using SyncroPatch 384i automated platform. ICV administrations were done using a stereotaxic frame of Harvard Apparatus, an infusion pump (Legatol30, KDScientific) and an anesthetic mask (Harvard Apparatus).Cloning of Recombinant DNA:Synthetic genes to prepare constructs SCNlA-948-CfaN-3FT (SEQ ID NO: 61), SCN1A-948- CfaCmut-3FT (SEQ ID NO: 62) were purchased and introduced into plasmid expression vectors using Kpnl and Notl restriction enzymes.Constructs SCN1 A-926-CfaN-3FT (SEQ ID NO: 59), SCN1 A-926-CfaCmut-3FT (SEQ ID NO: 60), SCNlA-1060-CfaN-3FT (SEQ ID NO: 63), SCNlA-1060-CfaCmut-3FT (SEQ ID NO: 64), SCNlA-1179-CfaN-3FT (SEQ ID NO: 65), SCNlA-1179-CfaCmut-3FT (SEQ ID NO: 66), SCN1A-3FT (SEQ ID NO: 67) 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.| | GKDEKAKGKDYKDHDGDYKDHDI DYKDDDDK | ~|Table 6: Protein sequences of constructs used in the examples. A first methionine can be added to the sequence to initiate protein synthesis. Split positions are numbered according to Navl-1 full-length sequence without methionine.Transfection of intein plasmids in HEK293T cells:HEK293T cells were maintained in DMEM with 10% FBS and antibiotics at 37°C in a 5% CO2 atmosphere. Cells were co-transfected at around 80% confluence using Lipofectamine 2000 and 1 pg of each plasmid in 6-well plate format. 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:SCN1A 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 phenylmethyl sulfonyl. After lysis, SCN1A samples were quantified by BCA protein assay kit. Samples with 40 pg of total protein were denatured at 37°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 150V. The antibodies used for immuno-blotting were either anti-flag tag, anti-HA tag or anti- Navl. l to detect the Navl. l protein and anti-P-tubulin as loading control. The quantification of Navl .1 bands detected by Western blot was performed using LLCOR Odyssey Infrared Imager.Confocal microscopy:HEK293T cells were co-transfected with 0.3 pg of each SCNIA-intein plasmid and as a negative control HEK293T cells were grown but they were not transfected. For the experiments where the plasmid encoded the full-length gene was used, a scramble plasmid was cotransfected with the full-length plasmid to achieve the same amount of DNA transfected when two intein plasmid were used. Cells were analyzed by confocal microscopy after 48 h posttransfection by specific antibodies to properly label flag tag and Navl.l protein. DAPI was used to counterstain nuclei.Patch clamp:HEK293T cells were co-transfected using PolyJET transfection reagent (SignaGen Laboratories) according to manufacturer’s guideline with 20 pg of each SCN1 A-intein plasmid or a plasmid encoding the SCN1A full-length gene in 175 cm2 flasks. Scramble plasmid was co-transfected with the full-length plasmid to achieve the same amount of DNA transfected when two intein plasmids were used. Electrophysiology analysis by patch clamp was performed 48h post-transfection using extracellular solution (140 mM NaCl, 4 mM KC1, 2 mM CaC12, 1 mM MgC12, 10 mM HEPES, 5 mM Glucose, pH 7.4 ajusted with NaOH, -298 mOsm / L) and intracellular solution (120 mM CsF, 10 mM NaCl, 10 mM EGTA, 10 mM HEPES, 2 mM NaATP, pH 7.2 adjusted with CsOH, -314 mOsm / 1). Alongside untransfected HEK293T cells and HEK-Navl. l stable cell line were used as controls. Activation I-V protocol was applied at room temperature, based on an initial held of the cells at -120 mV and stepped from -110 mV to +60 mV, in +10 mV increments, every 20 seconds using 50 ms sweeps. Voltage was recorded during one I-V control period followed by one I-V recording in the presence of 1 pM TTX. The voltage protocol generation and data collection were performed with PatchController384 V2.2.0 and Data Controller V2.2.0. The maximum inward current at each voltage pulse was measured for each cell and the maximum average current was calculated per condition. G / Gmax plots were obtained by normalizing the data using the equation G=I / (V-Erev) where I is the current amplitude, V is the test voltage and Erev is the reversal potential. A Boltzmann curve was fitted to the plots to obtain the activation V50.Animals:Wild-type C57B1 / 6J mice were purchased from Charles River (France) and maintained at PCB animal facility (Barcelona, Spain). Dravet syndrome (DS) mouse model was purchased from The Jackson Laboratory (stock no. 026133, conditional Scnla-A1783V mice; B6(Cg)- Scnlatml. lDsf / J) and maintained at CIMA animal facility (Navarra, Spain). Breading strategy for generation of DS mice used was described in Mora-Jimenez et. al., 2021. Briefly, breeding pairs consisted of heterozygous male Scnla-A1783V and homozygous female CMV-Cre (B6.C-Tg(CMV-Cre)lCgn / J, The Jackson Laboratory, stock no. 00605459). Details about allele modification and genotyping are described at https: / / www.jax.org / strain / 026133. All mice, both wild-type and DS, 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 intracerebroventricular (ICV) administration, mice aged P10-P15 days were anesthetized by inhalational isoflurane (induction with 3-4% isoflurane + 0.5L Ch / minute, maintenance with 1.5% isoflurane + 0.5L Ch / minute) anesthesia, whereas mice aged 4-5 weeks were anesthetized by intraperitoneal (IP) injection of ketamine (50-75 mg / kg) and medetomidine (0.5 -1.0 mg / kg). A 6-mm rostro-caudal incision was made in the skin of the posterior part of the head and animals were placed in a stereotaxic frame (Harvard Apparatus). Desired coordinates to reach ICV depending on the age of the animal were set up according to “The Mouse Brain Atlas”. For adult mice administration, a hole in the skull using a syringe was done before the introduction of the Hamilton, whereas for administration of pups the Hamilton was introduced directly. A total volume of 1.5-2.5 pL of vector dilution was injected into each lateral ventricle at a rate of 0.2 pL / minute using an infusion pump (Legatol30, KDScientific).For intravenous delivery through retroorbital administration, newborn pups aged P0-P3 days were used. Animals not anesthetized were laterally placed and 1 / 4- 1 / 3 of a needle (27-29G) was introduced at an angle of approximately 45° into the inferior pharynx or into the medial canthus. A little pressure was applied to overcome the conjunctiva and penetrate the retro-orbital sinus followed by the administration of 30 pL of solution per mice. Administration was done unilaterally in each animal.Thermal induction of seizures:Epileptogenic thermal threshold of mice was tested by induction of hyperthermia seizures as described in Mora-Jimenez et. al., 2021. Briefly, animals were assessed individually by placing them into a cylinder pre-warmed at 25°C, and then the temperature was gradually increased 0.5°C every 2 minutes until reaching 42.5°C as maximum or until the observation of a generalized seizure, time at which body temperature was immediately measured.2. Results and discussionIn 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 havesuperior properties than naturally occurring inteins. One of these inteins, termed Cfa, have 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 an engineered versions of naturally split inteins that possess greatly improved extein tolerance have been developed (Stevens et al., 2017). This intein is termed Cfamut.In order to demonstrate that Cfamut allow the reconstitution of proteins such as Navl. l which are sodium channels, via protein trans-splicing for gene therapy application, a study using different split sites was performed. Navl .1 is large protein, which is mutated in Dravet syndrome, and whose reconstitution 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 SCNIAgene 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 Site 1 (SEQ ID NO: 59 and 60). Briefly, Navl. l was first split at position 926 and the N-terminal fragment (residues 1-925, SIN) recombinantly fused to N-inteins, and the C-terminal fragment (residues 926-2008, SIC) 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. The results, see Figure 1, show that protein splicing worked in this split site.Three additional SCN1A constructs were cloned in which the SCN1A gene was split. Two of the sites did not contain a Phe on the position +2 of the split site. These three additional sites that were tested where position 948, which corresponds to a Gly at the +2 site, position 1060 which corresponds to a Met at the +2 site and position 1179 which corresponds to a Phe at the +2 site. Constructs were cloned and tested as shown above. Specifically, SCNlA(l-947)-CfaN, CfaCmut-SCNlA(948-2008) constructs were used to evaluate splicing SCNIA at position 948 (Site 2) (SEQ ID NO: 61 and 62), SCNlA(l-1059)-CfaN and CfaCmut-SCNlA(1060-2008) to evaluate splitting SCN1A at position 1060 (Site 3) (SEQ ID NO: 63 and 64), and SCN1A(1- 1178)-CfaN, CfaCmut-SCNlA(1179-2008) constructs were used to evaluate splicing SCN1Aat position 1179 (site 4) (SEQ ID NO: 65 and 66). Sites were selected based on the topological structure of Navl.l and taking into consideration the presence of folded domains. Sites were selected outside of well-defined folded domains in the intracellular side of Navl. l. These three different sites were tested, and it was observed for two of them provided high reconstitution yields (Figure 2 and 3).SCNlA(l-1059)-CfaN and CfaCmut-SCNlA(1060-2008) constructs (Site 3) and SCN1A(1- 1178)-CfaN, CfaCmut-SCNlA(l 179-2008) constructs (site 4) were co-transfected into HEK293T cells as well as full-length SCN1A (SEQ ID NO: 67) 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 show that the yield for these two sites are comparable to the protein obtained only with the full-length protein (Figure 3).The inventors also performed experiments to study the localization of the reconstituted protein using Site 3 and Site 4 and the full-length protein as a control. Results show that in both cases the protein was localized in the cell membrane which is the right localization of a sodium channel (Figure 4).To confirm that not only the protein had been reconstituted but that it was active, Patch clamp studies were performed. On this occasion, HEK293T cells were transfected with site 3 and site 4 and compared with non-transfected cells as well as cells transfected with the construction of the full-length protein. In addition to these controls, a stable cell line that expressed Navl.l was used, and the current amplitude was measured for all the conditions. Results show that the reconstituted protein using construct for site 3 and site 4 were a functional sodium channel and the results were comparable to both positive controls (stable cell line and full-length transfected cells) (Figure 5).To prevent the accumulation of undesired starting materials and to eliminate the excised intein fragments the inventors added a degron to the constructs with the general architecture shown in Figure 6.The results shown that when the inteins are combined with L9, a reduction on the expression levels of the C-terminal intein fragment is detected (Figure 7). Importantly, this does not affect the yields of the protein trans-splicing reaction (Figure 7).In order to assess whether protein trans-splicing with SCNIAgene efficiently occurs in central nervous system (CNS) after co-administration, AAV9 vectors coding for N- and C-split SCN1A gene under the control of different promoter were assessed through intracerebroventricular (ICV) injected into C57B1 / 6J WT mice aged 4-5 weeks. Results showed that reconstitution of the full-length Navl .1 protein was achieved in CNS in different brain areas after ICV injection into WT mice with all promoters tested that had different cell specificity (Figure 8). Moreover, same results of efficiently reconstitution of Navl.l protein by trans-splicing in WT mice were obtained when administering vectors ICV in younger mice aged PIO and Pl 5 days (Figure 9).The inventors not only provide evidence that the PTS technology successfully works in vivo, but also that the reconstituted Navl .1 protein behaves exactly as the endogenous Navl .1 protein of non-treated wild-type mice (Figure 10). It was observed that the size of the in vivo endogenous Navl. l protein band in western blot showed a higher migration that the one obtained from in vitro co-transfection sample, while reconstituted Navl.l protein by trans- splicing also migrated higher in the western blot, as the endogenous protein (Figure 10).To confirm that the in vivo band of the PTS reconstituted protein co-localized with the endogenous Navl. l protein of the treated WT mice, hippocampus samples were analyzed by western blot with co-incubation of a monoclonal antibody anti-HA tag and also with a monoclonal anti -Nav1.1 antibody. Results confirmed that the reconstituted Navl. l protein perfectly colocalized with wild-type Navl.l endogenous protein (Figure 11). Moreover, regarding unreacted starting material, barely any detection of N-fragment was observed in any of the animals analyzed when using and antibody that specifically recognize the tag (3FT) used in the N-fragment (Figure 12A), neither in animals that received both N- and C- vectors, nor in control animals that were injected only with AAV9-SCNlAN-CfaN. Regarding the C- fragment, same samples were reanalyzed using a polyclonal anti-Sodium Channel Pan antibody that specifically recognize an epitope located at C-terminus of Navprotein. Results confirmed that there was no unreacted C-fragment left (Figure 12B), being detected only a band corresponding to the full-length Navl .1 protein, a result expected due to the endogenous Navl .1 protein present in WT mice as well as due to the PTS AAV9-mediated treatment.The reconstitution of Navl.l protein by PTS in CNS was also evaluated at long-term. In vivo kinetic study of the expression of this protein was evaluated after bilateral ICV administration in 4-5 weeks old WT mice. Data showed that reconstitution of the full-length Navl. l protein was detected 3-months post-injection, at similar levels that at 1-month post-injection (Figure13). Sustained expression of PTS of Navl.l was detected 3-months post-injection in different brain areas, as hippocampus and cortex (Figure 13).The efficacy of the dual AAV approach was also evaluated in vivo in Dravet syndrome (DS) mouse model. To this end, newborn P0-P3 DS mice were retroorbital intravenously administered with therapeutic dual AAV9 approach. Littermates treated with saline solution were used as controls. The survival of the animals was monitored along the 3 months of the study. In addition, 2 months after administration, the epileptogenic thermal threshold of mice was evaluated by induction of hyperthermia seizures. Results indicate complete protection in survival (Figure 14A) and partial but statistically significant efficacy in reduction of thermal induced seizures (Figure 14B). The presence of reconstituted Navl. l protein was detected in different brain areas analyzed of DS mice treated intravenously with dual AAV-approach. (data not shown).

Claims

CLAIMS1. A combination of polynucleotides for use in the treatment of Sodium channel protein type 1 subunit alpha (SCN1A) associated disease in a subject in need thereof wherein the combination comprises: iii) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’ : a N-terminal fragment of Navl. l protein and N-split intein, fused directly or indirectly via a linker, iv) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-split intein and a C-terminal fragment of Navl. l protein, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in said subject generates Navl .1 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 Navl .1 protein is human Navl .1 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 Navl .1 protein up to residue 925 and the C-terminal fragment of Navl. l protein from residue 926 respectively,- the N-terminal fragment of Navl .1 protein up to residue 1059 and the C-terminal fragment of Navl. l protein from residue 1060 respectively; or- the N-terminal fragment of Navl .1 protein up to residue 1178 and the C-terminal fragment of Navl.l protein from residue 1179 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: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 22-277. 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: 28 to 58 or any functional variant thereof having at least 90% identity to any one of sequences SEQ ID NO: 28 to 58.

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, chimeric Synapsin I and CMV (Syn-CMV) promoter, minimal neuronalspecific enolase (NSEmin) promoter, chimeric murine SCN1A E2 enhancer and minimal CMV (E2min) 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 SCN1A associated disease is selected from the group consisting of Dravet syndrome, SCNIA-associated Non-dravet syndrome Epilepsy such as generalized epilepsy with febrile seizures-plus, Malignant migrating foal seizures of infancy, West syndrome, Lennox-Gastaut syndrome, Rett syndrome, Developmental and Epileptic, and SNC1 -associated nonepileptic disease such as familial hemiplegic migraine, Autism spectrum disorder, Arthrogryposis Multiplex Congenita, preferably wherein said disease is Dravet syndrome, preferably wherein said disease is Dravet syndrome, preferably wherein said combination is administered in subject by a parenteral route, more preferably by intravenous, intraarterial, intracerebral, intracerebral spinal fluid, intrathecal, intra-ci sterna magma or intracerebroventricular route.

14. A kit comprising:(i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N-terminal fragment of Nav1.1 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’a C-Cfa intein of SEQ ID NO: 2 or C-Cfamut 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 Nav1.1 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:- the N-terminal fragment of Navl .1 protein up to residue 925 and the C-terminal fragment of Navl. l protein from residue 926 respectively,- the N-terminal fragment of Navl .1 protein up to residue 1059 and the C-terminal fragment of Navl. l protein from residue 1060 respectively; or- the N-terminal fragment of Navl .1 protein up to residue 1178 and the C-terminal fragment of Navl. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Navl. l fragment and the C-terminal Navl. l 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 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 2115. 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: 22 and 23,- SEQ ID NO: 24 and 25, and- SEQ ID NO: 26 and 27.

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